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THE REGULATION AND FUNCTION OF PROGESTERONE RECEPTOR
ISOFORMS A AND B IN THE NORMAL MOUSE MAMMARY GLAND

By

Mark Douglas Aupperlee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Cell and Molecular Biology Program

2008

ABSTRACT

THE REGULATION AND FUNCTION OF PROGESTERONE RECEPTOR
ISOFORMS A AND B IN THE NORMAL MOUSE MAMMARY GLAND

By

Mark Douglas Aupperlee

Progesterone (P) is an important mitogen in the mammary gland, and it has been
implicated in increasing the risk of breast cancer. P acts through binding to its cognate
nuclear receptor, the progesterone receptor (PR), which exists as two isoforms, PRA and
PRB. The expression of PRA and PRB protein throughout BALB/c mouse mammary
gland development was performed by immunohistochemistry. PRA was the predominant
PR isoform in the virgin mammary gland, but during pregnancy PRA level decreased
while PRB level increased to significant levels. PRB was expressed in the majority of
proliferating cells during pregnancy, whereas PRA was expressed in few proliferating
cells during puberty or pregnancy.

To investigate the hormonal regulation of PR isoform expression and isoform-
specific functions, hormone ablation and replacement studies in adult BALB/c mice were
performed. Treatment with P produced extensive sidebranching and limited
alveologenesis that was enhanced by estrogen (E) + P treatment. PRA expression was
increased by E and decreased by P. PRB expression was increased by P or E+P
treatment. PRA was the predominant isoform expressed during sidebranching, and
proliferation during sidebranching primarily occurred in PRA negative cells. PRB was

predominantly expressed in alveoli, consistent with a role in alveologenesis.

In contrast to the BALB/c gland, the pregnant C57BL/6 mouse mammary gland
exhibits a delay in sidebranching and alveologenesis. It was hypothesized that this delay
was due to a reduced response to P in the C57BL/6 mammary gland. Therefore,
mammary gland development and in vivo proliferative responses to E and/or P in
BALB/c vs. C57BL/6 mice were analyzed. C57BL/6 glands have reduced sensitivity to
P as evidenced by reduced P-induced expression of PRB and Receptor Activator of NF-
KB Ligand protein expression, reduced nuclear localization of Id2, and significant
differences in nuclear cyclin D1 expression relative to BALB/c glands.

In summary, these studies characterized PR isoform expression throughout
development of the normal mouse mammary gland, determined the hormonal regulation
of PR isoforms in the adult mammary gland, and established a number of downstream

targets of P action in the mammary gland that are influenced by genetic background.

ACKNOWLEDGEMENTS

I would like to thank everyone involved with this dissertation. I would like to
thank my mentor and advisor, Dr. Sandra Haslam. She has taught me how to think
critically, to write clearly and concisely, to be a careful experimenter, to keep biological
relevance in mind in my research, and to pursue excellence. I have thoroughly enjoyed
my work with Dr. Haslam, and I have appreciated all the opportunities that Dr. Haslam
has provided me to write and to travel to conferences to present my work. I would also
like to thank the rest of my committee members, Drs. Susan Conrad, Michele Fluck, Will
Kopachik, and Donald Jump for their constructive criticism and support of my graduate
work. My committee members have done an excellent job of challenging me in my
development as a scientist, and I am grateful for that challenge.

The members of the Haslam lab, both past and present, have been wonderful to
work with, and I would like to thank them for their friendship and support. Jeff
Leipprandt, Kristen Bullard, and May Tan have provided technical support for my
studies, and I’d especially like to thank Kristen for her help in keeping the laboratory
organized. I love organization! Jianwei Xie and Anastasia Kariagina have been excellent
colleagues for the exchange ideas and in the critique of results. Kyle Smith has been a
wonderful friend and was instrumental in getting me into the Haslam lab and helping get
me started on my project; thank you for all the fun times and discussions both in and out
of the lab. Thanks to Gabriele Meyer for your support and willingness to have fun in the
lab, even when experiments are frustrating. Thanks to Alexis Drolet for your friendship,

your support, and for the great conversations in the lab that seemed to make the

iv

experiments go much faster. Although not an official Haslam lab member, I’d also like
to thank Sarah Santos for starting and finishing this whole process with me. It’s been
wonderful to share this experience with a quality scientist and person like Sarah.

Thanks to all the faculty and staff that have assisted me throughout graduate
school. Angie Zell, Christine VanDeuren, and Emily Zoeller have helped me navigate
the waters of assistantship papers, registrar forms, reimbursement papers, and insurance
papers, and have generally helped guide me through the bureaucracy at Michigan State. I
couldn’t have done it without you! Thanks to the faculty of the Breast Cancer and the
Environment Research Center for their interest and questions about my project.

I’d like to thank all of my fiiends and family who have supported me throughout
graduate school. Dan, Sandra, Nate, and Meg, thanks for fun experiences and helping me
to occasionally get out of the world of science. Thanks to all my friends at Sycamore
Creek Church for their support. Thanks to my brother, Todd, for not only being a caring
brother, but also a good friend. Thanks to my mother-in-law and father-in-law for their
love and support. I’d also like to thank my sister—in-law, Christina, for having fun with
me, supporting me, and caring about me. Mom and Dad, thank you for encouraging me
to pursue my dreams, for instilling in me the work ethic to pursue those dreams, and for
supporting and loving me along the way. Most importantly, thanks to my wife Jana for
her love, friendship, encouragement, and support. Jana is my best friend and most trusted
advisor and sharing graduate school together has been invaluable. Jana challenges me to

push myself to achieve more, and I am always grateful to be sharing life with her.

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................. viii
CHAPTER 1

Literature Review

Progesterone and Breast Cancer Risk ..................................................... 2
Progesterone Receptor: Structure ............................................................... 3
Progesterone Receptor: Regulation of Expression ............................................. 6
Progesterone Receptor: Nuclear Activity .......................................................... 7
Progesterone Receptor: Extranuclear Activity .......................................... 11
Progesterone Receptor: Detection of Expression ........................................... 12
Mammary Gland Development .................................................................... 14
The Role of Estrogen in the Mammary Gland ........................................... 21
Local mediators of Estrogen Action ............................................................. 23
The Role of Progesterone in the Mammary Gland ........................................... 25
Local mediators of Progesterone Action ................................................... 28
The Role of Pituitary Hormones in the Mammary Gland .................................. 31
The Influence of Mouse Strain on the Mammary Gland .................................. 32
Conclusion ............................................................................................. 34
References ........................................................................................ 35
CHAPTER 2

Progesterone receptor isoforms A and B: temporal and spatial differences in expression
during murine mammary gland development

Abstract ........................................................................................ 55
Introduction ....................................................................................... 56
Materials and Methods ...................................................................... 59
Results ................................................................................................ 65
Discussion ........................................................................................ 86
References ........................................................................................ 94
CHAPTER 3

Differential hormonal regulation and function of PR isoforms in normal adult mouse
mammary gland

Abstract ............................................................................................... 99
Introduction ......................................................................................... 100
Materials and Methods ............................................................................. 104
Results ............................................................................................... 108

vi

Discussion .......................................................................................... l 26
References .......................................................................................... l 36

CHAPTER 4

The mammary gland response to estrogen and/or progesterone: differential regulation of
proliferation is genetically determined

Abstract ............................................................................................. 140
Introduction ......................................................................................... 141
Materials and Methods ............................................................................. 143
Results ............................................................................................... 147
Discussion .......................................................................................... 168
References .......................................................................................... l 74
Concluding Remarks .............................................................................. 179

vii

LIST OF FIGURES

CHAPTER 1

Figure 1.1: Progesterone Receptors Structure ............................................ 5
Figure 1.2: Mammary gland cell types ................................................... 16
Figure 1.3: Mouse mammary gland development .......................................... 18
Figure 1.4: Terminal End Bud structure in the pubertal mammary gland ............... 19
CHAPTER 2

Figure 2.1: Quantitation of PRA at different stages of mammary gland development ...66

Figure 2.2:

Figure 2.3:

Figure 2.4:

Figure 2.5:
Figure 2.6:
Figure 2.7:
Figure 2.8:

Figure 2.9:

Immunoperoxidase localization of PRA at different stages of mammary. . . .67
gland development.

Quantitation of PRB at different stages of mammary gland development. ...69

Immunoperoxidase localization of PRB at different stages of mammary. . ...70
gland development

Immunodetection of PRA and PRB in wild type vs. PRA null mice .......... 72
Immunoblot analysis of PR in mammary gland ..................... _ ............. 7 4
Immunodetection of PRA by SC#538 anti-PR antibody ........................ 76
Colocalization of PRA and PRB in pregnancy .................................... 79
Quantitation of colocalization of PRB or PRA with BrdU in pregnant .......81

and virgin mammary glands

Figure 2.10: Detection of colocalization of PRB or PRA with BrdU in pregnant and ....82

virgin mammary glands.

Figure 2.11: Quantitation of colocalization of PRB or PRA with cyclin D1 in ............ 83

pregnant and virgin mammary glands

viii

Figure 2.12: Detection of colocalization of PRB or PRA with cyclin D1 in pregnant ...84

and virgin mammary glands

CHAPTER 3

Figure 3.1: Morphological response of the mammary gland to hormone treatment. . ...109

Figure 3.2: Cell type specific proliferation in response to hormone treatment ........... 111

Figure 3.3: Hormonal regulation of PRA expression in the adult mouse mammary .115
gland

Figure 3.4: Localization of PRA in proliferating cells ....................................... 116

Figure 3.5: Colocalization of PRA and cyclin D1 ............................................. 119

Figure 3.6: Hormonal regulation of PRB expression in the adult mouse mammary ....121
gland

Figure 3.7: Colocalization of PRB with BrdU or cyclin D1 ................................ 122

Figure 3.8: Colocalization of PRA and PRB with ERor ....................................... 125

CHAPTER 4

Figure 4.1: Mammary gland development in BALB/c and C57BL/6 mice .............. 148

Figure 4.2: Effect of E or P on morphology and proliferation in the BALB/c ........... 151
vs. C57BL/6 mammary gland

Figure 4.3: Effect of E + P treatment on morphology and proliferation in the .......... 153
BALB/c vs. C57BL/6 mammary gland

Figure 4.4: Hormonal regulation of PRA and PRB expression in the BALB/c ......... 156
vs. C57BL/6 mammary gland

Figure 4.5: Hormonal regulation of RANKL expression in the BALB/c vs. ............ 158
C57BL/6 mammary gland

Figure 4.6: Hormonal regulation of cyclin D1 in the BALB/c vs. C57BL/6 ............... 161

mammary gland

ix

Figure 4.7: Hormonal regulation of Id.2 in the BALB/c vs. C57BL/6 mammary ........ 163
gland

Figure 4.8: Expression and hormonal regulation of ERor in BALB/c and C57BL/6 ....165
mammary glands

Figure 4.9: Hormonal regulation of StatSa in BALB/c and C57BL/6 mammary ........ 167
glands

CHAPTER 1

LITERATURE REVIEW

LITERATURE REVIEW

Progesterone and Breast Cancer Risk

According to the 2007 information from the American Cancer Society, breast
cancer is the second most coMon cancer among American women, after skin cancer,
and it is the second leading cause of death from cancer in women, after lung cancer (1).
The chance of a woman developing invasive breast cancer during her life is about 1 in 8,
and the chance that breast cancer will cause a woman’s death is about 1 in 35. Thus, it is
important to understand the risk factors associated with the etiology and development of
breast cancer.

There are a number of reproductive factors that increase or decrease the risk of
breast cancer. These factors include the age of onset of menarche, the age at the first full
term pregnancy, the age at onset of menopause, and long-term menopausal hormone
therapy (2-6). The mechanism for these risk factors is not fully understood, but the
ovarian hormones estrogen (E) and progesterone (P) are believed to play an important
role. Henderson and Feigelson hypothesized that lifetime exposure to E is a crucial
factor increasing breast cancer risk due to the proliferative effects of E on the breast (7).
However, recent studies demonstrated that progestins, in combination with estrogens in
menopausal hormone replacement therapy (HRT), increase breast risk, whereas E alone
HRT is not associated with increased breast cancer risk (8-10). Understanding the
mechanisms of P action in the breast is particularly important since progestins are widely
used not only in HRT, but also in contraceptives and for suppression of ovarian function

in the treatment of certain pathological conditions (11, 12).

Progesterone has been shown to be an important mitogen in rodent models and in
the human breast (6, 13). In the adult human breast, DNA synthesis in the breast
epithelium, as measured by tritiated thymidine incorporation (14-16) or cell proliferation
as measured by PCNA or Ki67 expression (6) is increased during the luteal phase of the
menstrual cycle when P levels are highest. The greatest proliferative activity occurs in
the terminal duct lobular unit (TDLU), the site of origin of most breast cancers (6). The
fact that the highest proliferation during the menstrual cycle occurs during the luteal
phase indicates that P in combination with E promotes epithelial cell proliferation.
Furthermore, postmenopausal estrogen plus progestin hormonal therapy increases
proliferation in the breast above that of estrogen alone hormonal therapy (6). The ability
of P to stimulate proliferation in both the adult premenopausal and postmenopausal
human breast suggests that progestins have a potential role in the etiology of breast

cancer and in the growth of established tumors.

Progesterone Receptor: Structure

P exerts its effects through binding to the progesterone receptor (PR), which is a
member of the nuclear steroid receptor family (17, 18). PRs are activated by binding of
the ovarian hormone, P, and are classified as ligand-activated transcription factors that
regulate gene expression by binding directly or indirectly to DNA (19). When P is not
present, the PR is in a complex with several chaperone molecules, including heat shock
protein (hsp) 90, hsp70, hsp40, hsp-organizing protein (Hop) and p23 (20). Hsp70
interacts with hsp90 and this complex is involved in opening the progesterone binding

cleft of PR. p23 is a 23 kDa protein that binds to PR-hsp90 complexes once the PR has

been converted to the steroid-binding state and helps to stabilize that interaction. Hop
interacts with hsp70 and also plays a role in opening the progesterone binding cleft (20).
Upon ligand binding, the PR undergoes a conformational change, dimerization, and hsp
dissociation.

The PR is expressed as two isoforms, PRA and PRB, which are transcribed
from a single gene in both humans and rodents. Translation of PRA and PRB protein
initiates at two distinct AUG signals, which produces two proteins that are identical
except for an additional N-terminal 164 amino acids on PRB (Fig. 1.1.) (21). The
additional unique N-terminal portion of PRB is referred to as the B-upstream segment, or
BUS (22).

Regions common to both PRA and PRB include a central DNA binding domain
(DBD) and a carboxy-terrninal ligand binding domain (LBD) (Fig. 1.1). The LBD of
PRA and PRB is thought to bind P with equal affinity. PRA and PRB both contain
multiple activation function (AF) and inhibitory elements, which enhance or repress
transcriptional activation by association of these domains with transcriptional
coregulators (23). A hormone-inducible AF2 is located in the carboxy-terrninal LBD
(24). Hormone binding induces a conformational change in the PR that allows
association of AF 2 with cofactors, such as steroid receptor coactivator (SRC) SRC-l (25,
26), SRC-3, and CBP (27). A constitutively active AF 1 is located just upstream of the
DBD (24, 28). The unique region of PRB contains a third transcription activation
domain, AF3, in addition to AF 1 and AF2 that are common to PRA (29, 30). This
additional AF 3 allows the binding of a subset of coactivators to PRB that progestin-

bound PRA is unable to efficiently recruit (31). Another region common to both PRA

AF3 AFl AF 2

 

 

 

 

 

Figure 1.1. Progesterone Receptor Structure. PRB and PRA are identical steroid
hormone receptors except for an additional 164 amino acids on the N-terminus of PRB.
Both PRA and PRB contain a hormone binding domain (HBD), a hinge region (H), a
DNA-binding domain (DBD), and an inhibitory domain (ID). Activation functions (AF)
are regions that interact with co-activators. PRA and PRB contain a horrnone-
independent AF 1 and a hormone dependent AF2. The unique portion of PRB contains a

third activation function, AF3.

and PRB is an inhibitory domain at the N-terminal end of PRA and PRB upstream of
AF 1 (32). This inhibitory domain is active in PRA, and inhibits the transcriptional
activity of AFl and AF2, but is inactivated in PRB by the addition of the BUS and the
AF3 it contains (32). The BUS of PRB is thought to alter the structure of PRB and thus
its functional associations with other proteins, which leads to the difference in the

transcriptional activity between PRB and PRA (33).

Progesterone Receptor: Regulation of Expression

Studies in both the human (21) and rat (34) have shown that PRA and PRB are
produced from separate promoters. It is not known if there are two promoters in the
mouse as well. Most of what is known about the PR promoter has been learned through
examination of the human PR gene in vitro in cell lines. The MCF-7 breast cancer cell
line has been used to show that estrogen (E) upregulates expression of PR (35, 36).
However, neither the PRA nor PRB promoters contain a canonical estrogen response
element (ERE) (21). Further examination of the human PR promoter has identified a
number of sites that may mediate the estrogen responsiveness of the PR promoter. A
series of studies have found two Spl sites in the -80/-34 region (37), a +90 AP-l site
(38), and an ERE half site with two adjacent Spl sites from +571 to +595 (39, 40), that
confer estrogen receptor (ER)—mediated E responsiveness to the PR gene. In contrast to
the +90 AP-l site that increases transcription of PR, Petz et al. found an additional AP-l
site at +745 that decreases E-mediated transcription of the PR gene (41). The elements of

the PR promoter responsible for E-induced expression of PR are fairly well conserved

across species, but to date there have been no published studies examining the PR
promoter in mice.

Little is known about regulation of PR isoforms in the normal human breast. A
study examining PR in the postmenopausal breast found that PR expression is increased
by HRT with E relative to non-HRT users (6). However, that study did not address the
regulation of specific PR isoforms. Studies in breast cancer cell lines examining the
regulation of PR isoform expression by hormones have produced conflicting results. A
study by Graham et al. in T47D cells found that PRB was preferentially stimulated by E,
while both PR isoforms were downregulated by P treatment (42). However, a later report
by Vienon et al. showed stimulation of both PRA and PRB by E in T47D cells (43). This
same report also demonstrated preferential upregulation of PRA by E in MCF-7 cells and
preferential upregulation of PRB by E in ZR-75-1 cells (43). Thus, while E upregulates
PR expression in breast cancer cells, how E regulates PR isoforms expression in the
normal breast or in vivo in breast cancer is not well understood.

Studies in mice have also demonstrated an effect of E on PR expression (44, 45).
The loss of estrogen production through ovariectomy has been shown to decrease PR
levels (45), while the addition of E to an ovariectomized mouse increases PR levels (44,
45). However, the relative effect of hormones on PR isoform expression in the mouse

has not been determined.

Progesterone Receptor: Nuclear Activity
When P binds to PR it induces a conformational change that leads to dimerization,

localization to the nucleus, binding to a progesterone—responsive element (PRE), and the

recruitment of specific coregulators and general transcription factors (18). PR complexes
bound to DNA are able to increase target gene transcription through chromatin
remodeling and recruitment of the general transcription machinery to the promoter. PRA
and PRB dimers exist in three possible classes: AA, AB or B:B (46). The availability
of PRA and PRB to form dimers impacts dimer formation: a greater amount of one
isoform favors homodimer formation of that particular isoform. As mentioned above, the
region unique to PRB alters its transcriptional activity relative to PRA and PRA is
capable of inhibiting PRB, and thus the three classes of dimers can have different
transcriptional activity. Indeed a study of PRAzPRB heterodimers showed that in pure
heterodimers, PRA is a dominant negative inhibitor of PRB (46). In vitro studies have
shown that the two PR isoforms have very different effects on progestin-responsive
promoters (47, 48). PRA is a weaker activator of transcription (24) and in some contexts
PRA is also able to inhibit the activity of PRB and other nuclear receptors (48). PRB is a
stronger activator of transcription and can have transcriptional activity even in the
presence of antagonist (24, 49). The antagonist-bound transcriptional activity of PRB
appears to occur without direct binding to a PRE, but rather through interactions with
tethering proteins. In contrast to PRB, PRA is not transcriptionally active when bound by
antagonist (49).

Recent studies in breast cancer cell lines have shown that PRA and PRB generally
regulate different genes (50). Richer et al. used a PR-negative subclone of T47D cells
that were engineered to stably express either PRA or PRB to examine genes regulated by
PRA and PRB (50). There were 94 genes found to be regulated by P, and 65 of those

were regulated only by PRB, 4 were regulated only by PRA, and 25 genes were regulated

by both isoforms (50). These studies demonstrate that PRA and PRB regulate different
genes and due to the larger number of genes regulated uniquely by PRB, provide further
evidence that PRB is a more potent transcriptional activator than PRA. They also
highlight the importance of isoform-specific studies in determining PR function.
Phosphorylation of the PR has also been shown to modulate transcriptional
activity (51). In human PR, 14 constitutive and ligand-dependent phosphorylation sites
have been identified (51). Six phosphorylation sites are unique to PRB, implying that
transcriptional activity of PRB may be regulated differently by cellular kinase pathways
(52). Phosphorylation of Serines 81, 162, 190, and 400 is constitutive, even in the
absence of hormone (53). Phosphorylation of Serines 102, 294 and 345 is progestin-
dependent (51). Serine 294 has been specifically shown to be phosphorylated by
mitogen-activated protein kinase (MAPK) (54). In progestin treated T47D cells
expressing only PRB, Ser294 phosphorylation by MAPK stimulates proteasome-
dependent PR degradation (55). Paradoxically, such downregulation of PR protein
coincides with the highest PR transcriptional activity. The authors propose that
transcriptional activity of PR is tightly coupled with receptor turnover, which is regulated
by ligand-dependent phosphorylation on Ser294. In vitro, eight of the 14 sites (Serines
25, 162, 190, 213, 400, 554, 676, and Thr430) are phosphorylated by cyclin A/cyclin-
dependent protein kinase 2 (CDK2) complexes (53, 56). The function of PR
phosphorylation is not completely understood at this time, but it appears that
phosphorylation may affect interactions with co-regulators, influence nuclear localization

and receptor turnover, and alter hormone sensitivity (19).

Steroid receptor coactivators (SRC) bind the ligand-binding domain of steroid
receptors and enhance the transcriptional activities of steroid receptors. Several studies
have established that SRCs may modulate the functional activity of steroid receptors.
Studies in doubly transgenic mice expressing an indicator of PR activity and carrying the
genetically engineered disruption of either SRC-1 or SRC-3 have shown that the absence
of SRC-1 does not impair PR responses to E+P treatment in mammary epithelium,
whereas the absence of SRC-3 abrogates the PR responses after E+P treatment in
mammary gland (57). In another study, the genetically engineered loss of SRC-2
exclusively in cells expressing PR leads to impaired sidebranching and alveolar
formation after E+P treatment (58). Together, these results demonstrate that SRC
proteins modulate the physiological function of PR in mammary tissue in vivo. In the
human breast, SRC-2 is expressed in a distinct punctate nuclear pattern similar to the
pattern of PR expression (58). However the role of SRC-2 in the human breast is
currently unknown. It remains to be determined if different SRCs act in an isoform
specific manner.

The transcriptional activity of PR fluctuates throughout the cell cycle with the
highest transcriptional activity occurring during S phase (59, 60). Cyclin A/cyclin
dependent kinase 2 (cdk2) complexes have been shown to function as coactivators of PR-
dependent transcription. In HeLa cells, cyclin A increases transcriptional activity of PRA
and PRB independent of their phosphorylation status (61). In the S phase, the cyclin
A/cdk2 complex is recruited to PR bound to DNA and stimulates the additional
recruitment of coactivator SRC-1 (59). In the G1 phase when cyclin A is absent, PR

transcriptional activity and the recruitment of SRC-1 are diminished.

10

Interestingly, the majority of genes that are thought to be regulated by P do not
contain consensus PR binding sequences or PRES (50). Additionally, there are a number
of genes that are regulated by PR expression, but do so independently of P (62, 63).
Therefore, despite the depth of understanding of transcriptional activities of PRA and
PRB, the mechanism of action for the regulation of particular genes in response to P and
PR remains largely unknown. Importantly, the correlation of the regulation of specific

genes in response P and PR to changes in cell biology remains to be determined

Progesterone Receptor: Extranuclear Activity

In addition to its activity in the nucleus, PR has been shown to interact with
cytokine and growth factor signaling pathways at multiples levels to influence signaling
cascades that play important roles in mammary cell proliferation and differentiation. In
contrast to genomic effects of P treatment, which are delayed by several minutes to hours,
the nongenomic effects of P occur within minutes (19). In T47D cells expressing only
PRB, epidermal growth factor (EGF) and P act synergistically on promoters that drive the
cell-growth regulatory genes c-fos and p21, neither of which contains a PRB (64). This
synergy is believed to be mediated through a MAPK. Htunan PR has been shown to
mediate rapid activation of the Src/Ras/Raf/mitogen-activated protein kinase signaling
pathway through a Pro-Xaa-Xaa-Pro-Xaa—Arg motif located in the N-terminal domain of
both PRA and PRB (65). Mutation of the PRB DBD removed PR transcriptional activity,
but did not affect P-induced c-Src or MAPK kinase activation. Thus, the activation of

MAPK signaling pathways is distinct from PR transcriptional activity and is not

11

dependent upon the DBD of PR. Both PRA and PRB can bind to Src, but only PRB
produces strong stimulation of Src kinase activity and downstream effectors.

Another signaling pathway influenced by progesterone involves signal
transducers and activators of transcription (Stats) (64). Stat5 is important for normal
lobuloalveolar development and lactational function of the mammary gland (66).
Nuclear localization, DNA binding and regulation of target genes by Stat5 requires
phosphorylation that is induced by growth factors and cytokines, such as prolactin, via
Janus kinases. Studies in T47D cells expressing PRB only show that P acts to upregulate
Stat5 mRNA and that P/PRB action is also implicated in Stat5 nuclear localization. This
is believed to occur through a direct interaction of Stat5 and PR, and it is believed that

Stat5 is translocated to the nucleus as a companion with PR (64).

Progesterone Receptor: Detection of Expression

PR expression is primarily measured by immunoblots, real-time RT-PCR, and
immunohistochemistry. Of these methods, real time RT-PCR analysis is the most
sensitive for detection of PR isoforms. However, various studies have used primers that
claim to be specific for PRB mRNA, but are directed to the 5’ untranslated region (UTR)
of PRA mRNA, which overlaps with the PRB reading frame (67-69). In fact, such
primers amplify both PRB and PRA mRNAs since the 5’ UTR of PRA mRNA overlaps
with the PRB reading frame. Thus, the design of PRB-specific primers should be directed
against the 5’UTR of PRB mRNA. Additionally, when designing primers that detect
both PRA and PRB mRNA, the sequences between PRB and PRA translation start sites

should be used, instead of the sequences located in the DBD. Primers located in the DBD

12

may amplify PRC in addition to PRB and PRA mRNAs. For quantitative analysis, it is
additionally important that there be similar amplification efficiency of the primers used to
detect PRB vs. primers used to detect total PR (PRA+PRB) transcripts.

Biochemical methods to analyze PR isoform expression, such as immunoblot
analysis and immunoprecipitation, may provide important information about PR isoform
molecular sizes and post—translational modifications. They can provide quantitative
analysis of expression levels when used for homogeneous cell cultures, such as isolated
primary mammary epithelial cell cultures or breast cancer cell lines. However, when
used to quantify PR levels in protein extracts of whole mammary gland, an important
confounding factor is the unknown contribution of stromal proteins to the total protein in
extracts. This is particularly relevant in mammary tissues that exhibit changes in overall
epithelial content, such as the pubertal gland versus adult virgin gland versus pregnant
mammary gland. A further limitation to biochemical methods is sensitivity of detection.
For example, in the mouse whole mammary gland samples the dilution of PR present in
mammary epithelial cells by the stromal cell component that lacks PR often results in PR
levels below the limit of detection (70, 71).

Immunohistochemistry with antibodies that are specific for PRA or PRB is a
suitable method to determine the cell type-specific expression, intracellular distribution,
and colocalization of PR isoforms within the same cells. In some cases, interpretation of
studies of PR isoform expression has been confounded by lack of information about the
specificity of the antibody used to detect only PRA, only PRB or both PRA and PRB.
The study by Mote et al. in human breast tissue and cells has shown that of 11 antibodies

generated against human PR, 10 detect both PRA and PRB and 1 detects only PRB by

13

immunoblot analysis (72). However, in immunohistochemical analysis 8 of the
antibodies tested have detected only PRA, and 2 detect both PRA and PRB. Only one of
the antibodies tested has been specific for PRB. Prior to that study it has often been
assumed that antibodies that detect both PRA and PRB by immunoblot also detect both
isoforms by immunohistochemistry. Since a number of the commercially available anti-
PR antibodies detect only PRA or both PRA and PRB, the interpretation of
immunohistochemical analyses which draw conclusions about specific PR isoform
expression must be viewed in this context.

Studies in the adult human premenopausal breast using immunoblots have shown
that PRA and PRB are expressed in a 1:1 ratio (73). Immunohistochemical studies in
human breast (74), mouse (45) and rat mammary gland (75) showed that PR expression is
confined to the luminal epithelium. PR isoforms are expressed unevenly in different cell
types within the mammary gland. Analysis of PR isoform specific expression in the
normal human premenopausal breast has revealed that both PRA and PRB are expressed
in luminal cells and colocalized in the same cells (74). The average proportion of PR
positive epithelial cells in the normal human premenopausal breast is about 10-20%,
although individual ducts and lobules varied between 0 and 90% PR positivity (74). PR
isoforms have not been detected in human or mouse mammary stroma by

immunohistochemistry (45, 74).

Mammary Gland Development: Overview

Mammary gland development has been studied to learn more about the role of

hormones in vivo in the normal breast and how hormones are involved in the etiology of

14

breast cancer. The development of the mammary gland is unique because the majority of
mammary gland development occurs postnatally. With the exception of the emergence
of a primitive mammary epithelial rudiment established in the midgestational embryo,
mammary gland development primarin occurs in two distinct phases that are marked by
the onset of puberty and pregnancy (76). In humans, the mammary gland is present in
two breasts on the chest wall. In contrast, the mouse generally develops five pairs of
mammary glands. The first pair is located in the neck region near the salivary glands.
The second pair and third pair are located on the chest wall and are separated by a thin
layer of muscle. The fourth pair is located on the abdominal wall, and is the mammary
gland that is most commonly studied. The fifth pair is located in the inguinal region (77).
The mammary gland epithelium originates at the nipple and extends into the mammary
fat pad. In the human, a more complex structure is present that radiates out from the
nipple into five to ten ducts. The mouse contains a single duct that forms five to ten
secondary ducts.

The mammary gland is composed of numerous cell types that compose an
epithelial and a stromal component (Fig. 1.2). The ductal epithelium consists of two
distinct cell types: luminal epithelial cells that line the ducts and lobules, and
myoepithelial cells that form the contractile network surrounding the luminal epithelium.
Separating the epithelial component of the mammary gland from the stroma is a layer of
basement membrane. Immediately adjacent to this layer are fibroblasts, which interact
with the epithelium and secrete factors that influence epithelial cell proliferation and

migration. The adipocytes in the stroma comprise the major cell type of the mammary fat

15

 

Lil... \

epithelial .
cells Fibroblast Adrpocyte

      

Myoepithelial
cells

 

 

 

Figure 1.2. Mammary gland cell types. In this cross section of a mammary gland duct,
the luminal epithelial cells line the lumen of the duct and are directly surrounded by a
layer of contractile myoepithelial cells. The epithelimn is separated from the stroma by a
layer of basement membrane. Fibroblasts secrete the basement membrane and are
located adjacent to the basement membrane. Adipocytes fill the majority of the fat pad.
Not shown, but also present in the mammary gland, are cells found in blood and
lymphatic vessels or nerve bundles and wandering cells of the immune system.

pad. The stroma is also composed of a number of other cell types, such as those found in
blood and lymphatic vessels, nerve bundles and cells of the immune system.

The mouse is the most studied and best understood model of mammary gland
development (78) (Fig. 1.3). At birth the mammary gland exists as a small rudiment of
epithelium in the form of a ductal tree and for the first few weeks after birth, mammary
gland growth is isometric, or proportional to increases in body size (79). With the onset
of estrus cycles during the prepubertal growth period, extensive proliferation localized to
club—like structures called terminal end buds (TEB) (Fig. 1.4) drives allometric expansion
of the epithelium to fill the fat pad. Allometric growth of the mammary epithelium
generally commences at around 31 days of age (80). The TEB is composed of two cell
types. The outermost layer of the TEB is composed of cap cells, which interact with the
surrounding stroma through a thin basal lamina. The cap cells have a high proliferation
rate with very little apoptosis (81). Cap cells are thought to be progenitors of
myoepithelial cells, which are characterized by their expression of myosin (82-84). Cap
cells lack polarity and an organized cytoskeleton and are loosely adherent to one another
(85). The interior of the TEB is filled with body cells, which are thought to be more
luminal epithelial cell-like (81). In a TEB, the cap cell layer directly abuts the fat pad.
The neck of the end bud acquires more differentiated cells as ducts are formed. As the
ductal epithelium matures, extracellular matrix and fibroblasts are present in the
surrounding stromal compartment.

Hormones and growth factors control ductal proliferation at the terminal end bud
as well as bifurcation and trifurcation that lead to the formation of secondary and tertiary

branches. As the ductal network is formed, significant apoptosis occurs in the body cells

17

nipple
Pubertal fat pad

primary.
secondary,
Adult and tertiary
branching
sidebranches
Mature Adult
Pregnant
Lactating

 

Figure 1.3. Mouse mammary gland development. With the onset of puberty, the
production of estrogen and progesterone by the ovaries promotes ductal development,
which is driven by proliferation localized to bulbous structures called terminal end buds
(TEBs) at the ends of ducts. In the adult mammary gland, the ductal epithelium has
formed a network of primary, secondary, and tertiary ducts that extend to the limits of the
fat pad. In the mature adult mammary gland, successive estrous cycles induce the
formation of sidebranches that increase in number over time. Increased levels of
hormones, such as estrogen and progesterone, and the peptide hormone prolactin drive
proliferation and alveologenesis during pregnancy. The mammary gland achieves full
differentiation during lactation, when the mature luminal epithelial cells produce and
secrete milk into the alveoli that travels through the ductal network to the nipple. Upon
removal of the suckling stimulus, involution occurs and the mammary gland regressed to
a prepregnant—like state.

 

   

\ \ Basement Membrane \\/
Luminal EpitheVU Body

\ \
lial Cells
, Y

'_“__,I_ , -..94. -
I M x‘ O
- IA. ‘~ ‘
MYOepithelial .‘Qt’fi’AfiéLfio
Cells ...

Fibroblasts Adipocytes

 

 

 

Figure 1.4. Terminal End Bud structure in the pubertal mammary gland.

Illustration of a longitudinal section through a terminal end bud (TEB) showing the cap
cell layer and body cells at the leading edge of the TEB. Apoptosis in body cells farthest
away from the cap cells is critical for lumen formation. In the neck region of the end
bud, basement membrane and fibroblasts line the epithelial compartment that now
consists of differentiated luminal epithelial and myoepithelial cells.

of the TEB that is critical for normal ductal structure and lumen formation (81). As the
mammary epithelium reaches the limits of the fat pad, end buds regress and form duct
ends, and the mammary gland becomes proliferatively quiescent.

The adult mammary gland consists of a relatively simple ductal network of
primary, secondary, and tertiary ducts that has minimal morphological changes during
estrus cycles (Fig. 1.3) (77). In contrast, the adult human breast is primarily lobular,
with extensive lobuloalveolar structures present. However, there is an increase in
proliferation in the mammary gland during diestrus, when P levels are highest (86). With
each estrus cycle, the increase in proliferation is associated with the formation of
sidebranches, which are small branches off the secondary and tertiary ducts. These
sidebranches are the sites of alveolar formation. Successive estrus cycles over the
lifetime of the mouse increase sidebranching and alveolar content (Fig. 1.3) (86).

With the onset of pregnancy, levels of E and P increase (87) and the mammary
gland undergoes a second stage of proliferation. Proliferation in response to pregnancy
induces extensive sidebranching and alveologenesis. At mid-pregnancy (~day 14.5)
extensive alveoli are present throughout the mammary gland (Figure 1.3). Alveoli, the
milk-producing units of the mammary gland, are composed of luminal epithelial cells that
secrete lipids and proteins into a central lumen. The luminal epithelial cells of the alveoli
are surrounded by a basket-like structure of myoepithelial cells that surround the
alveolus. By day 16.5 of pregnancy, the alveoli have formed clusters, and dilate by the
pressure of secretions produced from the epithelial cells (88). The mammary gland
achieves fiill differentiation upon lactation (Fig. 1.3). Lactation is distinguished by two

stages (reviewed in (88)). The first stage begins at mid-pregnancy and is marked by a

20

sustained increase in the expression of genes involved in the synthesis of milk proteins
such as B-casein, lactalbumin, and whey acidic protein (WAP). During the first stage,
intracellular lipid droplets are visible. The second stage of lactation occurs around
parturition (day 21) and during this stage the expression of milk proteins increases
fiirther, the tight junctions between epithelial cells in the alveoli close, and the
cytoplasmic lipid droplets and casein are secreted into the alveolar lumen. Secreted lipids
and milk proteins travel through the ductal system to the nipple.

Mammary gland involution, an apoptotic—driven process, occurs upon removal of
the suckling stimulus, and the mammary gland regresses and loses alveolar structures.
However, the mammary gland remains permanently altered following full lactational
differentiation, as some residual alveolar structures and an increase in sidebranching

remain relative to the nulliparous mammary gland.

The Role of Estrogen in the Mammary Gland

Ductal elongation during pubertal development is largely dependent on E and
locally acting growth factors (89), while P may also play a role (80). Initial experiments
using ovariectomy to remove endogenous E production demonstrated an essential role for
ovarian hormones in ductal development and the formation of TEB structures (85). In
ovariectomized pubertal mice treated with E or progestins, E plays the essential role in
ductal elongation (90). Implantation of E pellets directly adjacent to the epithelium of
pubertal ovariectomized mice stimulates end bud formation only near the implants (91).

Interestingly, the overall changes in serum E levels are relatively minor during the

21

pubertal period when end buds are forming and active proliferation is present (80),
suggesting that factors other than E are also required for TEB formation.

E acts in the mammary gland through binding to its receptor, the estrogen receptor
(ER). There are two estrogen receptors, ERa and ERB (18). Similar to PR, estrogen
receptors generally act as ligand-activated transcription factors that regulate gene
expression by binding directly or indirectly to specific sites in the DNA (18). ER is also
thought to reside in the cell membrane or cytoplasm, and can initiate rapid responses
through interaction with various signaling pathways, such as the mitogen-activated
protein kinases (92). Studies examining the localization of ERa in end buds
demonstrated that nuclear ER was concentrated in stromal cells around end buds, but was
not present in the rapidly dividing cap cells (93).

A role in mammary gland development for E acting through ERa, has been
established using the ERa knockout (aERKO) mouse (94, 95). ERa is thought to be the
predominant receptor required to mediate the effects of E in the mouse mammary gland
(96). The mammary glands of adult aERKO mice appear similar to the glands of a
newborn. They fail to develop TEBs and ductal elongation fails, suggesting that ERa is
essential for ductal development (94, 95). Initial studies using tissue recombination of
mammary epithelium and stroma from wild-type (WT) and aERKO mice produced
contradictory results. An initial study using transplantation of neonatal tissue suggested a
necessary role for stromal ERa, whereas epithelial ERa was dispensable (94). A later
study using isolated adult mammary epithelial cells from WT and orERKO mice injected
into epithelial-free fat pads showed that both stromal and epithelial ERa are required for

mammary gland development (97, 98). These results demonstrated that neonatal and

22

adult mammary tissues use a different tissue-specific role for ERa. However, later
studies have shown that the genomic orERKO mice used in these studies are hypomorphic
for ERa (99). In these mice the ERa gene was disrupted through insertion of a neomycin
resistance gene into the first coding exon, but alternative splicing produced a variant ERa
protein that retained substantial ERa function (99). Therefore, circulating prolactin
levels were reduced in these mice (95) and restoration of prolactin was able to normalize
development. Additionally, treatment with exogenous E+P was able to induce ductal
elongation and TEB formation in these aERKO mice (98).

More recent studies using MMTV-Cre-mediated ablation of ERa that produces
no detectable ERa transcript have shown that epithelial ERa is also required for ductal
elongation and branching (100). In these same studies the use of whey acidic protein
(WAP)-Cre-mediated deletion of ERa reduced ductal sidebranching, alveologenesis, and
ductal dilation associated with pregnancy, suggesting that ERa may be not only
important for normal ductal development during puberty, but also plays an essential role
in alveologenesis during pregnancy (100). It is likely that both stromal and epithelial
ERa mediate proliferation in the mammary gland and this proliferation is induced
through a paracrine mechanism.

In contrast to ERa, which is expressed in a small subset of epithelial cells, ERB is
expressed in 70% of epithelial cells (101). Despite the high levels of ERB in the
mammary gland, ERB gene deleted mice develop normal ductal structures compared to
wild-type littermates. However, despite normal ductal development, mammary glands

from pregnant ERB gene deleted mice display increased proliferation and decreased

23

organization of epithelial cells during lactation compared to wild-type mammary glands
(102). These findings suggest a role for ERB in the terminal differentiation of the

mammary gland during lactation.

Local Mediators of Estrogen Action

During ductal growth, there are complex interactions between growth factors and
hormones leading to end bud formation and ductal elongation. Epidermal Growth Factor
(EGF) has been shown to be an essential mediator of the E response in ductal elongation.
Ankrapp et al. used neutralizing antibody to EGF to block E-induced stimulation of end
buds (103). Previous studies used ovariectomized mice treated with slow-release EGF
implants to demonstrate that EGF could substitute for E in the stimulation of end buds
(104). The EGF receptor (EGFR), a member of the ErbB receptor tyrosine kinase family
(105), is present on both epithelial and stromal cells (106). EGFR knockout mice display
inhibition of ductal growth and suggest an essential role for EGF in ductal elongation
(107). Additionally, transplant experiments placing EGFR -/- ducts into wild-type stroma
and vice versa, showed that while epithelial EGFR was dispensable for ductal outgrowth,
stromal EGFR was essential for ductal development (107). It has also been hypothesized
that stroma-derived EGF may regulate E-inducible PR in the mammary epithelium (78).

Amphiregulin is the major EGFR ligand expressed during puberty, and is
regulated by E (108). Amphiregulin is most highly expressed during puberty relative to
other EGFR ligands, and its expression localizes to epithelial cells in ducts and TEBs of
the pubertal mammary gland (109). Amphiregulin, like many other EGFR ligands, is

expressed as an inactive precursor molecule, and members of the ADAM family of

24

metalloproteinases have been shown to be responsible for the cleavage and activation of
amphiregulin in vitro (110). Studies using ADAM17 -/- mice revealed that ADAM17
plays a crucial role in mammary morphogenesis by releasing amphiregulin from
mammary epithelial cells (111). Recent studies have demonstrated that amphiregulin is
induced by E acting through ERa and that amphiregulin is required to induce
proliferation in the mammary epithelium. Similar to epithelial expression of ERor,
amphiregulin expression in the epithelium is necessary for normal epithelial cell
proliferation, TEB formation, and ductal development during puberty (108). Finally,
these studies also showed that amphiregulin is an important paracrine mediator of E
function during pubertal ductal development.

Hepatocyte Growth Factor (HGF), or scatter factor, has also been shown to be
involved in ductal elongation in the mammary gland (112). Interactions between stroma
and mammary epithelium have been studied using a minimally supplemented, serum-
free, three-dimensional collagen gel primary culture system (113). Using this system, it
has been shown that ER-expressing fibroblasts mediate E-induced epithelial cell
proliferation through HGF (114). Direct treatment of epithelial cells with B does not
produce a morphological or proliferative response. The effect of HGF on P-dependent
proliferation and alveologenesis was also examined using this system, and it was shown
that while HGF by itself induces tubule formation, HGF + progestin treatment reduces
tubule formation and induces the formation of multiltuninal alveolar-like structures
similar to those seen in vivo in response to E+P (115). Additionally, treatment with HGF
+ progestin also produces a synergistic increase in proliferation. In order to examine the

role of endogenous HGF in ductal development and alveologenesis in vivo, pellets

25

containing neutralizing antibody to HGF were implanted into the mammary glands of
pubertal or adult mice (113). In pubertal mice, HGF neutralizing antibody limited ductal
elongation, and in adult mice, ductal sidebranching was reduced by the anti-HGF
antibody. Therefore, it appears that HGF plays an important role in mediating the effects

of E in the pubertal and adult gland, and that P interacts with HGF.

The Role of Progesterone in the Mammary Gland

The pubertal mammary gland is generally less sensitive to P than the adult
mammary gland (116), and thus the primary role of P in mammary gland development is
in the adult during pregnancy, when P is essential for the induction of sidebranching and
alveologenesis (78). Studies in pubertal wild-type mice have shown that acute treatment
with E+P has only a minimal additional effect on proliferation and does not produce
sidebranching or alveologenesis, suggest that the pubertal mammary gland is less
sensitive to P than to E (90).

A requirement for P in the sidebranching response has been shown using
ovariectomized mice (89). In ovariectomized adult mice, acute treatment with E has only
a minor proliferative effect. In contrast, acute treatment with E + P significantly
increases proliferation resulting in sidebranching and the start of alveologenesis (90).
Thus, maturation of the gland in adulthood is accompanied by the acquisition of
responsiveness to P. Ovariectomy decreases PR expression and E treatment upregulates
PR expression (45). However, isoform-specific regulation of PR has not been
determined. In the adult mammary gland immunoblot studies have been used to

demonstrate that the ratio of PRA to PRB is about 3:1 (117). Immunohistochemical

26

studies of PR expression have not distinguished between PR isoforms, but suggest that
PR expression is localized to a subset of mammary epithelial cells (45, 118, 119). In the
adult gland treated with estrogen plus progesterone, it has been hypothesized that
estrogen upregulates PR expression and that proliferation is mediated by progesterone
acting through PR (78). It is thought that the early sidebranching and alveologenesis
response to pregnancy is primarily mediated by P acting through PR and that later
development of the alveoli is directed by prolactin (PRL) binding to the prolactin
receptor (PRLR) (120).

In order to examine the role of PR isoforms in mammary gland development,
mice lacking both PR isoforms (PRKO), lacking PRA (PRAKO), or lacking PRB
(PRBKO) have been generated (71 , 121, 122). The importance of P acting through PR
for sidebranching and alveologenesis has been demonstrated using the total PRKO mouse
(121). Tissue recombination studies using PRKO tissues revealed that epithelial PR
signaling is required for sidebranching and alveologenesis (123). In these same studies
chimeric epithelium composed of PR-/— cells in close vicinity to PR wild-type cells went
through complete alveolar development. Labeling of PR -/- epithelium in the chimeric
mixture showed that these cells contributed to alveolar development, suggesting that P
acts through a paracrine mechanism on a subset of mammary epithelial cells during
alveologenesis. Additionally, these results suggest that PR expression is only necessary
in a subset of epithelial cells for normal alveologenesis. Selective ablation of PRA
protein in the PRAKO mouse demonstrated that PRA is not essential for normal
mammary gland ductal development and alveologenesis. The PRAKO mouse does

contain severe abnormalities in ovarian and uterine function, so the role of PRA in

27

alveologenesis was studied through E+P treatment (122). PRB ablation did not effect
ovarian or uterine responses, but the mammary gland phenotype was similar to that of the
PRKO mouse (71). Thus, studies using the PRAKO and PRBKO mouse demonstrated
that PRB is essential for sidebranching and alveologenesis during pregnancy, whereas the
functional role of PRA was not determined (71, 122). While E plays a predominant role
in ductal elongation, careful examination of the PRKO mouse revealed a slight delay in
ductal elongation, suggesting that P also plays a role in ductal development during
puberty (124).

Transgenic mice overexpressing either PRA or PRB have also been used to
examine the role of PRA and PRB in the mammary gland (125, 126). These contain
either excess PRA (PRA transgenic) (126) or excess PRB (PRB transgenic) (125) driven
by a cytomegalovirus (CMV) promoter. PRA transgenic mice contain extensive
sidebranching in the adult mice, and also exhibit ductal hyperplasia and a disorganized
basement membrane. PRB transgenic mice were characterized by inappropriate alveolar
growth, as well as an inability of the ducts to completely fill the fat pad. Thus, the PRB
transgenic mouse further suggests a role for PRB during alveologenesis in the mammary
gland, whereas the PRA transgenic suggests a role for PRA in sidebranching. However,
expression of PR is usually only present in a subset of cells, and the inappropriate
targeting of PRA or PRB to cell types that usually do not express PR may confound the
influence of PRA or PRB overexpression on mammary gland phenotype.

In summary, P is important for sidebranching and alveologenesis during
pregnancy and these responses appear to be mediated through epithelial PRA and PRB.

A specific role for PRB in alveologenesis has been ascribed, but the role of PRA in the

28

mammary gland remains unclear. No studies have addressed the developmental and

hormonal regulation of PR isoforms during mammary gland development.

Local Mediators of Progesterone Action

There are a number of target genes that have been shown to mediate the responses
of P in viva. Msx-2, a homeobox containing transcription factor, has been shown to be
upregulated by P in T47D breast cancer cells (50) and plays a role in P-induced
branching during the peripubertal period (127). However, Msx-2 does not appear to be
involved in P-induced side branching and alveolar budding during pregnancy (127).

Calcitonin (CT), a peptide hormone involved in calcium homeostasis, has also
been shown to be regulated by P and is produced in luminal epithelial cells in the
mammary gland (128). Calcitonin acts through binding to the CT Receptor (CTR) Cla
subtype, which is a membrane-spanning G protein-coupled receptor (129). In the
mammary gland, the CTR is localized to myoepithelial cells, suggesting that CT
produced in luminal epithelial cells may influence myoepithelial cell proliferation and
organization (128). However, no direct link between CT and mammary gland
proliferation or organization has been established.

In response to P, Wnt-4 is released as a secreted factor that binds to the Frizzled
receptor (130). Originally, Wnt-4, along with Wnt-2, Wnt5a, Wnt5b, Wnt-6, and Wnt-7,
was shown to be expressed in the mammary gland, and Wnt-4 expression was reduced by
ovariectomy (131). In the canonical pathway, Wnt-4 binding to the Frizzled receptor

leads to the activation of B-catenin through stabilization, which causes accumulation in

the nucleus (132). Once in the nucleus, B-catenin is also able to activate transcription of

29

cyclin D1 (133). Overexpression of Wnt-4 in mammary epithelial cells was examined
through the use of a recombinant retrovirus to constitutively express Wnt-4 in mammary
epithelial cells transplanted into virgin animals (134). Wnt-4 overexpression results in
increased ductal sidebranching and the appearance of alveolar-like structures in these
virgin animals. The degree of mammary development in the epithelium overexpressing
Wnt-4 is similar to the development found after 10 days of pregnancy, which suggests
that Wnt-4 plays a role in mammary gland development during early pregnancy. In
agreement with this hypothesis, Wnt-4 knockout mice have been used to demonstrate an
essential role for Wnt-4 in the early sidebranching response to pregnancy in the
mammary gland (130).

Receptor Activator of Nuclear Factor kappa B (NFKB) Ligand (RANKL) has
been shown to be upregulated by P (71) and is essential for alveologenesis. RANKL is
produced by epithelial cells and secreted as a paracrine factor that can affect other
epithelial cells in an autocrine or paracrine manner through binding to Receptor Activator
of NFkB (RANK) (135). Studies using RANKL and RANK gene deleted mice have
shown an essential role for RANKL and RANK in the differentiation of mammary
alveolar cells during pregnancy. In the absence of either RANKL or RANK,
alveologenesis and lactation in the mammary gland is deficient (135). Binding of
RANKL to RANK induces the phosphorylation and degradation of IKKor and the
subsequent activation of NFkB (136). There are NFkB binding sites on the cyclin D1
promoter and NFkB activation leads to upregulation of cyclin D1 expression (137).

It has also been shown that RANKL is able to induce the nuclear translocation of

Id2, a basic helix—loop-helix (bHLH) inhibitor. RANKL does not affect Id2 protein

30

levels, but increases nuclear levels of the protein through phosphorylation of serine 5.
Interestingly, this nuclear translocation and accumulation of Id2 is impaired in RANKL
gene deleted mice (138). Id proteins, which lack a DNA binding domain but contain a
helix-loop-helix motif, act as negative regulators of bHLH transcription factors through
heterodimerization with bHLH partners (139). Once in the nucleus, Id2 is also capable of
repressing the cyclin-dependent kinase inhibitor (CDKI), p21, and thus is thought to play
a positive role in cell growth and cell cycle progression (138). While Id2 and cyclin D1
both stimulate cell cycle progression and tumorigenesis, overexpression of cyclin D1
does not rescue the defect of Id2 gene deficient mice in lobuloalveolar development
(140). Thus, the production of RANKL in response to P treatment may affect cell cycle
regulation through cyclin D1 and/or1d2.

Cyclin D1 has been proposed as a mediator of P-induced proliferation because
levels of cyclin D1 expression increase in response to P treatment and the increase is
associated with increased proliferation. Additionally, while B alone is able to increase
cyclin D1 levels and P further increases cyclin D1 levels, the increased level induced by
P is lost in the PRKO mouse, suggesting that P acting through PR is responsible for
increased cyclin D1 expression (141). Cyclin D1 is involved in cell cycle activation
through binding and activating Cdk4 and Cdk6 in the G1 phase of the cell cycle. Cdk4
and Cdk6, in turn, phosphorylated their downstream target, the retinoblastoma protein
Rb. Upon phosphorylation, pRB is inactive, allowing release of E2F and other
transcription factors, which activate the transcription of S-phase genes and cell cycle
progression (142). Studies examining the localization of cyclin D1 have demonstrated

that nuclear accumulation of cyclin D1 is required for S-phase entry (143). Cyclin D1 is

31

the primary D-type cyclin expressed in the mouse mammary gland, and its expression is
thought to primarily be controlled by mitogens. In situ hybridization has been used to
demonstrate a lack of cyclin D2 and D3 expression (144). Cyclin D1 gene deleted mice
develop normally through puberty, although a detailed examination of ductal elongation
has not been performed. During pregnancy, cyclin D1 gene deleted mice exhibit
dramatically reduced lobuloalveolar development (145, 146). As mentioned above,
increased cyclin D1 expression is linked to the Wnt4 pathway through B—catenin
activation and it is linked to the RANKL pathway through NFKB activation (133, 136).

However, the exact mechanism of how P increases cyclin D] levels is not known.

The Role of Pituitary Hormones in the Mammary Gland

Ductal elongation in the mammary gland requires the presence of E, but it also
requires the presence of the pituitary. Historical studies in ovariectomized mice showed
that the addition of growth hormone (GH) plus E was more effective at stimulating ductal
development than either hormone alone (147). More recently, mammary glands from GH
receptor gene deleted mice have been shown to fail to undergo ductal elongation (148).
GH acts on the mammary gland through local expression of insulin-like growth factor-I
(IGF-I), which has been shown by IGF-I substitution for the pituitary gland in promoting
ductal development and through increased IGF-I levels in response to GH treatment
(reviewed in (149)).

The pituitary hormone prolactin (PRL) has been called the master controller of
alveologenesis and lactogenic differentiation (88). Transplant studies using prolactin

receptor knockout mice (PRLR -/-), have shown that in the absence of PRL signaling, a

32

fully branched ductal system still develops and sidebranching and the formation of
alveolar buds occur in response to pregnancy. However, there is no lobuloalveolar
development and functional differentiation of mammary epithelial cells (150). Adding
support to the importance of PRL in alveologenesis is the similar phenotype of both janus
2 kinase (JAK2) and Signal transducer and activator of transcription 5a/b (StatSa/b) gene
deleted mice to the PRLR -/— mammary gland (151-154). JAK2 and STAT5 are both
principal downstream mediators of PRL signaling (88).

There are a number of local factors thought to play a role in mediating PRL action
in the mammary gland. Insulin-like grth factor 2 (IGF-2) has been shown to be
increased by PRL treatment both in vivo and in primary cultures of mouse mammary
epithelial cells (155). The addition of IGF2 to PRLR -/- mice restored alveologenesis,
and this is thought to occur through cyclin D1 because IGF-2 treatment induces cyclin D1
expression (155). RANKL is also thought to be a potential mediator of PRL effects in
the mammary gland. STATSa, an important mediator of PRL signaling, has been shown
to increase expression of RANKL (156). In BALB/c mice, E+P treatment induces
expression of activated nuclear STATSa, which is thought to be activated by PRL (157).
Therefore, PRL may have effects in the mammary gland that are indirect through IGF-2
or RANKL or direct through activation of JAK2 or STATS.

PRL also plays a role in the induction of P, as shown by reduced levels of P in
PRLR gene deleted mice (158). Treatment of PRL gene deleted mice with P results in
sidebranching, but not formation of alveolar buds (159). Additionally, P treatment of

PRLR gene deleted mice induces sidebranching, but not alveolar bud formation (120).

33

The Influence of Mouse Strain on the Mammary Gland

As described above, the mouse mammary gland has frequently been used as a
model for elucidating the role and function of P during normal mammary gland
development. In addition, it has been used to study the role of P in the etiology of
mammary cancer (160-162). Most in vivo studies of P action in genetically unaltered
mice have been carried out using BALB/c mice (80, 90, 116, 127, 163-166). Studies
showing that P-responsiveness is acquired when PR becomes inducible by E (90, 116),
that P is involved in branching (80, 127, 165), that P stimulates proliferation in the
mammary gland (164), and that P leads to sidebranching and alveologenesis in the adult
(90) have all been performed in BALB/c mice. More recently, however, insights into PR
isoform functions have been obtained from studies of total PR, PRA, or PRB-deficient
mice in a mixed C57BL/6 X 129SV genetic background (71, 121, 122). In fact, most
gene deleted mouse models used to study mammary gland development have been
created using this mixed genetic background (144, 150, 155, 167, 168).

Interestingly, strain-specific differences in mouse mammary gland development
(169), responsiveness to hormones, and susceptibility to carcinogenesis have been
reported (170). For example, C57BL/6 mice exhibit reduced P-induced sidebranching
and alveologenesis, and reduced susceptibility to carcinogen- and medroxyprogesterone
acetate-induced tumorigenesis compared to BALB/c mice (171, 172). There have been
few studies that examine the reduced hormonal responsiveness in the C57BL/6 mammary
gland. It has been reported that differences between C57BL/6 mice and mouse strains
exhibiting a more highly branched pattern may be attributed to different contributions of

the stroma (173). Additionally, a recent report suggests that the stroma plays a crucial

34

role in strain-specific differential hormone responsiveness (171). However, primary
culture of mammary epithelial cells from C57BL/6 mice have been shown to be less
responsive to treatment relative to BALB/c mammary epithelial cells (Leipprandt &
Haslam, unpublished observations), suggesting that the epithelium contains differences
between strains as well. Therefore, it remains to be determined what factor(s) are
responsible for differences in hormone responsiveness and tumorigenesis between mouse
strains.

It is important to consider mouse strain when examining the role of P and PR in
the normal mouse mammary gland, and a comparison of different mouse strains may

produce important information on P action in vivo.

Conclusion

Progesterone has been well established as an important mitogen in the mammary
gland. Numerous studies using genetically altered mice have identified P acting through
the PR as essential for sidebranching and alveologenesis in response to pregnancy.
However, little is known about PR isoform expression and function in the genetically
unaltered mouse mammary gland. The relative expression level and localization of PRA
and PRB protein throughout mammary gland development are not known. While a
functional role for PRB in alveologenesis has been described, a role for PRA in the
mammary gland has not been established. Both the developmental and hormonal
regulation of PRA and PRB in the mouse mammary gland are not known. Finally, the
downstream targets of PRA signaling have not been clearly elucidated. In order to more

carefully examine P action in genetically unaltered mice, the more hormonally sensitive

35

BALB/c mouse and the less sensitive C57BL/6 mouse were used to examine potential
downstream mediators of P in the mammary gland. The research presented in this thesis
establishes the developmental regulation of expression and localization of PRA and PRB,
proposes a function for PRA in early sidebranching, provides novel insight into the
regulation of PRA and PRB in vivo, and demonstrates that genetic background may play

an important role in determining P responses.

36

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105

53

CHAPTER TWO

PROGESTERONE RECEPTOR ISOFORMS A AND B: TEMPORAL AND
SPATIAL DIFFERENCES IN EXPRESSION DURING MURINE MAMMARY
GLAND DEVELOPMENT

Note: The contents of this chapter have been published in Aupperlee M.D., Smith K.T.,
Kariagina A., and Haslam S.Z. Progesterone receptor isoforms A and B: temporal and

spatial differences in expression during murine mammary gland development.
Endocrinology. (2005) Aug;146(8):3577-88.

54

ABSTRACT

Progesterone (P) is a potent mitogen in the mammary gland. Based on studies
using cells and animals engineered to express progesterone receptor (PR) isoforms A or
B, PRA and PRB are believed to have different functions. Using an
immunohistochemical approach with antibodies specific for PRA only or PRB only, we
show that PRA and PRB expression in mammary epithelial cells are temporally and
spatially separated during normal mammary gland development in the BALB/c mouse. In
the virgin mammary gland when ductal development is active the only PR protein
isoform expressed was PRA. PRA levels were significantly lower during pregnancy,
suggesting a minor role at this stage of development. PRB was abundantly expressed only
during pregnancy, during alveologenesis. PRA and PRB colocalization occurred in only a
small percentage of cells. During pregnancy there was extensive colocalization of PRB
with BrdU and cyclin D1; 95% of BrdU positive cells and 83% of cyclin D1 positive
cells expressed PRB. No colocalization of PRA with either BrdU or cyclin D1 was
observed at pregnancy. In the virgin gland PRA colocalization with BrdU or cyclin D1
was low; only 27% of BrdU positive cells and 4% of cyclin D1 positive cells expressed
PRA. The implication of these findings is that different actions of P are mediated in PRB
positive vs. PRA positive cells in vivo. The spatial and temporal separation of PR isoform
expression in mouse mammary gland provides a unique opportunity to determine the

specific functions of PRA vs. PRB in vivo.

55

INTRODUCTION

The relative roles of estrogen (E) and progesterone (P) in regulating epithelial cell
proliferation of the normal human breast and their contributions to breast cancer risk have
been controversial. Originally it was presumed that since P antagonizes E-induced
proliferation in the uterus, it would also antagonize E-induced proliferation in the breast
(1). However, P in combination with E has more potent proliferative activity than E
alone in the adult mammary gland in animal models (monkey and rodent) (2, 3) and in
the adult human breast (4). In humans this is the case for premenopausal cycling women
and for postmenopausal women receiving hormone replacement therapy (HRT). In
postmenopausal women, combined continuous E+P HRT is associated with the highest
proliferative index and the highest increase in breast epithelial density when compared to
no HRT or E alone HRT (4). Furthermore, a significantly greater breast cancer risk is
associated with E+P HRT (5-8). Thus, P can contribute significantly to breast cancer
risk.

Progesterone action is mediated through binding to the progesterone receptor
(PR). The progesterone receptor (PR) consists of two isoforms, PRA and PRB, which are
expressed from a single gene in both humans and rodents (9). Two promoters, one
specific for PRA and the other specific for PRB, have been identified for hmnan (10) and
rat (11) PR. Initiation of translation at two distinct AUG signals produces the B and A
forms of PR. PRB differs from PRA by an amino terminal extension of 164 amino acids.
Studies to identify the functional roles of PRA and PRB in the mammary gland have been

carried out in vivo using transgenic mice (PRA or PRB transgenes) (12, 13) and PR gene-

56

deleted mice [total PR (PRKO), PRA only (PRAKO) or PRB only (PRBKO)] (14-16).
From these studies it has been inferred that PRB is required for alveologenesis during
pregnancy. The specific function of PRA has not yet been identified. In vitro studies
using cell lines have shown that the unique amino terminal region of PRB encodes a
transactivation function that plays an important role in specifying target genes that can be
activated by PRB but not by PRA (17). Therefore, PRA and PRB can have different
functions in the same cell, and the activity of the individual isoforms of the receptor may
also vary among different types of cells.

The mouse is currently the most extensively studied and best understood model of
progesterone action in the normal mammary gland. Genetically altered mice have
provided some insights into the functions of the two PR isoforms in mouse mammary
gland. These genetically altered mice have an altered mammary gland phenotype (12-16);
this suggests that mammary gland development is abnormal. Our approach in the present
study was to investigate specific PR isoforms in mammary gland of genetically unaltered,
wildtype mice as a function of development.

Biochemical methods to analyze PR isoform expression and function in the
mouse mammary gland have provided limited information about the functional roles of
PRA and PRB because they do not provide insight into the cellular distribution or
colocalization of the isoforms. The most direct approach to address this question is
immunohistochemical analysis of PR isoform-specific expression. It was generally
assumed that if an anti-PR antibody detected both isoforms in immunoblot analysis, then
it also detected both isoforms in immunohistochemical analysis (16, 18, 19). The report

of Mote et al. (20) showed that this assumption is not correct. Mote et al. (20) analyzed a

57

panel of 11 anti-human PR antibodies for their ability to detect PRA and/or PRB in
human cells engineered to express specific isoforms of PR. To determine antibody
specificity, MCF-7 breast cancer cell sublines that express only PRA, only PRB or both
PRA and PRB were analyzed (20). By immunoblot analysis, 10 of the antibodies detected
both PRA and PRB; only one antibody detected only PRB. By contrast, by
immunohistochemistry, eight of the antibodies detected only PRA. These 8 antibodies
were unable to detect PRB in MCF-7 cells expressing only PRB. Two of the antibodies
detected both PRA and PRB. Only one antibody detected PRB only.

The findings of Mote el al. demonstrate the importance of using anti-PR
antibodies with well defined immunohistochemical PRA or PRB isoform specificity.
Previous studies of PR in mouse mammary gland used anti-PR antibodies that had not
been characterized for immunohistochemical PR isoform specificity (16, 18, 19). The
purpose of the present study was to determine the in vivo expression pattern of PRA and
PRB proteins in mouse mammary gland by immunohistochemistry using well
characterized, PR isoform-specific antibodies. We have used antibodies that detect only
PRA or only PRB by immunohistochemistry in human tissues and have also been shown
to have the same isoform specificity in mouse ovary (21). Using these PR isoforrn-
specific antibodies we analyzed PR isoform expression and colocalization in various
structures of the normal mouse mammary gland (ducts, end buds, side branches, alveoli)
at different developmental stages that are known to exhibit different proliferative and
morphological responses to progesterone (22-24). We also investigated colocalization of

PRA, PRB, BrdU and cyclin D1.

58

MATERIALS AND METHODS

Animals:

BALB/c female mice from our own colony were the source of mammary glands
at the following ages and developmental stages: virgin immature (3 or 6 weeks), virgin
adult (10-12 or 17-20 weeks), pregnant (7 or 14 days), lactating (10 days), or postpartum
involuting (9 weeks). To simulate mammary gland development during pregnancy, ovary
intact virgin mice received subcutaneous beeswax pellets containing 17B-estradiol (20
pg) plus progesterone (20 mg) (E+P) for 13 days. C57BL PRA null mice were obtained
from Dr. Orla Conneely (Baylor College of Medicine). All animal experimentation was
conducted in accord with accepted standards of humane animal care, and approved by the

All University Committee on Animal Use and Care at Michigan State University.

Immunohistochemistry with anti-PR isoform-specific antibodies:

Mouse monoclonal antibodies specific in immunohistochemistry for PRA only
(hPRa7; referred to as anti-PRA antibody) or PRB only (hPRa6; referred to as anti-PRB
antibody) (20, 21) were a generous gift from Dr. Christine Clark (University of Sydney)
or were purchased from Neomarkers (Fremont, CA). Mammary tissues were fixed in
10% phosphate buffered formalin (0.4% sodium phosphate monobasic and 0.65% sodium

phosphate dibasic (anhydrous) in 10% formalin) overnight at 4 C, dehydrated, cleared

59

and embedded in paraffin. Five um sections were mounted onto coverslips to which 3-
arninopropyl triethoxysilane (APES) had been applied, and allowed to dry for 24 hours at
room temperature. Tissue sections were immersed in 10 mM sodium citrate solution
(pH 6.0) and exposed to a combination of heat and pressure for antigen retrieval as
previously described (25). The protocol used to detect PRA or PRB in mouse mammary
gland was similar to that used in human breast tissue (26) and mouse ovary (21) as
described. To block non-specific background staining, sections were incubated with goat
anti-mouse IgG Fab fragments (Jackson Laboratories, West Grove, PA) (1:100 in
phosphate buffered saline (PBS) containing 1% BSA (1% PBSA), 60 min), rinsed with
PBS, and then blocked with normal goat serum (Vector Laboratories, Burlingame, CA)
(121 dil in PBS, 30 min). Incubation with primary mouse anti-PRA or anti-PRB
monoclonal antibody (12100 dil in PBS/0.5% Triton-X 100) was for 1 hr followed by 30
min with a biotinylated goat anti-mouse antibody (Dako, Carpinteria, CA) (1:400) and
ABC reagent (Vector Laboratories, Burlingame, CA). Two PBS rinses were performed
between incubation with each antibody. Immunoperoxidase localization of antibody
staining was obtained using 3'-3'- diaminobenzidene (DAB). The sections were
counterstained with hematoxylin. Sections were visualized using a Nikon Eclipse 400
microscope and a SPOT RT color camera with SPOT software (Diagnostic Instruments,

Sterling Heights, MI).

60

Double labeling with PRA and PRB isoform-specific antibodies:

When we labeled with either anti-PRA or anti-PRB antibody alone, virgin and
pregnant mammary glands yielded the same isoform-specific staining patterns whether
detection was by immunoperoxidase or immunofluorescence. However, when we double-
labeled virgin or pregnant mammary gland with the anti-PRA antibody plus anti-PRB
antibody, the PRA- and PRB-specific patterns were not maintained and all PR positive
cells were positive for both PRA and PRB. We overcame this antibody staining artifact in
double labeling experiments, by using a rabbit polyclonal anti-PR antibody, SC#538
(Santa Cruz Biotechnology, Santa Cruz, CA) that we demonstrated in this study
recognizes only PRA (see Fig. 2.7). With this method the PR isoform-specific patterns
were maintained in double labeling experiments. After antigen retrieval, sections were
incubated overnight at 4 C with SC#538 (1:400 in 2% PBSA), rinsed twice with PBS,
and incubated with goat anti-rabbit antibody conjugated to Alexa 488 (green), (Molecular
Probes, Eugene, OR) (12100 in PBS, 30 min). Sections were then blocked with goat anti-
mouse IgG Fab fragments (Jackson Laboratories, West Grove, PA) (12100 in 1% PBSA,
60 min), blocked with normal goat serum (Vector Laboratories) (1:1 in PBS, 30 min),
and incubated overnight at 4 C with mouse monoclonal primary antibody (anti-PRB,1 :50
in PBS-0.5% Triton-X 100). PRB localization was detected with goat anti-mouse
secondary antibody conjugated to Alexa 546 (red) (Molecular Probes, Eugene, OR)
( 1:100 in PBS, 30 min). In some experiments the fluorochromes used to detect PRA and

PRB were reversed. Nuclei were counterstained with TOPRO-3 Iodide (blue) (Molecular

61

Probes, Eugene, OR) and sections were visualized and images captured using a Zeiss

Pascal laser scanning confocal microscope.

Immunoblot analysis:

In the 6-week-old virgin mammary gland there is a high ratio of stroma to
epithelium. To overcome the problem of dilution of epithelial cell proteins, mammary
epithelial cells were obtained from pooled mammary glands of seven 6-week-old mice
and enriched by an enzymatic dissociation method used to obtain epithelial cells for
primary culture, as previously described (27). Whole mammary glands were obtained
from l4-day pregnant mice. Uteri were obtained from 6-week-old virgin mice. Whole
mammary glands or uteri were minced and homogenized in PEMTG buffer (50 mM
potassium phosphate pH 7.0, 10 nM EGTA, 10 mM sodium molybdate, 12 mM
thioglycerol, 10% glycerol) (lml/gm mammary tissue, 0.5 ml/uterus) containing protease
inhibitor cocktail (Sigma, St. Louis, MO) using a Polytron homogenizer. Epithelial cells
were sonicated in 400 pl PEMTG buffer. Homogenates were centrifuged at l4000g for
30 min and supernatants were used for immunoblots. Mammary gland extract (35 pl) or
uterine extract (15 pl) was mixed with NuPAGE LDS sample buffer and NuPAGE
Sample Reducing reagent (Invitrogen, Carlsbad, CA) according to the manufacturer
instruction and boiled for 10 min at 70° C. Protein samples were resolved on 4-
20%NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA) under denaturing conditions and
transferred onto Protran nitrocellulose membranes (Schleicher&Schuell, Keene, NH).

Membranes were treated with Qentix Western Blot Signal Enhancer (Pierce, Rockford,

62

IL), blocked in 5% milk in Tris-Buffered Saline with 0.5% Tween-20 overnight at 4°C
and incubated with primary antibodies for at least 2 hrs at room temperature. To detect
PR, mouse monoclonal anti-human PR hPRa7 (dil 1:100) or hPRa6 (dil 1:100)
(Neomarkers, Fremont, CA) or rabbit polyclonal anti-human PR SC#538+SC#539 (dil
1:100 for each) (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies were
used. The combination of SC#538+SC#539 was used for immunoblot analysis of
pregnant mammary gland in an attempt to enhance detection of PRA, because PRA
expression was reduced during pregnancy. The secondary antibodies were horseradish
peroxidase labeled sheep anti-mouse antibody (di1122000) (Amersham, UK) or donkey
anti-rabbit antibody (dil 1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA),
respectively. After 1 hr incubation with secondary antibodies membranes were washed,
incubated with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford,

IL) and exposed to X-ray film for 2-10 min.

Colocalization of PRA, PRB, cyclin D1 and BrdU:

For these studies mouse monoclonal anti-BrdU antibody (provided as a kit from
Amersham Biosciences, Piscataway, NJ) and mouse monoclonal anti-cyclin D1 antibody
(Cell Signaling Technology, Beverly, MA) were used. After antigen retrieval tissue
sections were incubated overnight at 4°C with mouse monoclonal anti-PRA or anti-PRB
antibody. PRA or PRB localization was detected with goat anti-mouse secondary
antibody conjugated to Alexa 546 (red) (Molecular Probes, Eugene, OR) (12100 in PBS,

30 min). Sections were then blocked with goat anti-mouse IgG Fab fragments (Jackson

63

Laboratories, West Grove, PA) (12200 in 1% PBSA, 60 min), blocked with normal goat
serum (Vector Laboratories) (1:1 in PBS, 30 min), and incubated for one hour at RT with
anti-BrdU antibody or overnight at 4 C with the anti-cyclin D1 antibody ( 1:200 in 2%
PBSA). BrdU and cyclin D1 localization were detected with a biotinylated goat anti-
mouse secondary antibody (Dako, Carpinteria, CA) (12400 in PBS, 30 min), which was
recognized by streptavidin-conjugated Alexa 488 (green) (Molecular Probes, Eugene,
OR) (1:100 in PBS, 45 min).

For all dual immunofluorescent labeling, nuclei were counterstained with
TOPRO-3 Iodide (blue) (Molecular Probes, Eugene, OR) and sections were visualized

and images captured using a Zeiss Pascal laser scanning confocal microscope.

PR quantitation and statistical analyses:

Sections treated for PRA and/or PRB detection by immunoperoxidase or
immunofluorescence methods were quantitated for the number of PRA and/or PRB
positive cells with the aid of a light microscope (immunoperoxidase) or from captured
images (immunofluorescence). Three to 10 mice per developmental stage were analyzed;
a minimum of 1000 total cells and 3 independent sections per mouse were analyzed. PR
positive cells are expressed as a percentage of total epithelial cells counted. Results are
expressed as mean i SEM and differences are considered significant at P < 0.05 by using
Student’s t test or ANOVA where appropriate.

Images in this dissertation are presented in color.

64

RESULTS

Immunoperoxidase localization and quantitation of PRA at different stages of
mammary gland development

The earliest age examined for PRA expression was 3 weeks of age. At this age
ovarian cycles have not yet started and the pre-pubertal mammary gland exists as a small
epithelial rudiment similar to the one present at birth; the percentage of PRA positive
cells was 55 d: 2% (Fig. 2.1). By 6 weeks of age, ovarian cycles have started and in the
pubertal 6-week-old virgin mammary gland 58 i 3% of mammary epithelial cells were
PRA positive (Fig. 2.1). At 6 weeks of age, the PRA positive cells were observed in end
buds (Fig. 2.2A, E) and in ducts (Fig 2.28, F). PRA positive cells in end buds were
localized in the internal layer of cells; the cap cell layer of end buds was negative for
PRA (Fig. 2.2E). At 10-12 week of age the mammary glands of most mice had grown to
the limits of the fat pad; however the glands of some mice (23%) still contained endbuds.
In the mammary glands of 10 to 12-week-old virgin mice, the percentage of PRA positive
epithelial cells in ducts was 50 d: 2 % (Figs. 2.1, 2.2C,G), which was not significantly
different from 3- or 6-week-old virgin. We also examined the effect of estrus cycle stage
on PRA expression; no difference in the percentage of PRA positive cells was observed
at estrus vs. diestrus (52 i 3% estrus vs. 51 :h 4% diestrus). At 17-20 weeks of age, in all
cases, endbuds were no longer detected and the ductal tree had grown to the limits of the
mammary fat pad. The percentage of PRA positive cells decreased significantly to 28 i 3
% (p < 0.05) (Fig. 2.1). At 7 days of pregnancy PRA was detected in 25 i 1 % of cells

(Fig. 2.1). However, at 14 days of pregnancy, PRA was detected in only 11 i 2 % of

65

 

 

 

 

 

100 _ D Duct
p < 0.05
30 z [—1 p< 0.01 I Alveolus
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60 I l—l

Percent PRA positive cells

 

 

 

“a“ lo «it x a
0' "v“
\P' {V
l l l I I l _1
Virgin Preg. Lact Invol.

Figure 2.1. Quantitation of PRA at different stages of mammary gland development.
Immunoperoxidase localization of PRA was carried out using anti-PRA antibody on
tissue sections from 3, 6, 10-12 or 17-20-week-old virgin, 7 or 14 day pregnant, 10-day
lactating mice, and at 9 weeks post weaning (lactational involution) as described in the
Methods section. The values represent the mean + SEM from 3-5 mice per group with a
minimum of 1000 cells per mouse analyzed. PRA positive cells decreased significantly
with age (3 and 6 wk > 10-12 wk > 17-20 wk) in virgin mice and further during
pregnancy and lactation (7d > 14 d > lact). The 9 week involuted mammary gland had
fewer PRA positive cells than age-matched virgin mammary gland (l7-20-week-old) (p<
0.01). No PRA staining was detected (ND) during lactation.

 

 

 

 

 

Figure 2.2. Immunoperoxidase localization of PRA at different stages of mammary
gland development. Representative sections from 6-week-old immature (A, E end bud,
B,F duct), 12-week-old adult (C, G duct), and 14 day pregnant (D, H alveoli) mouse
mammary gland were treated with anti-PRA antibody (A-H) as described in the Methods
section; control sections without antibody (1 immature end bud, J immature duct, K adult
duct, L pregnant alveoli). Higher magnification images of boxed areas in A-D are shown
in E-H. Brown stained PRA positive nuclei are indicated by black arrowheads and PRA
negative cells by red arrowheads. End bud cap cells (E) or myoepithelial cells (F, G) are
indicated by arrows (Scale bar, 50 pm).

67

ductal epithelial cells and 6.0 i 0.3% of alveolar cells (p< 0.001)(Figs. 2.1, 2.2D,H). No
PRA positive cells were detected during lactation (Fig. 2.1). After lactational involution,
at 9 weeks post-weaning, PRA was detected in 12 i 1% of the ductal cells and 10 d: 1%
regressed alveolar cells (Fig. 2.1). Notably, the percentage of PRA positive cells was
significantly lower after pregnancy compared to age-matched virgin mice at l7- 20 weeks
of age (p <0.01) (Fig. 2.1). Antibody staining of PRA was always localized to the
nucleus of epithelial cells and was not detected in myoepithelial cells or stromal cells at

any of the developmental stages studied (Fig. 2.2).

Immunoperoxidase localization and quantitation of PRB at different stages of

mammary gland development

No PRB positive cells were detected in 3, 6, 10-12 or 17-20 week-old virgin mammary
glands (Figs. 2.3, 2.4A,B,D,E). During pregnancy, no PRB was detected at 7 days, but
PRB was abundantly expressed by 14 days, in 48 :l: 4% of epithelial cells (Fig. 2.3). PRB
was localized mainly in alveolar cells (Fig. 2.4C,F). PRB staining was seen in both the
cytoplasm and nucleus of epithelial cells (Fig. 2.4F). PRB was not detected in
myoepithelial cells or stromal cells (Fig. 2.4F). No PRB was detected in the lactating
mammary gland. Afier lactational involution PRB staining was observed in 6 :t 1 % of
cells in remaining alveolar structures (Fig. 2.3); less than 1% of ductal cells were PRB

positive.

68

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ND ND ND ND ND ND
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3wk 6wk 10-12 17-20 7D 14D 10D 9wk
I wk wk J l J l J
Virgin Preg. Lact. Invol.

Figure 2.3. Quantitation of PRB at different stages of mammary gland
development. Immunofluorescence localization of PRB was carried out using anti-PRB
antibody on tissue sections from 3, 6, 10-12, or 17-20-week-old virgin, 7 or 14 day
pregnant, 10-day lactating mice, and at 9 weeks post weaning (lactational involution) as
described in the Methods section. The values represent the mean i SEM from 3-5 mice
per group with a minimum of 1000 cells per mouse analyzed. No PRB staining was
detected (N D) in the virgin mammary gland (3, 6, 10-12, or l7-20-week old), in the 7 day
pregnant mammary gland or during lactation. PRB was detected at 14 days of pregnancy
and in a smaller percentage of cells in the 9 wk involuted mammary gland (p<0.001).

69

 

 

 

 

 

 

Figure 2.4. Immunoperoxidase localization of PRB at different stages of mammary
gland development. Representative sections fi'om 6-week-old immature (A, D end bud),
12-week-old adult (B, E duct), and 14 day pregnant (C, F alveoli) mice were treated with
anti-PRB antibody (A-F) as described in the Methods section; control sections without
antibody (G immature, end bud; H adult, duct; I pregnant, alveoli). Higher magnification
images of boxed areas in A,B are shown in D,E and a higher magnification of pregnant
mammary gland is shown in F. Brown stained PRB positive nuclei (F) are indicated by
black arrowheads and PRB positive cytoplasmic staining (F) by double arrows; PRB
negative nuclei by red arrowheads. End bud cap cells (D) are indicated by arrows (Scale
bar, 50 pm).

70

PR isoform specificity of antibodies for immunohistochemistry

Although Mote et al.(20) had already demonstrated PRA and PRB isoform
specificity of monoclonal anti- PRA (hPRa7) and anti-PRB (hPRa6) antibodies,
respectively, we sought further confirmation using PRA gene-deleted mice (PRAKO)
(15). Fig. 2.5A shows that no staining was detected with the anti-PRA antibody in virgin
8-week-old PRAKO mice, whereas PRA positive cells were detected in wildtype 8-week-
old virgin mice. PRAKO mice cannot become pregnant; however, E+P treatment induces
pregnancy-like lobuloalveolar development (15). Fig. 2.5A also shows that no PRA
staining was detected in E+P-treated PRAKO mice, whereas PRA staining was detected
in E+P-treated wildtype mice.

Fig. 258 shows that PRB staining was observed in 12-week-old E+P-treated
PRAKO mice with the anti-PRB antibody, and the pattern of staining was the same as
seen in wild type E+P-treated mice. No PRB staining was detected in 8-week-old virgin
PRAKO or wildtype mice (Fig. 2.5B). These results demonstrate that the anti-PRB
antibody detects PRB only in PRAKO mice (under conditions of simulated pregnancy)
similar to wildtype mice. Thus, the staining patterns obtained in PRAKO mice confirmed
the specificity of the anti-PRA antibody to detect only PRA and the specificity of the

anti-PRB antibody to detect only PRB.

71

>
m

Anti-PRA Anti-PRB
8 week virgin 12 week 13d E+P 8 week virgin 12 week 13d E+P

PRAKO

Wl'

l——I

 

>
AAH T

Figure 2.5. Immunodetection of PRA and PRB in wild type vs. PRA null mice.
Immunofluorescence localization of (A) PRA or (B) PRB was carried out on sections
from 8-week-old virgin or 13 day E+P-treated 12-week-old virgin wild type (WT) and
PRA null (PRAKO) mice. Antibody staining was carried out with anti-PRA antibody (red
nuclei) or anti-PRB antibody (light blue nuclei); nuclei were counter-stained with
TOPRO-3 (dark blue nuclei). Positive staining is indicated by white arrowheads and
negative nuclei are indicated by yellow arrowheads. (Scale bar, 20 pm).

Immunoblot analysis of PRA and PRB expression

The apparent absence of PRB in virgin mammary gland, based upon
immunohistochemistry, was explored further by immunoblot analysis, using an antibody
that detects both PRA and PRB isoforms (Fig. 2.6A). As expected both PRA and PRB
were detected in mouse uterus immunoblot (lane 1) which is known to express both
isoforms (28). By contrast, this antibody detected only PRA in virgin mammary gland
(Fig. 2.6A, lane 2). PRB was not detected in virgin mammary gland using antibody
specific for PRB (hPRa6) (Fig. 2.6B, lane 2); the same antibody detected PRB in mouse
uterus (Fig. 2.68, lane 1). These findings are consistent with our immunohistochemical
finding of only PRA in virgin mammary gland, and indicate that absence of PRB is not
due to epitope masking.

The low level of PRA in pregnant mammary gland, based on
immunohistochemistry, was explored further by immunoblot analysis using antibody that
detects both isoforms (Fig. 2.63). Immunoblot analysis showed only a PRB band (Fig.
2.68, lane 2), consistent with immunohistochemistry showing a predominance of PRB
over PRA. Failure to detect a PRA band is likely due to the low percentage of PRA

positive cells in pregnant mammary gland.

Immunofluorescence colocalization of PRA and PRB

The different patterns of PRA and PRB expression observed during pregnancy

suggested that PRA and PRB are present in different cells. To test this hypothesis we

73

 

111 kD —> "- “ PRB
80 kD —>
C 1 2
80kD —> lie-- 2 g < PRA

Figure 2.6. Immunoblot analysis of PR in mammary gland. A. Extracts from uterus
(lanes 1) and isolated epithelial cells from 6-week-old mammary glands (lane 2) were
subjected to SDS-PAGE and blots were probed with hPRa7 anti-PR antibody as
described in the Methods section. PRA was detected a single band at 91 kD in uterus and
isolated epithelial cells (lanes 1,2); PRB was detected as a single band at 119 kD in uterus
only (lanes 1). B. Extracts from uterus (lanes 1) and isolated epithelial cells from 6-
week-old mammary glands (lane 2) were subjected to SDS-PAGE and blots were probed
with hPRa6 anti-PR antibody, which detects only PRB, as described in the Methods
section. PRB was detected as single band at 119 kD in uterus (lane 1); no PRB was
detected in isolated epithelial cells (lane 2). C. Extracts from uterus (lanes 1) and whole
14-day pregnant mammary glands (lane 2) were subjected to SDS-PAGE and blots were
probed with a mixture of SC#538 and SC#539 anti-PR antibodies described in the
Methods section. PRA was detected a single band at 91 kD in uterus only (lane 1); PRB
was detected as a single band at 119 kD in uterus and mammary gland (lanes 1, 2).

74

undertook colocalization studies with the anti-PRA and anti-PRB antibodies.
Immunofluorescent labeling of virgin and pregnant mammary glands with either anti-
PRA or anti-PRB antibody alone yielded the same isoform-specific staining patterns that
were obtained by immunoperoxidase detection. However, when we double-labeled virgin
or pregnant mammary gland with the anti-PRA antibody plus anti-PRB antibody, the
PRA- and PRB-specific patterns were not maintained and all PR positive cells were
positive for both PRA and PRB. To overcome this artifact, we sought to identify another
antibody that was specific for only PRA in immunohistochemistry.

The pattern of PRA expression that we observed in the virgin mammary gland
with the anti-PRA antibody was similar to antibody staining patterns reported by others
who used the SC#538 anti-PR antibody (18). This led us to surmise that the SC#538
antibody might in fact be PRA-isoform specific. To directly test this hypothesis, we
carried out double labeling experiments with the anti-PRA antibody plus SC#538
antibody, and used immunofluorescence confocal microscopy to investigate
colocalization of the antibodies. The results presented in Fig. 2.7A show complete
colocalization of the anti-PRA antibody with the SC#538 anti-PR antibody in the virgin
mammary gland. In the 14-day pregnant gland (Fig. 2.7B) the SC#538 antibody also
showed complete colocalization with the anti-PRA antibody and the same low level of
expression (relative to the virgin) that was observed with the monoclonal anti-PRA
antibody (Fig 2.2D,H). PRA was exclusively localized in the nucleus with the SC#538
antibody in both virgin and pregnant mammary glands. Thus, it appears that the SC#538
antibody is specific for the PRA isoform in immunohistochemistry. The SC#538 has also

been shown to be specific for PRA in immunohistochemistry in human cells (20).

75

       

B
SC#538 Ab anti-PRA Ab SC#538 Ab anti-PRA Ab
nuclei nuclei I merge I

Figure 2.7. Immunodetection of PRA by SC#538 anti-PR antibody. Tissue sections
from (A) 6-week-old virgin or (B) 14 day pregnant mammary glands were double
labeled with anti-PRA antibody (red nuclei) and SC#538 antibody (green nuclei); nuclei
were counterstained with TOPRO-3 (dark blue). In the virgin and pregnant gland the anti-
PRA and SC#538 antibody staining show complete colocalization and are visualized as
white nuclei in the merged images. (Scale bar, 20 pm)

 
   

76

Having established the specificity of SC#538 to detect only PRA, we carried out
colocalization studies of PRA and PRB in double labeling experiments with SC#538 and
the anti-PRB antibody. At 14 days of pregnancy, three subsets of cells were found: cells
that expressed PRA only, PRB only, or both PRA and PRB (Fig. 2.8A, B). Forty-three
percent of cells were positively labeled for PRB (Fig. 2.8A). Of the PRA positive cells
(8%), about half were also positive for PRB (Fig. 2.8A). Thus, colocalization of PRA

and PRB occurred in only 4% of cells during pregnancy.

PR isoform expression and colocalization with cyclin D1 or BrdU

A role for P has been implicated in ductal development in the virgin mammary gland
(19). PRB and cyclin D1 are required for alveologenesis during pregnancy (16, 29).
Epithelial cell proliferation is common to both ductal development and alveologenesis.
Having found that PRA and PRB are present in different cells and at different stages of
mammary gland development, it was of interest to determine how PR isoform expression
was related to proliferation and cyclin D1 expression. To accomplish this, mammary
glands were obtained from 14 day pregnant and 6-week-old mice injected with a pulse of
BrdU 2 hours prior to sacrifice, to label cells in S-phase. Tissue sections were double
labeled with anti-BrdU plus anti-PRA antibody or with anti-BrdU antibody plus anti-PRB
antibody. Additional tissue sections were also double labeled with anti-cyclin D1
antibody plus anti-PRA or anti-PRB antibody. Immunofluorescence confocal microscopy
was used to determine the colocalization of PRA and/or PRB with BrdU or with cyclin

D1.

77

Figure 2.8. Colocalization of PRA and PRB in pregnancy. Dual immunofluorescence
detection of PRA and PRB was carried out and visualized by laser scanning confocal
microscopy as described in the Methods section. A. Quantitation of PRA and PRB
colocalization; the values represent the mean :t SEM of the percentage of epithelial cells
expressing one isoform only (PRA or PRB) or both isoforms (PRAB);values were
obtained using 5 mice with a minimum of 1000 cells per mouse analyzed. B.
Photomicrograph of PRA and PRB colocalization. PRA (green nuclei) and PRB (red
nuclei); nuclei were counterstained with TOPRO-3 (blue nuclei). Three subsets of PR
positive cells are seen in the merged image: those expressing both isoforms (white nuclei
in square), PRA only (green nucleus in circle) or PRB only (red nuclei in oval). (Scale
bar, 20 pm).

78

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PRB only PRA only PRAB
PR isoform colocalization

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79

In the pregnant mammary gland 16% of cells were BrdU positive at 2 h post
BrdU injection, and 46% of cells were PRB positive (Fig. 2.9A). Fifteen percent of cells
were BrdU and PRB positive; thus 95% of BrdU positive cells were PRB positive (Figs
2.9A, 2.10A). In pregnant mammary gland PRA and BrdU were not colocalized in the
same cells (Figs. 2.9A, 2.108).

We also analyzed PRA and BrdU colocalization in the 6 week-old, virgin
mammary gland. We chose this age and stage of development because there is extensive
proliferation and a high percentage of PRA positive cells in the virgin mammary gland.
We found that 15% of cells were BrdU positive, 56% of cells were PRA positive and 4%
were PRA and BrdU positive (Fig. 2.98). Thus only 27% of BrdU positive cells were
PRA positive and only 7% of PRA positive cells were BrdU positive. Most BrdU
positive cells were located in the cap cell layer of end buds (Fig. 2.10D), which is a
region of the end bud that is devoid of PRA positive cells (Fig. 2.2A,E). Fewer BrdU
positive cells were present in ducts (Fig. 2.10C).

In the pregnant mammary gland 56% of cells were cyclin D1 positive and 49%
were PRB positive (Fig. 2.11A). Forty-six percent of cells were positive for both PRB
and cyclin D1; thus 83% of cyclin D1 positive cells were PRB positive and 94% of PRB
positive cells were cyclin D1 positive (Fig. 2.12A). There was no colocalization of PRA
with cyclin D1 (Figs. 2.11A, 2.128).

In the 6-week-old virgin mammary gland the percentage of cyclin D1 positive
cells was significantly less than in pregnant mammary gland (18% vs. 56%; p<0.05)

(Fig2.11A,B). Fifty-four percent of cells were PRA positive, and cyclin D1 and PRA

80

A 100 B 100

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LII
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fill

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(It
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Percent posrt1ve cells

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Pregnant Virgin

Figure 2.9. Quantitation of colocalization of PRB or PRA with BrdU in pregnant
and virgin mammary glands. Dual immunofluorescence detection of PRB or PRA and
BrdU was carried out on tissue sections from (A) 14 day pregnant and (B) 6-week-old
virgin mammary glands and visualized by laser scanning confocal microscopy as
described in the Methods section. A minimum of 1000 cells were counted for each
antibody combination tested i.e., PRB and BrdU or PRA and BrdU in pregnant mammary
gland and PRA and BrdU in virgin mammary gland. The values represent the mean +
SEM from 3-5 mice with a minimum of 1000 cells per mouse analyzed.

81

 

Figure 2.10. Detection of colocalization of PRB or PRA with BrdU in pregnant and
virgin mammary glands. Dual immunofluorescence detection was carried out in
(A,B)14 day pregnant or (C,D) 6-week-old virgin mammary gland using anti-PRB (A) or
anti-PRA (B,C,D) antibodies and TOPRO-3 nuclear stain and were visualized by laser
scanning confocal microscopy as described in the Methods section. A. PRB (red nuclei,
white arrows) and BrdU (green nuclei, white arrowheads) staining were extensively
colocalized (white nuclei, yellow arrows) in merged images. B. PRA (red nuclei, white
arrows) and BrdU (green nuclei, white arrowheads) staining did not colocalize in merged
images and were seen as red (white arrows) and light blue (white arrowheads) nuclei. C.
In 6-week-old virgin mammary gland duct PRA (green nuclei, white arrow), BrdU (red
nuclei, white arrowhead) staining did not colocalize in merged images and were seen as
red (white arrowheads) and light blue (white arrows) nuclei. D. In 6-week-old mammary
gland end bud most PRA (green nuclei, white arrow), BrdU (red nuclei, white arrowhead)
staining did not colocalize and in merged images and were seen as red (white
arrowheads) and light blue (white arrows) nuclei. End bud cap cells were prominently
labeled by BrdU (red nuclei, white arrowheads). Instances of colocalization of PRA and
BrdU are seen in merged image as white nuclei (yellow arrows). (Scale bar, 20pm).

82

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Pregnant Virgin

Figure 2.11. Quantitation of colocalization of PRB or PRA with cyclin D1 in
pregnant and virgin mammary glands. Dual immunofluorescence detection of PRB or
PRA and cyclin D1 was carried out on tissue sections from (A) 14 day pregnant and (B)
6-week-old virgin mammary glands and visualized by laser scanning confocal
microscopy as described in the Methods section. The values represent the mean + SEM
from 3 mice per group (virgin and pregnant) with a minimum of 1000 cells per mouse
analyzed for each antibody combination tested i.e., PRB and cyclin D1 or PRA and
cyclin D1 in pregnant mammary gland and PRA and cyclin D1 in virgin mammary gland.

83

Vbi“ ‘? :4'

Ali-.1213:

 

Figure 2.12. Detection of colocalization of PRB or PRA with cyclin D1 in pregnant
and virgin mammary glands. Dual immunofluorescence detection of PRB or PRA and
cyclin D1 was carried out on tissue sections from (A,B) 14 day pregnant and (C,D) 6-
week-old virgin mammary glands and visualized by laser scanning confocal microscopy
as described in the Methods section. Nuclei were counterstained with TOPRO-3 (A-D,
blue). Examples of PRB (A; red nuclei) or PRA (B,C,D; red nuclei) positive cells are
indicated with white arrows, and examples of cyclin D1 positive cells (A, B,C; green
nuclei) are indicated with white arrowheads. PRB and cyclin D1 colocalization is seen as
white nuclei in the merged image (A) and examples are indicated with yellow arrows. In
pregnant mammary gland (B) there was no colocalization of PRA and cyclin D1 in the
merged image and PRA positive nuclei stain red (white arrows) and cyclin D1 positive
nuclei stain light blue (white arrowheads). In virgin mammary gland (C) when
colocalization of PRA and cyclin D] was observed it was seen as white nuclei in the
merged image; examples are indicated with yellow arrows. (D) An example of a virgin
duct without cyclin D1 positive cells. (Scale bar, 20pm)

84

were colocalized in 1% of cells; thus 4% of cyclin D1 positive cells were also PRA
positive and 2% of PRA positive cells were cyclin D1 positive (Fig. 2.1 1B). Fig. 2.12C
illustrates colocalization in a duct that is cyclin D1 positive. Many ducts had no cyclin

D1 positive cells, yet PRA was highly expressed (Fig. 2.12D).

85

DISCUSSION

The results presented in this paper demonstrate that PRA and PRB expression are
temporally and spatially separated during murine mammary gland development. Only
PRA was highly expressed in the immature and adult virgin mammary gland. By
contrast, PRB was seen only during pregnancy, mainly in alveolar epithelial cells. During
pregnancy, the majority of PR positive cells contained only PRB and colocalization of
PRA and PRB occurred in a small proportion of epithelial cells. During pregnancy PRB
colocalized extensively with the proliferation marker BrdU and with cyclin D1. In
contrast PRA did not colocalize with BrdU or cyclin D1 during pregnancy and was
infrequently colocalized with BrdU or cyclin D1 in the virgin gland. The implication of
these findings is that different actions of P are mediated in PRB positive vs. PRA positive

cells in vivo.

Progesterone action in the virgin mammary gland: predominant role of PRA

In the 6-week-old immature virgin gland while 54 % of epithelial cells were PRA
positive, only 2% of PRA positive cells were cyclin D1 positive and only 4% of PRA
positive cells were BrdU positive. These results indicate that the majority of PRA
positive cells were not in S-phase during our 2 hour labeling period. Most BrdU positive
cells were in the cap cell layer of end buds, which is recognized to be a major growth
point. The cap cell layer was devoid of PRA positive cells, supporting the concept that

PRA positive cells do not constitute the major pool of proliferating cells. We cannot rule

86

out the possibility that P may play a role in proliferation via a paracrine mechanism in
which PRA positive cells produce a factor that affects the proliferation of neighboring PR
negative cap cells.

Proliferation leading to ductal elongation occurs via cap cell proliferation and is
mediated by E and growth factors such as EGF, HGF and IGF-1 (3, 30). The requirement
for E is supported by the complete absence of ductal elongation in ERa gene-deleted
mice (31). In contrast, ductal elongation does occur in total PR gene-deleted (PRKO)
mice (14). These results indicate that the presence of PR is not an absolute requirement
for ductal elongation in the virgin gland.

Organogenesis during embryonic development results from the net effect of the
precise spatial patterning of proliferation and apoptosis. Similarly, postnatal ductal
development in the mammary gland can be considered to be the result of spatially
organized proliferation and apoptosis. Proliferation occurs in the cap cell layer of the
endbud, giving rise to a multilayered internal mass of cells below the cap cell layer (32).
Formation of the ductal lumen requires the removal of this internal cell mass. Apoptotic
cells have been observed in this internal layer of cells of the end bud (32), suggesting that
apoptosis may play a key role in lumen formation in ducts. We have previously shown in
vitro that mammary organoids derived from virgin mammary gland respond to the
synthetic progestin, R5020, by forming a lumen (30). Treatment of organoids with
R5020 induces apoptosis that is spatially localized within mammary organoids and
centrally within luminal structures; R5020 does not induce proliferation in these
organoids (30). Based on these observations we have hypothesized that one of the

actions of P in mammary gland development is to facilitate lumen formation through P-

87

induced apoptosis (30). In the present study we showed that only PRA was expressed in
the virgin gland, and within endbuds PRA positive cells were localized in the internal
layer of cells. This raises the possibility that one way that P promotes ductal development
in the virgin gland, at least in part is by facilitating lumen formation through a pro-
apoptotic mechanism mediated by PRA. The observation that ductal development can
occur in total PR deleted as well as PRA gene-deleted mice indicates that there are
additional mechanisms that promote lumen formation, and that these mechanisms are

operative in PR gene-deleted mice and may compensate for the lack of PR.

Progesterone action in pregnancy: predominant role of PRB

PRB positive cells were seen only in mammary glands of pregnant mice (Figs.
2.4C, 2.8B,C) or in alveolar structures of adult E+P-treated mice (Fig. 2.58). In pregnant
mice PRB was abundantly expressed and the PRB positive cells were localized mainly in
alveolar structures. We found extensive colocalization of PRB with BrdU and cyclin D1
in pregnant mammary gland. This indicates that PR8 positive cells are in the proliferative
pool of cells and express cyclin D1. Our results indicate that PR8 has the primary role in
inducing alveologenesis. Other studies have inferred the same conclusion based upon
different approaches, namely, that there is no defect in alveologenesis in the PRAKO
mouse (15), that there is a lack of alveologenesis in the PRBKO mouse (16), and that
precocious alveologenesis occurs in PRB over-expressing transgenic mice (13).

In contrast to PRB, there was no PRA colocalization with either BrdU or cyclin

D1 in the pregnant gland, suggesting that PRA positive cells do not constitute the major

88

proliferative pool in the pregnant mammary gland. These observations do not discount
the possibility that PRA nevertheless plays a role in pregnancy since expansion of the
epithelium and sidebranching are detected as early as day 7 of pregnancy (unpublished
observations, Aupperlee & Haslam), when 27% of the epithelial cells were PRA positive
and none were PRB positive (Figs. 2.1,2.3).

Previous studies have reported a lack of colocalization of PR with markers of
proliferation (16, 33). However, in those studies the PR isoform specificity of the
antibody used (DAKO A0098) was not identified. We have determined that the DAKO
A0098 anti-PR antibody colocalizes with PRA and not with PRB. This was determined in
studies carried out as shown for the SC#538 anti-PR antibody (Fig.2.7) (unpublished
observations, Aupperlee & Haslam). Our own studies using PR isoform-specific
antibodies demonstrate a lack of colocalization of PRA with BrdU in pregnant mammary
gland, but we find extensive colocalization of PRB with BrdU and cyclin D1 in pregnant
mammary gland.

The lack of PRA and PRB staining during lactation is in agreement with previous
reports of the absence of specific P ligand binding and lack of detectable PR mRNA in
lactating mouse mammary gland (28, 34). Although PRA positive cells were detected
again post-involution, the percentage of PRA positive cells never returned to the pre-
pregnancy virgin level. This was not due to aging since the percent of PRA positive cells
was significantly higher in 20-week-old virgin mammary glands than age-matched parous
mice. A low level of PRB (6 % PRB positive cells) was detected in alveolar structures
after lactational involution, but not in age-matched virgin mammary gland. These results

demonstrate that expression of both PRA and PRB is permanently altered by pregnancy.

89

Pregnancy is protective against carcinogen-induced mammary tumors in mice and rats
(3 5). Our results show two important changes caused by pregnancy: a reduction in PRA
positive cells relative to age-matched virgins, and presence of PRB post lactation relative
to the virgin state. Further studies to elucidate the specific functional roles of PR isoforms
in the mammary gland before, during and after pregnancy may provide new insights
about the mechanism(s) underlying differences in susceptibility to tumorigenesis of

virgin vs. parous mice.

PR isoform subcellular localization and progesterone action

Previous studies using cell lines have shown that if expressed in the same cells,
PRA and PRB proteins can dimerize and bind to DNA as three different species: AA or
88 homodimers or A8 heterodimers (9). The specific contribution of each of the dimers
to the effects of P may be dependent on the transactivation properties contributed to the
complexes by the PRB-specific domain. It has also been reported that PR8
transcriptional activity is inhibited by PRA. During pregnancy we found that the vast
majority of PRB positive cells contained only PRB and only a small percentage of cells
(4%) contained both PRA and PRB. Our results indicate that the prevailing situation in
the mouse mammary gland is that cells contain AA or 88 homodimers, and that the
potential for A8 heterodimer formation is limited to a small number of cells during
pregnancy. This suggests that in the mouse, heterodimer formation does not play a major

role in progesterone action in the mammary gland.

90

In our study, PRB was detected primarily in the nucleus and in some cells faintly
in the cytoplasm (see Fig. 2.4F). In contrast, using the anti-PRA and SC#538 anti-PR
antibodies, PRA was detected only in the nucleus. We cannot rule out the possibility that
there may also be a cytoplasmic form of PRA not detected by the PR antibodies we used.
PR localization in the mammary gland in both cytoplasm and nucleus has been detected
using a PR antibody of unknown PR isoform specificity (18) and in human T47D breast
cancer cells overexpressing either PRA or PRB (36). It is conceivable that different anti-
PR antibodies may detect epitopes that are exposed on the cytoplasmic, nuclear, or both

forms of the receptor.

PR isoform expression in the human vs. mouse

In the human breast, immunohistochemical analysis of PR isoform expression has
been carried out on normal tissue from premenopausal cycling women (26). In that study
PRA or PRB expression and colocalization were determined by dual
immunofluorescence with the same antibodies used herein (26). PRA vs. PRB were
expressed at a ratio of 1:1, and patterns of expression were similar. The proportion of PR
positive cells was 10-20% with marked variability throughout a section, with PR
positivity in individual ducts or lobules ranging from 0-90%. Dual immunofluorescence
studies revealed uniform colocalization of PRA with PRB (26). Our study indicates an
interesting difference in PR isoform expression between the mouse and the human
mammary gland. In the mouse there is PRB expression only during pregnancy, and

colocalization of PRA with PR8 occurs in only a small percentage of cells. One possible

91

explanation for this difference may be the predominance of a ductal organization of
mammary epithelium in the adult non-pregnant mouse. This is particularly true in
BALB/c strain mice, used in our study. In contrast, in the adult non-pregnant human
there is a higher ratio of lobules to ducts. Studies of PRB null mice have shown that PRB
expression is required for alveologenesis and lobule formation (16). Therefore, PRB
expression may be a defining characteristic of mammary lobule formation and/or
maintenance and may explain why PRB positive cells are more abundant in the human
breast. In this regard, the maintenance of some alveolar structures in mouse mammary
gland after pregnancy may also be due to the continued, albeit reduced, expression of
PR8 after pregnancy. Analysis of other mouse strains, such as the C3H strain, which
develop a more lobular morphology in the virgin state (compared to BALB/c strain) may
provide additional insights into the relationship between alveolar morphogenesis and
PRB expression. It is also important to note that PR isoform expression in the human has
only been studied in the adult premenopausal breast. There is no information on PR
isoform expression at other stages of human breast development such as puberty or
pregnancy. It remains to be seen what analysis of these other stages may reveal about the
pattern of PR isoform expression and/or colocalization in the human breast. Clearly more
information is needed about PR isoform expression in the human breast.

Understanding the specific functions of PRA and PRB isoforms in vivo is critical
to understanding their respective roles in the normal breast and in the etiology of breast
cancer. The spatial and temporal separation of PRA and PRB isoform expression in
mouse mammary gland offers a unique opportunity to explore further the specific

functions and mechanisms of action PRA vs. PRB in vivo.

92

ACKNOWLEDGEMENTS

The authors thank Alexis Drolet, Jeff Leipprandt, Nityanand Sunil, and Yvette Gross for
technical support of these studies. This work was supported by the Breast Cancer and the
Environment Research Centers Grant U01 ES/CA 012800 from the National Institute of
Environment Health Science (NIEHS) and the National Cancer Institute (NCI), National
Institutes of Health, Department of Health and Human Services (to S.Z.H.). Its contents
are solely the responsibility of the authors and do not necessarily represent the official
views of the NIEHS or NCI, NIH. This work was also supported by Department of
Defense Breast Cancer Research Program Fellowships DAMD17-03-1-0605 (to M.D.A.)

and DAMD17-02-1-0488 (to K.T.S.).

93

10.

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CHAPTER 3

DIFFERENTIAL HORMONAL REGULATION AND FUNCTION OF PR
ISOFORMS IN NORMAL ADULT MOUSE MAMMARY GLAND

Note: The contents of this chapter have been published in Aupperlee, MD. and Haslam,
S.Z. Differential hormonal regulation and function of PR isoforms in normal adult
mouse mammary gland. Endocrinology. (2007) May;l48(5)22290-300.

98

ABSTRACT

In normal mouse mammary gland, the mitogenic action of progesterone (P) is
mediated by two progesterone receptor (PR) isoforms, PRA and PRB. PRA is
predominantly expressed in the adult virgin, and PRB is predominantly expressed during
pregnancy. To investigate hormonal regulation of PR isoform expression and isofonn-
specific functions in vivo, adult ovariectomized BALB/c mice were treated for 3, 5, or 10
days with estrogen (E), P, or E+P. Using an immunohistochemical approach with
isoform-specific antibodies, we investigated hormonal regulation of PRA and PRB and
their functional roles in proliferation and morphogenesis. Significant E-induced
proliferation was only observed after 5 days at the distal tips of ducts; there was no
sidebranching or alveologenesis. P induced proliferation that resulted in sidebranching
and alveologenesis, but E+P treatment produced more proliferation sooner and more
extensive sidebranching and alveologenesis. PRA levels were increased by E and
decreased by P. Increased PRB levels were induced by treatment with P or E+P and
coincided with the formation of alveoli. PRA was the predominant PR isoform expressed
during sidebranching, and colocalization of PRA with BrdU revealed that proliferation of
PRA positive and negative cells were responsible for P-induced sidebranching. PRB was
the predominant PR isoform expressed during alveologenesis, and colocalization of PRB
with BrdU showed that both PRB positive and negative cells proliferated during alveolar
expansion. These results demonstrate different hormonal regulation of PRA and PRB
levels in vivo and suggest that P can induce proliferation through either PRA or PRB via

direct and paracrine mechanisms.

99

INTRODUCTION

Progesterone (P) plays an important role in regulating proliferation and
differentiation in the normal mammary gland (1). Proliferation is regulated by P in the
adult human breast (2) as well as in rodent and monkey models (1, 3). Progesterone acts
through binding to its cognate nuclear receptor, the progesterone receptor (PR), and PR
exists as two isoforms, PRA and PRB, which are identical except for a 164 amino acid N-
terminal extension on PRB. PRA and PRB can regulate different genes and have
different functions (4). Studies using PRB knockout mice, PRB transgenic mice, and
immunohistochemical analysis of PRB expression in wild-type mice indicate a role for
PRB in alveologenesis (5-7). The role of PRA in the mammary gland is less clear, but
studies with PRA transgenic mice and immunohistochemical analysis of PRA expression
in wild-type mice indicate that PRA may be involved in ductal development and
sidebranching (7, 8).

To date, PR expression and progesterone action in the normal mammary gland
have been most extensively studied in the mouse model. PRA and PRB expression are
temporally and spatially separated during murine mammary gland development from
puberty through pregnancy, lactation, and involution (7). Our previous studies of
developmental expression of PR isoforms found that PRA is predominantly expressed in
the virgin mouse mammary gland, which is primarily composed of ducts, whereas
expression of PRB is induced in alveolar structures upon lobuloalveolar development
during pregnancy along with a concomitant decrease in PRA expression (7).

Additionally, PRA and PRB are infrequently expressed in the same cell. In contrast, in

100

the normal adult premenopausal human breast PRA and PRB are coexpressed in the same
cells and the ratio of PRA:PRB in individual PR positive cells is 1:1 (9). The pattern and
level of PRA and PRB expression at other stages of human mammary gland development
are not known. However, breast cancers exhibit an altered PRAzPRB ratio with a higher
PRAzPRB ratio associated with less differentiated and more aggressive tumors (9). The
mechanism(s) that regulate the relative expression of PRA and PRB in breast cancer is
not known.

Little is currently known about the hormonal regulation of PRA and PRB
expression in the normal breast. Studies using human breast cancer cell lines have shown
that E induces expression of PR, and P has been shown to downregulate expression of PR
(10, 11). However, it is not clear how individual PR isoforms are regulated. It has been
reported that ovariectomy reduces PR expression in the mouse mammary gland (12).
While a role for estrogen (E) in regulation of PR has been determined (12, 13), isoforrn-
specific regulation of PRA and PRB has not been examined. The effects of E and/or P on
PR isoforms in vivo in the normal human breast have not been well studied. Since
alterations in PRAzPRB ratios are associated with breast cancer progression (9),
understanding the normal regulation of PRA and PRB may provide insight into the
deregulation that occurs in breast cancer.

Biochemical analyses of PR expression, such as immunoblots, can provide useful
information about the molecular sizes of protein isoforms or protein post-translational
modifications. However with regard to analysis of whole mammary gland extracts,
immunoblots can be limited in their sensitivity and accuracy for PR detection and

quantitation due to the dilution of epithelial proteins by stroma-derived proteins. This

101

occurs because PR is expressed in the epithelial compartment of the gland, whereas the
stromal compartment is PR negative (4, 7). This is particularly relevant for quantitation
of relative expression of PRA or PRB in mammary tissues that exhibit changes in overall
epithelial content, such as after ovariectomy versus treatment with pregnancy levels of E
+ P. In the first case, the gland exists as a rudimentary ductal system with a
predominance of stroma, whereas after E+P treatment there is a proliferative expansion
of the epithelium in the form of sidebranches and alveoli and an overall increase in the
ratio of epithelium to stroma. Thus, the same amount of protein from mammary gland
extracts obtained at different physiological states represents different amounts of
mammary epithelium.

In the present study, we used an immunohistochemical approach with antibodies
specific for PRA or PRB (7, 14) to examine the hormonal regulation of PRA and PRB
and the roles of PRA and PRB in mediating proliferative and morphological responses in
the adult mouse mammary gland. One advantage of this approach is that it allowed
analysis and quantitation of the cellular distribution of PR isoforms and their
colocalization with proliferation markers. We found that PRA expression was increased
by E and decreased by P. The initial proliferative response to P, leading to sidebranching,
was mediated by PRA. Proliferation and PRB expression were induced by P alone, but
were accelerated and enhanced by the combination of E+P. Induction of PRB expression
coincided with decreased PRA levels and the onset of alveologenesis. Analysis of ERa
expression revealed that only PRA was extensively colocalized with ERor. These studies

demonstrate differences in the hormonal regulation of PRA and PRB and isoform-

102

specific roles in mediating proliferation and differentiation in the normal mouse

mammary gland.

103

MATERIALS AND METHODS

Animals:

Mammary glands were obtained from adult (19 to 22-week-old) BALB/c female
mice purchased from Harlan (Indianapolis, IN). Hormone treated adult virgin mice were
ovariectomized (OVX) and one week after OVX animals were injected for 3, 5, or 10
days with saline control (C), l7-|3-estradiol (E) (1 pg/injection), progesterone (P) (l
mg/injection), or E+P (1 pg + 1 mg respectively/injection) administered subcutaneously.
Two hours prior to sacrifice mice were injected with 5-bromo-2’-deoxyuridine (BrdU)
(70 pg/g of body weight) to label proliferating cells. All animal experimentation was
conducted in accord with accepted standards of humane animal care and approved by the
All University Committee on Animal Use and Care at Michigan State University.
Mammary tissues were fixed and processed as whole mounts (15) or formalin fixed and

paraffin-embedded for immunohistochemistry as previously described (7).

Immunohistochemistry with anti-PR isoform specific antibodies:

The protocol used to detect PRA and PRB was the same as previously described
(7). Tissue sections were treated with a combination of heat and pressure for antigen
retrieval and then blocked with goat anti-mouse IgG Fab Fragments (Jackson
ImmunoResearch Laboratories, West Grove, PA) [12100 in PBS containing 1% BSA (1%

PBSA), 60 min], blocked with normal goat serum (Vector Laboratories, Burlingame,

104

CA)(1:1 in PBS, 30 min), and then incubated with primary antibody against PRA
(hPRa7, Neomarkers, Fremont, CA) (1250 in PBS/0.5% Triton X-100, overnight, 4 C) or
against PRB (hPRa6, Neomarkers) (1:50 in PBS/0.5% Triton X-100, overnight).
Sections were rinsed with PBS/0.5% Triton X-100 and the primary antibody was
recognized by goat anti-mouse antibody conjugated to Alexa 488 (Molecular Probes,
Eugene, OR) (12200 in PBS, 30 min).

When immunostaining to recognize PRA, nuclei were counterstained with 4’,6-
diamidino-2-phenylindole, dilactate (DAPI) (Molecular Probes) (1:10,000 in H20), and
sections were visualized and images captured using a Nikon inverted epifluorescence
microscope (Mager Scientific, Dexter, MI) with MetaMorph software (Molecular
Devices Corporation, Downington, PA). When immunostaining to recognize PRB,
nuclei were counterstained with TOPRO-3 Iodide (Molecular Probes) (121000 in
fluorescent mounting media) and sections were visualized and images captured using a

Zeiss Pascal laser scanning confocal microscope (Zeiss, Thornwood, NY).

Colocalization of PRA or PRB with BrdU, cyclin D1, or ERa:

Double labeling of PRA or PRB with BrdU and cyclin D1 was performed as
previously described (7). Briefly, after antigen retrieval, sections were blocked and
incubated with anti-PRA or PRB antibody (overnight, 4 C) as described above.

For colocalization with BrdU, sections were first stained for PRA or PRB, and

then PRA or PRB localization was detected with a goat anti-mouse secondary conjugated

to Alexa 488 (Molecular Probes) (1:200 in PBS, 30 min). Next, sections were blocked

105

with goat anti-mouse IgG Fab fragments (Jackson Immunoresearch Laboratories) (1:100
in 1% PBSA, 60 min), blocked with normal goat serum (Vector Laboratories) (1:1 in
PBS, 30 min) and incubated for 60 min at room temperature with anti-BrdU antibody (kit
from Amersham Biosciences, Piscataway, NJ). BrdU localization was detected with a
biotinylated goat anti-mouse secondary (Dako, Carpinteria, CA) (12400 in PBS, 30 min),
which was recognized by streptavidin-conjugated Alexa 546 (Molecular Probes) (1:100
in PBS, 30 min).

For colocalization with cyclin D1, sections were first stained for PRA or PR8,
and then PRA or PRB localization was detected with a goat anti-mouse secondary
conjugated to Alexa 546 (Molecular Probes) (12200 in PBS, 30 min). Sections were then
blocked with 2% PBSA for 30 min and incubated overnight at 4 C with rabbit polyclonal
anti-cyclin D1 antibody (Biosource, Camarillo, CA) (1:100 in 2% PBSA). Cyclin D1
localization was detected with a goat anti-rabbit antibody conjugated to Alexa 488
(Molecular Probes) (1:200 in PBS, 30 min).

For colocalization with ERa, sections were first stained for PRA or PRB, and
then PRA or PRB localization was detected with a goat anti-mouse secondary conjugated
to Alexa 488 (Molecular Probes) (1:200 in PBS, 30 min). Next, sections were blocked
with goat anti-mouse IgG Fab fragments (Jackson Immunoresearch Laboratories) (1:100
in 1% PBSA, 60 min), blocked with normal goat serum (Vector Laboratories) (1:1 in
PBS, 30 min) and incubated overnight at 4 C with mouse monoclonal anti-ERa antibody
(NCL-L-ER-6Fll) (Novocastra, Newcastle, United Kingdom). ERa localization was

detected with a biotinylated goat anti-mouse secondary (Dako, Carpinteria, CA) (12400 in

106

PBS, 30 min), which was recognized by streptavidin-conjugated Alexa 546 (Molecular
Probes) (1:100 in PBS, 30 min).

For all dual-immunofluorescence labeling, nuclei were counterstained with DAPI
(Molecular Probes) (1 :10,000 in H20), and sections were visualized and images captured
using a Nikon inverted epifluorescence microscope (Mager Scientific, Dexter, MI) with

MetaMorph software (Molecular Devices Corporation, Downington, PA).

Quantitation of fluorescence and statistical analyses:

Sections treated for detection of PRA, PRB, BrdU, or cyclin D1 by
immunofluorescence methods were quantitated for the number of positive luminal
epithelial cell nuclei from captured images using MetaMorph software. Positive nuclei
displayed staining above luminal epithelial cytoplasmic background. To analyze
fluorescence intensity, the average pixel intensity of all positively stained nuclei within
the ductal epithelium was determined. Images were thresholded to exclude background
fluorescence and gated to include intensity measurements only from positively staining
epithelial cells. Six mice per treatment group were analyzed; a minimum of 1000 total
cells and three independent sections per mouse were analyzed. Results are expressed as
mean +/- SEM, and differences are considered significant at P < 0.05 by using Student’s t
test.

Images in this dissertation are presented in color.

107

RESULTS

The most dramatic changes in mammary gland morphology, proliferation and PRA
and PRB expression occur in the adult mammary gland in response to pregnancy (7).
Thus, we treated adult ovariectomized mice with pregnancy levels of estrogen (E),
progesterone (P) or E + P for a total of 3, 5, or 10 days, and studied the hormonal
regulation of PRA and PRB expression and their relationship to hormonal regulation of

proliferation and alveolar morphogenesis.

Morphological responses of the mammary gland to hormone treatments

Morphological responses to ovariectomy and hormonal treatments are shown in
Fig. 3.1. Ovariectomy and control treatment resulted in a reduction in the size of the
ducts and duct ends, and mammary gland morphology was similar after ovariectomy and
3, 5, or 10 days of control treatment. Treatment with E produced a transient enlargement
of the distal tips of ducts and dilation of ducts; this response was maximal after 5 days
and decreased by 10 days. Treatment with E+P produced morphological changes that
increased with treatment length. After 3 days of E+P, sidebranching and some dilation of
the ducts were observed. Treatment for 5 days with E+P produced more extensive
sidebranching and the start of alveologenesis, as defined by the presence of multi-luminal
structures at the ends of sidebranches. A close examination of sidebranches revealed
bulb shaped structures that appeared to pinch off into alveolar units. After 10 days of
E+P treatment, there was more extensive alveolar development; all the sidebranches

produced lobular structures with multiple alveoli. After 3 days of treatment with P alone,

108

10 days

 

Figure 3.1. Morphological response of the mammary gland to hormone treatment.
Mammary gland whole mounts were prepared from adult BALB/c ovariectomized mice
treated for 3, 5, or 10 days with saline (control, C), estrogen (E), progesterone (P) or E +
P. Proliferation in response to E occurred afier 5 days at the distal tips of ducts (black
arrow indicates the distal tip of a duct), but the distal tips regressed by 10 days. A higher
magnification inset of stimulation of the distal tips at the ends of ducts is shown for 5
days E. Afier 3 days of E+P or 5 days of P, sidebranching was present (black
arrowheads). Alveologenesis started after 5 days of E+P or 10 days of P (open
arrowheads). A higher magnification inset of alveologenesis is shown for 5 days E+P.
Expansion of alveoli occurred after 10 days of E+P (open arrowhead) (scale bar, 1 mm).

109

no change in morphology was visible. However, after 5 days of P treatment, there was
extensive sidebranching throughout the mammary gland. Treatment with P for 10 days
produced the start of alveologenesis. These results show that treatment with P alone
caused sidebranching and the initiation of alveologenesis. Sidebranching and
alveologenesis were accelerated and enhanced by the addition of E in the E+P treated

mice.

Proliferative responses to hormone treatments

The morphological changes described above are associated with proliferation. To
determine the relationships among hormone treatments, proliferation, and changes in
morphology, mice were treated with a pulse of 5-Bromo-2’-deoxyuridine (BrdU) two
hours prior to sacrifice. Since the mammary epithelium is composed of two cell types,
luminal epithelial cells and myoepithelial cells, double labeling with BrdU and (rt-smooth
muscle actin (SMA), a specific marker of myoepithelial cells, was carried out to
determine the proliferative response in the two mammary cell types.

Analysis of proliferation in response to the various hormone treatments is shown
in Fig. 3.2A,B. No proliferation was observed in luminal or myoepithelial cells in
ovariectomized, control-treated animals. Treatment with E produced a significant but
transient increase in proliferation after 5 days; proliferation occurred in luminal epithelial
cells and myoepithelial cells and was specifically localized to the distal tips of ducts.

Surprisingly, 3 days of treatment with P alone also increased proliferation of

luminal and myoepithelial cells relative to OVX controls. Luminal epithelial cell and

110

>

50% «

40% . D Luminal Epithelial
I Myoepithelial

Percent BrdU+ cells

 

 

Figure 3.2. Cell type specific proliferation in response to hormone treatment. Dual
immunofluorescence detection of BrdU and or-SMA was performed on mammary gland
sections from adult OVX mice treated for 3, 5, or 10 days with saline (control, C),
estrogen (E), progesterone (P), or E+P. A. Quantitation of the percent BrdU positive
myoepithelial cells and luminal epithelial cells was determined. Proliferation after 5 days
of E treatment was significantly increased only in the distal tips of ducts. Proliferation
was highest after 3 or 10 days of E+P treatment. Treatment with P produced sustained
proliferation after 3, 5, or 10 days of treatment. Proliferation was induced in both luminal
and myoepithelial cells. The values represent the mean i SEM from three to five mice
per group with a minimum of 1000 cells/mouse analyzed. 8. After 3 days of E+P
treatment, proliferating cells were recognized using an anti-BrdU antibody (teal),
myoepithelial cells were distinguished by anti-or-SMA staining (red), and nuclei were
counterstained with DAPI (blue). Proliferation occurred in both myoepithelial (solid
white arrowheads) and luminal epithelial cells (open arrowheads) (scale bar, 25 pm).

111

myoepithelial cell proliferation were also observed in ducts, sidebranches and alveoli
after 5 and 10 days of P treatment. From 7-1 1% in luminal epithelial cells were BrdU
positive (BrdU+) and 4-12% myoepithelial cells were BrdU+ between 3 and 10 days of
treatment.

Overall, the greatest proliferation throughout the gland was observed in response
to E+P treatment. Proliferation occurred in ducts, sidebranches and alveoli. After 3 days
of E+P treatment, 26% of luminal epithelial cells were BrdU+ and 23% of myoepithelial
cells were BrdU+. Proliferation decreased after 5 days of E+P treatment to 7% in
luminal epithelial cells and 6% in myoepithelial cells. After 10 days of E+P treatment,
proliferation increased and 17% of luminal epithelial cells in both ducts and alveolar
structures were BrdU+; a smaller percentage of myoepithelial cells (7%) were BrdU+.

These results demonstrate that treatment with E alone produced a transient burst
of proliferation in luminal and myoepithelial cells that was restricted to duct ends. In
contrast, treatment with P alone produced a low level of sustained proliferation in
myoepithelial and luminal epithelial cells throughout the 10 day treatment period. Thus, P
by itself, in the absence of E, was capable of inducing proliferation in both cell types.
Treatment with E+P resulted in a biphasic proliferative response that was high at day 3,
decreased at day 5, and was high again at 10 days of treatment. Notably, E enhanced

overall proliferation when combined with P.

Hormonal regulation of PRA

The hormonal regulation of PRA was investigated by immunofluorescence

staining with antibody specific for PRA. The effects of ovariectomy and hormone

112

treatments on the percentage of PRA positive (PRA+) cells are shown in Fig. 3.3A.
Ovariectomy did not change the percentage of PRA+ cells compared to the ovary intact
control. Similarly, no effect of treatment with E on the percentage of PRA+ cells was
observed. To determine if the cellular content of PRA was affected by the various
treatments, analysis of immunofluorescence staining intensity was performed using
software that measures pixel intensity in positive cells (Fig. 3.38, C). This analysis
revealed that the level of PRA protein was significantly decreased by ovariectomy
compared to ovary-intact controls. Treatment with E increased PRA content in PRA+
cells relative to ovariectomized controls, but did not restore PRA content to the level of
the ovary-intact control. Treatment with P for 5 or 10 days significantly reduced the
percentage of PRA+ cells and significantly decreased PRA levels below that of
ovariectomized controls. Treatment for 10 days with E+P produced the largest decrease
in the percentage of PRA+ cells. Treatment with E + P did not increase PRA content
above that in ovariectomized controls. These results demonstrate that E upregulates the

levels of PRA, whereas P downregulates PRA levels and blunts upregulation by E.

The relationship between PRA level and proliferation during morphogenesis

PRA and PRB are detected only in luminal epithelial cells (7); therefore, analysis
of the relationship between PR isoforms and proliferation was determined in luminal
epithelial cells. The relationship between PRA, progesterone, and proliferation was
examined in E+P or P treated mice by studying the colocalization of PRA with the
proliferation marker BrdU by dual immunofluorescence (Fig. 3.4). After 3 days of E+P

treatment, 41% of luminal epithelial cells were PRA+, 25% were BrdU+ and 4% were

113

Figure 3.3. Hormonal regulation of PRA expression in the adult mouse mammary
gland. PRA was detected by immunofluorescence with an anti-PRA antibody on
mammary gland sections from adult intact or ovariectomized mice treated for 3, 5 or 10
days with control (C), estrogen (E), progesterone (P) or E+P. A. Quantitation of the
percent PRA positive luminal epithelial cells. The values represent the mean :1: SEM from
three to five mice per group with a minimum of 1000 cells/mouse analyzed. *, p<0.05 10
day E+P was significantly less than all other groups. #, p<0.05 5 and 10 day P were
significantly less than intact, all C and E treated groups and 3d E+P group (p<0.05). 8.
Representative sections of PRA immunofluorescence staining from adult intact or
ovariectomized mice treated for 10 days with C, E, P, and E+P. The percent PRA
positive cells decreased in the E+P or P treated group. PRA intensity was highest in the
intact mouse and following E treatment of ovariectomized mice and lowest in the P
treated ovariectomized mice (scale bar, 25 pm). C. Quantitation of PRA
immunofluorescence staining intensity. Values represent the mean pixel intensity in PRA
positive cells :t SEM from three to five mice per group with a minimum of 1000
cells/mouse analyzed. Intensity of PRA staining was significantly decreased after
ovariectomy and treatment with E significantly increased the intensity of PRA staining
compared to ovariectomized controls. Treatment with E+P did not increase PRA staining
and treatment with P alone fiirther decreased the intensity of PRA staining compared to
the ovariectomized control groups.

114

>

Percent Posltlve Cells

0

Reletlve Fluorescence Intensity

60%
50%
40%
30%

20% .
10% 1

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115

 

Intact

OVX
10d C

OVX
10d E

OVX
10d E+P

OVX
10d P

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PRA

   

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ovx 8
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3dayP 5dayP 10dayP
Treatment

Figure 3.4. Localization of PRA in proliferating cells. A. Dual immunofluorescence
detection of PRA and BrdU was performed on mammary gland sections from adult
ovariectomized mice treated for 3, 5, or 10 days with E+P or P alone. In the
representative images shown, PRA+ cells are teal, BrdU+ cells are pink, PRA and BrdU+
cells are white, and nuclei counterstained with DAPI are blue. After 3 days of E+P, a
small population of proliferating cells express PRA (open arrowhead), whereas after 5 or
10 days of E+P most proliferating cells are PRA negative (yellow arrowhead). Examples
of cells expressing PRA only are indicated with white arrowheads (scale bar, 25 pm). 8.
Quantitation of the percent PRA+ cells, BrdU+ cells, and colocalization of PRA and
BrdU in E+P treated mammary glands. C. Quantitation of the percent PRA+ cells,
BrdU+ cells, and colocalization of PRA and BrdU in P treated mammary glands. The
values represent the mean i SEM from three to five mice per group with a minimum of
1000 cells/mouse analyzed.

116

positive for both PRA and BrdU. Of the total proliferating cells, 16% expressed PRA
(Fig. 3.4A, 8). Thus, a subpopulation of PRA+ cells, in addition to PRA negative
(PRA-) cells, was proliferating coincident with the formation of sidebranches (Fig. 3.1).
After 5 days of E+P treatment, only 5% of the luminal epithelial cells were proliferating
and now only 5% of proliferating cells were PRA+. The lower percentage of cells
positive for both PRA and BrdU was not due to a decrease in the percentage of PRA+
cells since 42% of luminal epithelial cells were still PRA+. After 10 days of treatment
with E+P, 13% of luminal epithelial cells were proliferating. Since there was a
significant increase in the total number of epithelial cells after 10 days of E+P treatment
(Fig. 3.1), the total number of proliferating cells was likely higher at 10 days compared
with 5 days of E+P. The percentage of PRA+ cells was significantly reduced to 20%,
and only 3% of BrdU+ cells were PRA+. The reduced percentage of PRA+ cells and
reduced colocalization of PRA and BrdU coincided with increased alveologenesis.
Overall, proliferation induced by P alone was lower than that induced by E+P
(Fig. 3.4C). The percentage of BrdU+ cells was 9%, 7%, and 4% after 3, 5, and 10 days
of P treatment, respectively. In P-treated mice, the development of sidebranches was
delayed and observed only after 5 days of treatment compared with after 3 days of E+P
treatment. This is likely due to the lower amount of proliferation induced by P alone.
BrdU incorporation in PRA+ cells was lower than observed with E+P treatment. After 3
or 5 days of P treatment, only 2% of proliferating cells were PRA+. After 10 days of P
treatment when alveolar development was observed, no PRA+ cells were proliferating.
Taken together, the results obtained in P and E+P treated mice suggest that alveolar

development and expansion are not associated with the proliferation of PRA+ cells.

117

Nuclear localization of cyclin D1 is associated with the induction of cell cycle
progression toward S-phase and cyclin D1 expression is believed to be regulated by P
(16). Additionally, cyclin D1 expression is believed to be required for alveologenesis
during pregnancy (17). Thus, we also examined colocalization of PRA with cyclin D1
after treatment with E+P or P by dual immunofluorescent labeling (Fig. 3.5). After 3
days of E+P treatment, nuclear cyclin D1 was expressed in 51% of luminal cells, 42% of
cells were PRA+, and 42% of cyclin D1 positive (cyclin D1+) cells were also PRA+ (Fig.
3.58). After 5 days of E+P treatment, the percentage of cells expressing nuclear cyclin
D1 was reduced to 34% and only 13% of these cyclin D1+ cells were PRA+. Thus, while
the percentage of PRA+ cells after 3 or 5 days E+P treatment was similar, colocalization
of PRA with cyclin D1 significantly decreased after 5 days of treatment. Following 10
days of E+P treatment, nuclear cyclin D1 was expressed in 37% of luminal epithelial
cells and only 3% of cyclin Dl+ cells were PRA+. Thus, PRA colocalized with cyclin
D1 during sidebranching after 3 days of E+P treatment, but not during maximal
alveologenesis observed after 10 days of E+P treatment.

In mice treated with P alone for 3 days, the percentage of cyclin D1+ cells (37%)
was lower than observed with E+P treatment and 29% of cyclin Dl+ cells were PRA+
(Fig. 3.5C). The percentage of cyclin Dl+ cells and colocalization with PRA did not
change significantly after 5 or 10 days of treatment and PRA colocalized with cyclin D1

with a similar frequency at 3, 5 and 10 days of P treatment.

118

B
60%" [:1 Cyclin D1

      

    

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g I PRA + Cyclin 01
OVX 8 40%_
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0.
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10 day E+P
OVX C Treatment
5d E+P
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509'..- PRA
% I PRA + Cyclin D1
(3 40%“
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Treatment

Figure 3.5. Colocalization of PRA and cyclin B]. A. Dual immunofluorescence
detection of PRA and cyclin D1 was performed on mammary gland sections from adult
ovariectomized mice treated for 3, 5, or 10 days with E+P or P alone. In the
representative merged images PRA+ cells are teal, cyclin D1+ cells are pink, PRA and
cyclin D1+ cells are white, and nuclei counterstained with DAPI are blue. Specific
examples of PRA+ cells (white arrowhead), cyclin D1+ nuclei (yellow arrowhead), and
colocalization of PRA and cyclin D1 (open arrowhead) are shown. Colocalization of
PRA with cyclin D1 was greatest after 3 days E+P and decreased after 5 and 10 days of
E+P treatment (scale bar, 25 pm). 8. Quantitation of percent PRA+ cells, cyclin Dl+
cells, and colocalization of PRA and cyclin D1 in E+P treated mammary glands. The
percent cyclin Dl+ cells increases in E+P treated mammary glands relative to saline
control treatment. C. Quantitation of percent PRA+ cells, cyclin D1+ cells, and
colocalization of PRA and cyclin D1 in P treated mammary glands. The percent cyclin
D1+ positive cells increases in E+P treated mammary glands relative to saline control
treatment. The values represent the mean :t SEM from three to five mice per group with
a minimum of 1000 cells/mouse analyzed.

119

Hormonal regulation of PRB

High levels of PRB are detected mainly during pregnancy and after involution (7).
PRB expression during pregnancy is primarily associated with the formation of alveolar
structures, and PRB levels are lower in ducts (7). During pregnancy, levels of estrogen
and progesterone are much higher than during normal estrus cycles (18, 19).
Additionally, these high hormone levels are maintained during pregnancy, so there is
greater continuous exposure to hormones during pregnancy than during the estrus cycle
in the adult virgin (18). Based on these previous findings, we hypothesized that
regulation of PRB would differ from that of PRA and would be upregulated by P.

The regulation of PRB expression was examined in ovariectomized mice by
immunofluorescence using an antibody specific for PRB (Figs. 3.6, 3.7). PR8 was not
detected after ovariectomy or after treatment with E (data not shown). PRB levels were
increased by 5 days of E+P or by 10 days of P treatment, but at a very low level, so
accurate determination of the percent positive cells was not feasible (Fig. 3.6). PR8
levels rose to a clearly detectable level in 25% of epithelial cells only after 10 days of
E+P treatment (Fig. 3.7). Thus, PRB was only detected after prolonged treatment with P
or E+P. Additionally, the increase in PRB levels coincided with the appearance of

alveolar structures (Fig. 3.1).

The relationship between PRB level and proliferation during morphogenesis

Colocalization of PRB with proliferation was determined by dual

immunofluorescence with antibodies specific for PRB and BrdU. This analysis was

120

3 day E+P 5 day E+P 10 day E+P

PRB
<
merge ..-

3dayP 5dayP 10dayP

PRB ..-
merge I.-

Figure 3.6. Hormonal regulation of PRB expression in the adult mouse mammary
gland. Immunofluorescence detection of PRB was performed on mammary gland
sections from adult OVX mice treated for 3, 5, or 10 days with C, E, P or E+P. PRB was
only detected after 5 days E+P or 10 days of P treatment. PRB (green nuclei, white
arrowheads) was faintly detected after 5 days of E+P or 10 days of P treatment, but was
most strongly expressed after 10 days of E+P treatment. Nuclei were counterstained with
DAPI (blue)(scale bar, 20 pm).

 

          

121

 

 

 

 

 

A

g 60' 10 day E+P treatment

<1.)

0 50‘

E 40-

g 30-

E 20‘

8

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o. O .

B BrdU PRB PRB +

BrdU

60‘ 10 day E+P treatment

00

 

00

Percent positive cells
A N 8 h 01

 

 

 

   

O

 

Cyclin 01' PRB PRB+
or

Figure 3.7. Colocalization of PRB with BrdU or cyclin D1. Dual
immunofluorescence detection of PRB colocalization with BrdU or with cyclin D1 was
performed on mammary gland sections from adult OVX mice treated for 10 days with
estrogen + progesterone (E+P). A. Quantitation of percent PRB+ cells, BrdU+ cells and
colocalization of PRB and BrdU. B. Quantitation of percent PRB+ cells, cyclin D1+
cells and colocalization of PRB and cyclin D1. The values represent the mean :1: SEM
from three to five mice per group with a minimum of 1000 cells/mouse analyzed.

122

carried out only in the 10 day E+P treatment group because this was the only treatment
that resulted in clearly detectable and quantifiable levels of PRB (Fig. 3.7A). After 10
days of E+P treatment, 10% of luminal epithelial cells were BrdU+, 30% were PRB+ and
49% of the BrdU+ cells were PRB+. Thus, about half of proliferating cells were PRB+
and since increased PRB levels coincided with the development of alveoli, this suggests
that alveoli are formed by proliferating PRB+ cells. This is in contrast to the finding that
only 3% of proliferating cells were PRA+ during the time of maximal alveolar
development at 10 days of E+P treatment (Fig. 3.48)

Dual immunofluorescence detection of PRB and cyclin D1 was also performed in
the 10 day E+P-treated mammary gland (Fig. 3.78). Nuclear cyclin D1 expression was
detected in 35% of luminal epithelial cells and 30% of cyclin D1+ cells were PRB+. This
is in contrast to the 7% of cyclin D1+ cells that were PRA+ (Fig. 3.58). Thus, after 10
days of E+P treatment PRB was the predominant isoform expressed and was more
frequently colocalized with BrdU and cyclin D1 than PRA. Taken together, the frequent
colocalization of PRB with BrdU and with cyclin D1 at the time of extensive alveolar

development further suggests that PRB+ cells proliferate to form alveoli.

Colocalization of PRA and PRB with ER

As shown above, the levels of both PRA and PRB were regulated by estrogen,
which acts through binding to the estrogen receptor (ER). Estrogen increased PRA levels
and enhanced P-induced increase in PRB levels. ERa, and not ERB, is required for ductal

development in the mammary gland (20, 21). ERE appears to play a role during lactation,

123

when neither PRA nor PR8 are expressed (21). Thus, to further address the role of E in
the regulation of PR isoforms, we used dual immunofluorescence to analyze ERa
expression with PRA or PRB expression.

ERa was expressed in all the treatment groups through day 10 (Fig. 3.8A).
However, intensity of staining with anti-ERa antibody was lower in E and E+P treated
mammary glands than in control or P- treated glands, indicating that E downregulates
ERa levels (Fig. 3.8A). Notably, PRA and ERa were coexpressed in the same cell under
all treatment conditions (Fig. 3.88). Increased PRB levels coincided with decreased ERa
levels and the majority of PRB+ cells were ERa negative (Fig. 3.88). The co-expression
of ERa and PRA suggests that E acting through ERa may directly regulate PRA
expression. Conversely, the lack of significant co-expression of PRB with ERa suggests

that E acts indirectly to enhance P-induced upregulation of PRB.

124

nuclei merge

10d
C

merge
10d
E
10d
E+P

Figure 3.8. Colocalization of PRA and PRB with ERa. Dual immunofluorescence
detection of ERa and ERa colocalization with PRA or with PRB was performed on
mammary gland sections from adult OVX mice treated for 3, 5, or 10 days with saline
control (C), E, P, or E+P. A. Representative immunofluorescence images of ERa
staining. ERor (red) expression was detected in all groups, but was decreased by
treatment with E or E+P. Nuclei were counterstained with DAPI (blue)(scale
bar, 25 pm). 8. Representative images of PRA (green) or PRB (green) colocalization
with ERa (red) are shown. In merged images, colocalized cells are yellow. There was a
high degree of PRA and ERa expression colocalization. The majority of PRB positive
cells (white arrowheads) did not colocalize with ERa. Instances of PRB and ERa
colocalization are shown with yellow arrowheads (scale bar, 20 pm).

   

10d
p

 

125

DISCUSSION

We have previously reported that PRA is predominantly expressed in the virgin
gland, whereas PR8 is predominantly expressed during pregnancy. In this report we
have examined the hormonal basis for the differential expression of the two PR isoforms.
Since the greatest proliferative and morphological responses to P occur during pregnancy
we have focused our study on the mature, adult mammary gland and the effect of
pregnancy levels of E and P on PR isoform levels. Additionally, we have analyzed the
relationship between regulation of PR isoform expression, proliferation and alveolar

development.

PRA is upregulated by estrogen and downregulated by progesterone

We found that while ovariectomy did not affect the percentage of PRA+ cells, it
dramatically reduced the level of PRA protein. PRA levels could be restored by treatment
with E. In contrast, treatment with P alone or E+P caused a reduction in the percentage
of PRA+ cells. Additionally, P treatment decreased PRA levels below that observed after
ovariectomy. Furthermore, when P was combined with E, it blunted the upregulation of
PRA by B. These results are in agreement with previous studies that have shown
estrogenic regulation of PR in the adult virgin mouse mammary gland (12) and our
results demonstrate that PRA is the predominant isoform that is regulated by estrogen.

Based on in vitro studies, progestins are reported to downregulate PR (11). Our studies

126

confirm downregulation of PR levels by P and show that this effect is specific for the

PRA isoform in the adult virgin mouse mammary gland.

PRA mediates sidebranching

Alveolar development proceeds through a specific sequence of proliferative and
morphological events. During pregnancy, the earliest event in alveolar development is the
production of ductal sidebranches. Our studies indicate that ductal sidebranching can be
effectively induced by P in ovariectomized mice and does not require estrogen. We have
previously shown that PRA is the predominant isoform expressed in the nulliparous
mouse mammary gland (7), and a recent study using the same anti-PRA antibody
confirmed this result (22). Since ductal sidebranching is induced at a time when PRA is
the predominant isoform expressed we conclude that this process is mediated by
progesterone acting through PRA.

Interestingly, sidebranching can be induced by E+P treatment in the PRA gene-
deleted mouse (PRAKO) (23). The PRAKO mouse studies were carried out in the
C57Bl/6 genetic background. C57Bl/6 adult wild-type mice have less developed
mammary glands when compared to other strains, such as BALB/c (24). Additionally,
the C5781/6 strain is less responsive to hormones than the BALB/c strain in which our
studies were carried out (25). In particular, we have found C57Bl/6 mice to be much less
responsive to progesterone than BALB/c mice and exhibit delayed sidebranching during
pregnancy (Aupperlee & Haslam, unpublished observations). Therefore, it is possible

that additional mechanisms that promote ductal sidebranching may be operative in the

127

C57Bl/6 strain and might explain the lack of a phenotype in the C57BL/6 PRAKO
mouse.

Ductal sidebranching is accelerated in mice treated with E+P and since E
increases PRA levels, it is likely that E contributes to ductal sidebranching through its
positive effect on the level of PRA. Three days of E+P treatment produced the greatest
colocalization of PRA with BrdU and PRA with nuclear cyclin D1, indicating that a
subset of PRA+ cells were proliferating in response to E+P treatment at this time.
However, in both P and E+P treated mice, the majority of cells that proliferate and form
sidebranches were PRA-. The delayed development of sidebranches in P-treated mice
most likely reflects the overall lower proliferation observed after P treatment compared
with E+P treatment. Interestingly, although a significant percentage of PRA+ cells
colocalized with nuclear cyclin D1 after P treatment, only a small percentage of PRA+
cells were BrdU+. This suggests that overall fewer PRA+ cells proliferated after P
treatment. Alternatively, it is possible that treatment with P alone leads to slower
progress through the G1 phase of the cell cycle, which is reflected by the difference in
colocalization with PRA and nuclear cyclin D1, a G1 phase marker, or BrdU, an S phase

marker.

PRB upregulation by progesterone

We found that PRB was expressed at a detectable level only after sustained

exposure to P. E alone did not result in upregulation of PRB; however, E accelerated the

upregulation by P. The earliest detection of significant PRB levels coincided with the

128

development of alveoli at 5 days of E+P or 10 days of P treatment. Why prolonged
treatment with P was required to obtain increased PR8 levels is not entirely clear. The
coincident timing of the decreased levels of PRA, the initiation of alveologenesis, and
PRB upregulation suggest that these events are linked.

One possible explanation for the requirement of prolonged P treatment to
upregulate PRB is that PRA inhibits PRB expression. In this case, one would expect that
the upregulation of PRB by P would occur when PRA levels are at their lowest.
However, this explanation is not compatible with the observation that PRA levels are
decreased faster and to a lower level after treatment with P alone (Fig. 3.1A), yet PRB
expression is upregulated later and less robustly in P treated glands compared with E+P
treated glands (Fig 3.6).

There are two significant differences between P and E+P treated glands that may
affect the upregulation of PRB: 1) the overall lower amount of proliferation and 2) the
longer time required for ductal sidebranching and the development of alveoli to occur in
the P treated glands compared with E+P treated glands. It has been hypothesized by
others that the adult virgin mammary gland contains progenitor cells that give rise to
three different cell lineages: ductal luminal cells, alveolar luminal cells, and
myoepithelial cells (26). It is possible that the cells that proliferate to form the
sidebranches in response to P are derived from progenitor cells committed to the alveolar
luminal cell lineage and that it is a property of these cells to express PRB and form
alveoli. Once PRB expression is induced then P acting through PRB may form a positive

regulatory loop to further increase PRB expression and the expansion of alveolar cells.

129

The role of estrogen and ERa

Estrogen upregulation of PRA was correlated with the co-expression of ERa and
PRA within the same cells. Based on this observation it is likely that E acting though
ERa regulates PRA through a direct mechanism in PRA+ cells. We also observed that E
downregulated ERa, which may indicate that decreases in PRA following prolonged E+P
treatment are partially due to a loss of ERa. Surprisingly, we found that ERa was not
expressed in the majority of PRB+ cells. Although E enhanced the upregulation of PRB,
it does not appear to be due to a direct, ER-mediated effect in PRB+ cells. We propose
that B may facilitate the upregulation of PRB through the maintenance of PRA and lead
to the enhancement of sidebranching and the expansion of the putative alveolar cell
lineage in which PRB is then induced. Additionally, we considered the possibility that E
could enhance PRB upregulation indirectly through a systemic effect by increasing
plasma prolactin (Prl) levels. However, treatment of ovariectomized adult mice for 5
days with Prl alone had no stimulatory effect on mammary gland morphology. Also, the
morphology of the mammary gland after treatment for 5 days with P+Prl was not
different from that observed after treatment with P alone (unpublished observations,

Aupperlee & Haslam).

130

Progesterone induces sidebranching and alveologenesis through direct and

paracrine mechanisms

It has been previously reported that in the virgin mouse mammary gland
proliferating cells are PR negative (5, 27). This has been interpreted to mean that P
induces proliferation through a paracrine mechanism(s) (5, 27). In the present study, at
least 84% of the cells proliferating at the time of ductal sidebranching were PRA- (Fig.
3.48). These results suggest that P acting on PRA+ cells may induce a paracrine factor
that stimulates the proliferation of PRA- cells. Studies by others have implicated Wnt4 as
a paracrine mediator of P-induced sidebranching (28). Thus, our study provides further
evidence for a paracrine mechanism of P action and indicates that this mechanism is
operative in PRA+ cells during sidebranching. We also have made the novel observation
that in addition to increasing proliferation in epithelial cells, P also increased proliferation
in myoepithelial cells. Proliferation of myoepithelial cells was observed during both
ductal sidebranching and alveologenesis. Since myoepithelial cells lack both PRA and
PRB, this indicates that their proliferation is mediated through an indirect effect of P. The
mechanisms operative in P-induced proliferation of PR- luminal epithelial cells and
myoepithelial cells are currently under investigation.

Cyclin D1 expression has been shown to be regulated by progesterone (16) and is
believed to be essential for alveolar development leading to lactation (17). We have
shown that cyclin D1 levels were increased by treatment with P or E+P. The highest
percentage of cyclin Dl+ cells was observed after 3 days of E+P treatment and coincided

with the development of sidebranches. Colocalization of cyclin D1 with PRA and

131

colocalization of PRA with BrdU were also highest at this time point. However, 60% of
cyclin D1+ cells were PR-. These results also indicate that cyclin D1 may be regulated by
P in PRA- cells through a paracrine mechanism(s) and that the upregulation of cyclin D1
by P in both PRA+ and PR- cells promotes the proliferation that produces sidebranching.
During alveologenesis and alveolar expansion at 5 or 10 days of E+P treatment,
colocalization of PRA with cyclin D1 or BrdU was significantly decreased. The decrease
in colocalization of PRA with cyclin D1 after 5 or 10 days of E+P treatment is similar to
the decreased colocalization during extensive alveolar expansion at day 14 of pregnancy
(7). In contrast, cyclin D1 and PRB were highly colocalized after 10 days of E+P during
alveolar expansion and similar to the level of colocalization at day 14 of pregnancy (7).
Of the total cyclin Dl+ cells, 40 % were PRB+, less than 5% were PRA+ and about 55%
of cyclin D1+ cells were PR-. This suggests that the upregulation of cyclin D1 by P in
both PRB+ and PR- cells likely promotes the proliferation required for alveolar
expansion. Thus, P acting on either PRA+ or PRB+ cells appears to produce indirect

effects on PR- cells that promote sidebranching and alveologenesis, respectively.

Estrogen enhances progesterone-induced sidebranching and alveologenesis

We propose the following 2 models that integrate the effects of E and P to explain
the observed differences in PRA and PRB expression, proliferation, ductal sidebranching
and alveologenesis in P vs. E+P treated mice. The first model describes the sequence of
events resulting from E+P treatment. In this case, we propose that the high proliferation

index observed after 3 days of E+P treatment is due to the combination of 1) robust

132

stimulation of proliferation of PR- cells by paracrine factors induced by P in PRA+ cells,
and 2) proliferation of a subpopulation of PRA+ cells, both facilitated by maintenance of
PRA levels by E. Longer treatment with E+P, after 5 and 10 days, results in the
downregulation of ERa by E that together with P leads to downregulation of PRA.
Proliferation of putative alveolar progenitor cells in sidebranches and sustained P
exposure leads to the increase in PRB levels. An earlier increase in PRB, by 5 days,
results in earlier and more extensive alveolar development.

The second model describes the sequence of events resulting from treatment with
P alone. The lower amount of proliferation after 3 days of P treatment is due to 1) a less
robust paracrine induction of proliferation in PRA- cells, and 2) reduced proliferation of
PRA+ cells, both due to the lower level of PRA in the absence of the 8. Thus, in the
absence of E, 5 days of P treatment are required to produce an amount of ductal
sidebranching comparable to that observed by 3 days of E+P. Subsequent to the reduced
proliferation of the putative alveolar progenitor cells during ductal sidebranching,
upregulation of PRB and alveologenesis are delayed.

In summary, we have shown that PRA and PRB are differentially regulated. PRA
is upregulated by E and downregulated by P whereas PRB is upregulated by P. ERa
colocalizes with PRA and this suggests that E directly upregulates PRA through an ER
mediated mechanism. PRB does not colocalize significantly with ER and if E has a role
in PRB regulation it likely occurs through an indirect mechanism. The proliferative and
morphological changes in the mammary gland that occur during pregnancy were
mimicked in our experiments by continuous treatment with either P or E+P. We have

shown that P acting on PRA+ cells caused ductal sidebranching by promoting

133

proliferation of both PRA+ and PRA- cells. This was followed temporally by the
induction of PRB and P action in PRB+ and PRB- cells to cause the formation and

expansion of alveoli.

134

ACKNOWLEDGEMENTS

This work was supported by the Breast Cancer and the Environment Research Centers
Grant U01 ES/CA 012800 from the National Institute of Environment Health Science and
the National Cancer Institute, National Institutes of Health, Department of Health and
Human Services. Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the National Institute of Environment Health
Science or National Cancer Institute, National Institutes of Health. This work was also
supported by Department of Defense Breast Cancer Research Program Fellowship

DAMD17-03-1-0605 to M.D.A.

135

10.

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138

CHAPTER FOUR .

THE MAMMARY GLAND RESPONSE TO ESTROGEN AND/OR
PROGESTERONE: DIFFERENTIAL REGULATION OF PROLIFERATION IS
GENETICALLY DETERMINED

Note: The contents of this chapter have been submitted for publication in
Endocrinology. Aupperlee M.D., Drolet A.A., Durairaj S., Schwartz RC, and Haslam

S.Z. The mammary gland response to estrogen and/or progesterone: differential
regulation of proliferation is genetically determined.

139

ABSTRACT

Progesterone (P) promotion of proliferation in the normal mammary gland is implicated
in the etiology of mammary cancer in mice and humans. BALB/c and C57BL/6 mice
exhibit strain-specific differences in mammary gland development and response to
hormones that may be determining factors of susceptibility to mammary carcinogenesis.
To elucidate the basis for these inherent differences, we analyzed mammary gland
development and in vivo proliferative responses to estrogen (E) and/or P. C57BL/6 mice
exhibited a hypoplastic ductal phenotype in the virgin gland and delayed alveolar
development during pregnancy. In comparison to BALB/c mammary glands, we found
that C57BL/6 glands exhibited reduced sensitivity to P as evidenced by reduced P-
induced expression of progesterone receptor isoform 8 and Receptor Activator of NF-KB
Ligand protein expression, reduced nuclear localization of Id2, and significant differences
in nuclear cyclin D1 expression. In contrast, E responsiveness was greater in C57BL/6
than in BALB/c glands. These observations suggest that in human populations with
heterogeneous genetic backgrounds, individuals may respond differentially to the same
hormone, and thus, genetic diversity may have a role in determining the effects of P in

mammary tumori genesis.

140

INTRODUCTION

Progesterone (P) plays an important role in the proliferation and differentiation of
the normal mammary gland in rodents and humans (1-3). P is also implicated in the
etiology of breast cancer and breast cancer progression (4-7). Breast cancer risk is
increased in women receiving combined estrogen plus progestin menopausal hormone
replacement therapy (HRT) (3, 8-12). Recent decreases in breast cancer incidence have
been attributed to reduced use of HRT (13). P acts through binding to its cognate steroid
receptor, the progesterone receptor (PR), which exists as two isoforms, PRA and PRB,
that are functionally distinct transcriptional regulators (14-16). Alteration in the ratio of
PRA to PRB has been associated with breast cancer progression (4-6). The dysregulation
of PR expression associated with BRCA-l and BRCA-2 mutations (17, 18) further
highlights the potential importance of progesterone signaling pathways in the etiology of
breast cancer.

The mouse mammary gland is a frequently used model system for elucidating the
role of P in normal development and function, as well as in the etiology of mammary
cancer. Many studies of progesterone action were carried out using BALB/c mice (1,
19-27). Additional insights into PR isoform functions were obtained from studies of total
PR-, PRA-, or PRB-deficient mice in a mixed C57BL/6 X 129SV genetic background
(28-30). These studies show that PRB is essential for alveologenesis, whereas the
specific function(s) of PRA in mammary gland development are not well defined.

Mouse strain-specific differences in mammary gland development (31), response

to hormones, and susceptibility to carcinogenesis have been reported (32) C57BL/6 mice

141

exhibit reduced P-induced sidebranching and alveologenesis, and susceptibility to
carcinogen— and medroxyprogesterone acetate-induced tumorigenesis compared to
BALB/c mice (33, 34). These findings suggest that an analysis of P action in different
mouse strains may provide important information about mechanism(s) of P action in the
normal mammary gland and in mammary cancer development

We have analyzed mammary gland development and in vivo responses to
exogenous hormones in wild-type BALB/c and C57BL/6 mice because these mouse
strains have been widely used to study P action in normal mammary gland development
and in mammary tumorigenesis (1, 19-30, 35). We found a hypoplastic ductal phenotype
in the virgin gland and delayed alveolar development during pregnancy in C57BL/6
mice. We found reduced P-induced PRB and RANKL protein expression, reduced
nuclear localization of Id2, and significant differences in nuclear cyclin D1 expression
between the two strains. These findings indicate alternative mechanisms of P-regulated
proliferation that are reflected in strain-specific differences in mammary development.
The identification of strain-specific determinants of progesterone-regulated proliferation
has implications for the role of genetic diversity in determining the effect of P in

mammary tumorigenesis.

142

MATERIALS AND METHODS

Animals:

Mammary glands were obtained from BALB/c (Harlan, Indianapolis, IN) and
C57BL/6 (Jackson Laboratory (Bar Harbor, ME) pubertal (6-wk-old), adult (19 to 22-wk-
old), and 7-, 10-, and 14-day pregnant mice. Adult virgin mice were ovariectomized
(OVX) and one week later were injected subcutaneously for 3, 5, or 10 days with saline
control (C), 17-B-estradiol (E) (1 pg), progesterone (P) (1 mg), or E+P (1 pg E + 1 mg P).
Two h prior to gland removal, mice were injected with 5-bromo-2’-deoxyuridine (BrdU)
(70 pg/g of body weight). Mammary tissues were fixed and processed as whole mounts
(36) or paraffin-embedded for immunohistochemistry as previously described (19).

Mixed C57BL/6 X 129SV genetic background cyclin D1 'l' (D1 'l') mice were a
gift from Dr. Piotr Sicinski (Dana Farber Cancer Institute, Boston, Massachusetts). To
generate D1 "' mice in a BALB/c genetic background, these mice were backcrossed to
BALB/c mice for 6 generations. All animal experimentation was conducted according to
standards approved by the All University Committee on Animal Use and Care at

Michigan State University.

Immunofluorescence:

The protocol used to detect PRA, PRB, ERa and Stat5a, using anti-PRA (1:50;

hPRa7), anti-PRB (1:50; hPRa6) (Neomarkers, Fremont, CA)), anti-StatSa (12300; 8D

143

Biosciences, San Jose, CA), or anti-ERa (1:10; Novocastra, Newcastle, United Kingdom)
antibodies,was described previously (19). Primary antibodies were detected by goat anti-
mouse antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR). To detect
RAN KL, sections were first stained for PRA and than incubated with goat anti-RANKL
(R&D Biosystems, Minneapolis, MN) (1:500 in PBS/0.5% Triton X-100, ovemight, 4'
C). RANKL antibody was detected by rabbit anti-goat antibody conjugated to Alexa 488.
Id2 was detected with rabbit anti-Id2 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA),
followed by goat anti-rabbit antibody conjugated to Alexa 488. For PRA, RANKL, Id2,
or STATSa immunostaining, nuclei were counterstained with 4’,6-diamidino-2-
phenylindole, dilactate (DAPI) (1210,000; Molecular Probes). For PRB immunostaining,
nuclei were counterstained with TOPRO—3 Iodide (121000; Molecular Probes), and
sections were visualized and images captured using a Zeiss Pascal laser scanning

confocal microscope (Zeiss, Thomwood, NY).

Dual Immunoflourescence:

Double labeling of PRA with BrdU or cyclin D1 was described previously (19).
For colocalization with BrdU, sections were first stained for PRA, followed by goat anti-
mouse antibody conjugated to Alexa 488. After blocking with goat anti-mouse IgG Fab
fragments (1:100; Jackson Immunoresearch Laboratories) sections were incubated with
anti-BrdU antibody (kit from Amersham Biosciences, Piscataway, NJ) detected with a
biotinylated goat anti-mouse secondary antibody (1:400; Dako, Carpinteria, CA)

followed by streptavidin-conjugated Alexa 546 (1:100). For colocalization with cyclin

144

D1, rabbit polyclonal anti-cyclin D1 (1:100; Biosource, Camarillo, CA) was used. Cyclin
D1 was detected with a goat anti-rabbit antibody conjugated to Alexa 488. Nuclei were
counterstained with DAPI. Sections were visualized and images captured using a Nikon
inverted epifluorescence microscope (Mager Scientific, Dexter, MI) with MetaMorph

software (Molecular Devices Corporation, Downington, PA).

Western blot detection of RANKL and Id2:

Whole mammary glands obtained from OVX and hormone treated BALB/c and
C57BL/6 mice were homogenized in H8 [H8 : 50mM Tris-HCI (pH 7.2), 6 mM MgC12,
1 mM EDTA, 10% sucrose (w/v), protease inhibitors (2.5 pg /ml leupeptin, 0.5 mM
phenylmethylsulfonyl fluoride in DMSO, 5 pg/ml aprotinin, and 5 pg/ml antipain] (
0.5ml H8 / 100mg tissue), and centrifuged at 2000 x g for 5 min at 4' C; the supernatant
was used as the cytoplasmic extract. For nuclear extracts, the pellet was washed twice in
0.5 ml H8, centrifuged at 2000 x g for 5 min at 4°C, and resuspended in HPB (HP8220
mM HEPES [pH 7.9], 25% glycerol (v/v), 420 mM NaCl, 1.5 mM MgCl, 0.2 mM
EDTA, 0.5 mM dithiothreitol, protease inhibitors) by vigorous vortexing for 15 min at 40
C. After centrifugation at 14,000 x g for 15 min at 4°C, supematants were frozen at -800
C. Western transfers were then performed as previously described (37). Detections were
performed with anti-RANKL (BioLegend Co., San Diego, CA; Cat.# 510007)(121000
dilution) or anti-Id2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; SC-489)(1:1000

dilution), using horseradish peroxidase-conjugated anti-goat IgG (Promega, Madison,

145

WI) (1210,000 dilution) or anti-rabbit IgG (Promega) (1210,000 dilution), and Western

Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Waltham, MA).

Quantitation and statistical analyses:

BrdU, PRA, ERa or cyclin D1 were quantitated for the number of positive
luminal epithelial cell nuclei from captured images using MetaMorph software as
previously described (1). To analyze fluorescence intensity the average pixel intensity of
all positively stained nuclei within the ductal epithelium was determined. Image
thresholds were set to exclude background fluorescence and gated to include intensity
measurements only from positively staining epithelial cells. Background staining in
epithelial cells was determined through setting thresholds to exclude positive staining,
and the level of positive staining above background was calculated by subtracting
background fluorescence intensity. A minimum of 3 mice per treatment group and a
minimum of 1000 cells in three independent sections per mouse were analyzed. Results
are expressed as mean i SEM, and differences are considered significant at P < 0.05 by
using Student’s t test or ANOVA as appropriate.

Images in this dissertation are presented in color.

146

RESULTS

C57BL/6 mouse mammary glands exhibit a delay in sidebranching compared to

BALB/c mice

Ductal development, the degree of ductal branching, and end bud number and size
were similar between the two strains in 6-week-old pubertal glands (Fig. 4.1). The adult
BALB/c mammary gland contained a well-arborized ductal tree with sidebranching,
whereas the C57BL/6 mammary gland was comprised of a simple ductal network with
little sidebranching. During pregnancy, sidebranching and alveologenesis were delayed
in the C57BL/6 mouse and less extensive at 7 and 10 days of pregnancy compared to the
BALB/c mouse. However, by 14 days, the degree of sidebranching and alveologenesis

in the two strains was indistinguishable.

Differences in hormone-induced proliferation and morphology between strains

We considered that delayed sidebranching and alveologenesis in the C57BL/6
mammary gland might be due to differential responsiveness to P. Since the rate of
mammary gland development during early pregnancy was a major difference between the
two strains, we examined the effect of E and/or P doses commonly used to induce

pregnancy-like alveologenesis in OVX adult mice (1).

147

 

Figure 4.1. Mammary gland development in BALB/c and C57BL/6 mice. Mammary
gland whole mounts were prepared from 6-wk-old immature (A, B, G, H), 20-wk-old
adult (C, I), 7 d (D, J), 10 d (E, K), and 14 d (F, L) pregnant BALB/c (A-F) and C57BL/6
(G-L) mice as described in Materials and Methods. Lower (A, B) and higher
magnification (G, H) images are shown for 6-wk-old immature mammary glands; all
other images are higher magnification images to show sidebranching and alveologenesis.
Black arrowheads indicate examples of sidebranching in the adult BALB/c mouse (C).
Scale bar =1 mm.

148

Response to estrogen

In both strains, OVX resulted in reduced size of ducts and duct ends compared to
intact animals (Fig 4.2A). Treatment with E caused enlargement of the distal tips of
ducts and ductal dilation in both strains that was maximal after 5 days and decreased by
10 days. Notably, C57BL/6 mice maintained more enlarged distal tip structures after 10
days of treatment. (Fig. 4.28)

To determine the relationship between E-induced morphological changes and
proliferation, BrdU incorporation was examined by immunohistochemistry (Fig 4.2C). E
treatment produced a significant increase in proliferation in both strains that was maximal
after 5 days and was specifically localized to the distal tips of ducts. While the percentage
of BrdU positive (BrdU+) cells in enlarged distal tips was similar in C57BL/6 and
BALB/c mice after 10 days, there were significantly fewer enlarged distal tip structures

in the BALB/c mammary gland (Fig 4.2A, 8).

Response to progesterone

P treatment produced sidebranching and alveologenesis in the OVX BALB/c
mammary gland that was maximal after 10 days treatment (Fig 4.2D). P treatment of
C57BL/6 mice produced neither sidebranching nor alveologenesis. In BALB/c mice, P
treatment increased the percentage of BrdU+ cells in ducts after 3 days and in ducts,
sidebranches, and alveoli after 5 and 10 days (Fig 4.2E). In C57BL/6 mice, P treatment

produced minimal proliferation, which was restricted to ducts.

149

Figure 4.2. Effect of E or P on morphology and proliferation in the BALB/c vs.
C57BL/6 mammary gland. A. Representative mammary gland whole mounts from
intact and OVX 3d C, and 5d and 10d E-treated BALB/c and C57BL/6 OVX mice. White
arrowheads indicate E stimulation of the distal tips of ducts. Scale bar = 1 mm. B.
Quantitation of stimulated distal tips of ducts. The number of stimulated distal tips was
counted per square cm for BALB/c and C57BL/6 adult OVX mice treated for 5 or 10
days with E. A stimulated distal tip was defined as having an area 2 0.003 mmz, which
represents the area of an unstimulated duct end. The number of stimulated distal tips in
the BALB/c was significantly less than in the C57BL/6 after 10 d E (P < 0.05). C.
Quantitation of proliferating luminal epithelial cells after B treatment. The percentage of
BrdU+ cells in ducts and distal tips (DT) was determined by immunofluorescent
detection of BrdU in mammary gland sections from adult BALB/c and C57BL/6 OVX
mice treated with 10 d C and 3, 5, or 10 d E. D. Representative mammary gland whole
mounts from adult BALB/c and C57BL/6 OVX mice treated with P for 5 or 10 days.
White arrowheads indicate sidebranching in the BALB/c gland. Scale bar = 1 m. E.
Quantitation of the proliferating lmninal epithelial cells after P treatment.
Immunofluorescent detection of BrdU was performed on mammary gland sections from
adult BALB/c and C57BL/6 OVX mice treated with 10 d C and 3, 5, or 10 d P. The
values for B, C and E represent the mean :t SEM from three mice per treatment group
with a minimum of 1000 cells per mouse analyzed.

150

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Figure 4.2. Effect of E or P on morphology and proliferation in the BALB/c vs.
C57BL/6 mammary gland. A. Representative mammary gland whole mounts from
intact and OVX 3d C, and 5d and 10d E-treated BALB/c and C57BL/6 OVX mice. White
arrowheads indicate E stimulation of the distal tips of ducts. Scale bar = 1 mm. B.
Quantitation of stimulated distal tips of ducts. The number of stimulated distal tips was
counted per square cm for BALB/c and C57BL/6 adult OVX mice treated for 5 or 10
days with E. A stimulated distal tip was defined as having an area 2 0.003 mmz, which
represents the area of an unstimulated duct end. The number of stimulated distal tips in
the BALB/c was significantly less than in the C57BL/6 after 10 d E (P < 0.05). C.
Quantitation of proliferating luminal epithelial cells after B treatment. The percentage of
BrdU+ cells in ducts and distal tips (DT) was determined by immunofluorescent
detection of BrdU in mammary gland sections from adult BALB/c and C57BL/6 OVX
mice treated with 10 d C and 3, 5, or 10 d E. D. Representative mammary gland whole
mounts from adult BALB/c and C57BL/6 OVX mice treated with P for 5 or 10 days.
White arrowheads indicate sidebranching in the BALB/c gland. Scale bar = 1 m. E.
Quantitation of the proliferating luminal epithelial cells after P treatment.
Immunofluorescent detection of BrdU was performed on mammary gland sections from
adult BALB/c and C57BL/6 OVX mice treated with 10 d C and 3, 5, or 10 d P. The
values for 8, C and E represent the mean i SEM from three mice per treatment group
with a minimum of 1000 cells per mouse analyzed.

150

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151

Response to estrogen + progesterone

In BALB/C mice, E+P treatment produced extensive sidebranching and
alveologenesis that was maximal by 10 days (Fig. 4.3A). Even after 10 days of
treatment, very little sidebranching or alveologenesis was observed in C57BL/6 mice.
The C57BL/6 mammary gland mainly displayed enlarged duct ends, similar to the
response observed after E treatment alone (Fig 4.3A).

A high percentage of BrdU+ cells were present in both ducts and sidebranches in
the E+P-treated BALB/c gland. The percentage of BrdU+ cells was maximal after 3 days,
decreased after 5 days, and was increased again after 10 days (Fig 4.38). In the
C57BL/6 gland, maximal proliferation was also observed after 3 days, was maintained at
a higher level after 5 days and decreased after 10 days. Proliferation in the C57BL/6
gland was localized to ducts and enlarged distal tips, and was similar to the response to E

alone.

PRA and PRB regulation in the C57BL/6 and BALB/c mammary gland

PRA is the predominant isoform expressed in the adult virgin mouse mammary
gland and is thought to mediate the initial stages of sidebranching (1, 33). In the
C57BL/6 gland, there were significantly more PRA positive (PRA+) cells (58% C57BL/6
vs. 43% BALB/c; p<0.05) (Fig 4.4A). There was no difference in the percentage of
PRA+ cells between the two strains after OVX or in E-treated glands. P or E+P

treatment for 5 or 10 days decreased the percentage PRA+ cells in the BALB/c gland

152

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Figure 4.3. Effect of E + P treatment on morphology and proliferation in the
BALB/c vs. C57BL/6 mammary gland. A. Representative mammary gland whole
mounts from adult BALB/c and C57BL/6 OVX mice treated with E+P for 3, 5 or 10
days. Black arrowheads indicate sidebranching in the BALB/c gland White arrowheads
indicate stimulation of the distal tips of ducts in the C57BL/6 gland Scale bar =1 mm. B.
Quantitation of proliferating luminal epithelial cells. Immunofluorescent detection of
BrdU was performed on mammary gland sections from adult BALB/c and C57BL/6
OVX mice treated with 10 d C and 3, 5, or 10 d E+P. The values represent the mean :1:
SEM from three mice per group with a minimum of 1000 cells per mouse analyzed.

153

(p<0.05). However, in the C57BL/6 gland, PRA+ cells were significantly decreased only
after 10 days E+P treatment (p<0.05). Examination of the fluorescence intensity of PRA
immunostaining, as a semiquantitative measure of PRA level, revealed no difference in
PRA levels per cell between the two strains under any treatment (data not shown),
indicating that the decrease of PRA+ cells in the BALB/c gland was not due to
preferential downregulation of PRA protein, but rather was due to the dilution of PRA+
cells by proliferation of PRA negative (PRA-) cells. Indeed, the majority of proliferating
cells in the P or E+P-treated BALB/c glands were PRA- (Fig. 4.48). In contrast,
significant proliferation in the C57BL/6 gland occurred only after E+P treatment.
However, significantly fewer PRA- C57BL/6 cells proliferated compared to BALB/c
cells.

Induction of PRB expression occurs in response to P or E+P treatment of OVX
adult BALB/c mice and is correlated with alveolar development (Aupperlee & Haslam
2007). In the C57BL/6 mammary gland, no PRB expression was detected after either 10
days P or E+P treatment (Fig. 4.4C); the lack of PRB expression corresponded with a

lack of alveologenesis (Fig. 4.38).

RANKL is not induced by P alone in C57BL/6 mice

Since most proliferating cells in the BALB/c mammary gland were PRA-, we
considered that P-induced proliferation, as has been previously suggested, must occur
through a paracrine mechanism (29, 38). Receptor Activator of NF-KB Ligand (RANKL)

has been implicated as one such paracrine mediator of P-induced proliferation (29).

154

Figure 4.4. Hormonal regulation of PRA and PRB expression in the BALB/c vs.
C57BL/6 mammary gland. A. Quantitation of the percentage PRA+ luminal epithelial
cells. PRA was detected by immunofluorescence in mammary gland sections from adult
intact or OVX BALB/c and C57BL/6 mice treated for 5 or 10 d with control (C), E, P, or
E+P. PRA+ cells were significantly less abundant in 5 and 10 d P and 10 d E+P-treated
groups than in intact, C, E, and 5 d E+P-treated groups in the BALB/c mammary gland
(*, P<0.05). PRA+ cells were significantly more abundant in intact C57BL/6than in intact
BALB/c glands (#, P<0.05). PRA+ cells were significantly less abundant in 10 d E+P-
treated C57BL/6 glands than in all other C57BL/6 groups (§, P<0.05). B. Quantitation of
the percentage BrdU+ and BrdU+/PRA- luminal epithelial cells. Dual
immunofluorescent detection of PRA and BrdU was performed on mammary gland
sections from adult OVX BALB/c and C57BL/6 mice treated for 5 and 10 d with P or
E+P. The values (A, 8) represent the mean i SEM from three mice per group with a
minimum of 1000 cells per mouse analyzed. C. Immunofluorescent detection of PRB
was performed on mammary gland sections from adult BALB/c and C57BL/6 OVX mice
treated for 3, 5, or 10 d with C, E, P, or E+P. Representative PRB staining in 10 d E+P-
treated C57BL/6 and BALB/c mammary gland. PRB (green nuclei, white arrowheads)
was detected in the BALB/c mammary gland after 5 or 10 d E+P, and 10 d P treatment.
Nuclei were counterstained with DAPI (blue). PRB was not detected in the C57BL/6

mammary gland under any treatment condition. Scale bar = 25 pm.

155

70. D BALB/C

'03
110‘

 

 

0’Intact c E 5dP10dP 5d 10d
E+P E+P

lls
N
O

I BrdU+PRA-
EI Total BrdU+

_‘L A
0 0|
1

Percent positive ce
O 01

 

 

BALB/c CS7BL/6

C57BU6 BALB/c

156

 

 

Since treatment with P by itself was insufficient to induce proliferation and sidebranching
in the C57BL/6 gland, we considered that this might be due to a lack of RANKL
induction. Little RANKL expression was detected after OVX (Fig 4.5A) or E treatment
in both mouse strains (data not shown). RANKL expression was induced by P treatment
in the BALB/c gland, but not in the C57BL/6 gland (Fig 4.5A,B). RANKL expression
was induced in both BALB/c and C57BL/6 mice after 5 days E+P treatment, but was
more strongly induced in BALB/c mice. RANKL and PRA were colocalized in the same

cells in both strains (Fig 4.58).

Cyclin D1 is regulated differently in C57BL/6 and BALB/c mammary glands

Another mediator of P-induced proliferation is cyclin D1 (D1), which increases in
expression and nuclear localization in response to P treatment (1, 39). D1 is also
downstream of RANKL signaling in the mammary gland (40). The lack of P-induced
proliferation or induction of RANKL in C57BL/6 mice suggested that nuclear
localization of D1 might also be decreased or absent. Surprisingly, nuclear D1 was
present in a significantly higher percentage of cells in the OVX C57BL/6 gland (Fig.
4.6A) and there was a significantly higher percentage of cells co-expressing nuclear D1
and PRA (Fig. 4.68). E treatment did not affect nuclear D1 levels in either strain (data
not shown). Nuclear D1 was increased in PRA- cells only after E+P treatment and

coincided with an E+P-induced increase in RANKL (Fig. 4.58).

157

A BALB/c C57BL/6
c P E+P c P E+P
RANKL —- — - 28 kD

 

B—actin —— — — _ —

B BALB/c CS7BL/6

 

Figure 4.5. Hormonal regulation of RANKL expression in the BALB/c vs. C57BL/6
mammary gland. A. Immunoblot analysis of RANKL in mammary glands from adult
OVX BALB/c and C57BL/6 treated for 5 d with C, P, or E+P. Cytoplasmic extracts
from mammary gland were subjected to SDS-PAGE and RANKL detected in a western
blot as described in Materials and Methods. RANKL was detected as a 28 kDa species
following P and E+P treatment in the BALB/c mammary gland, but only after E+P
treatment in the C57BL/6 mammary gland; B-actin served as a loading control. 8. Dual
immunofluorescent detection of RANKL (red) and PRA (green) in mammary gland
sections from adult BALB/c and C57BL/6 OVX mice treated for 5 d with P or E+P.
RANKL was expressed in the cytoplasm (red) in PRA+ cells (green nuclei). Scale bar=
25 pm.

158

In the BALB/c gland, both P and E+P treatment increased nuclear D1 expression,
indicating that nuclear localization of D1 was P-dependent (Fig. 4.6). Increased nuclear
D1 expression occurred predominantly in PRA- cells (Fig. 4.68). We hypothesize that
RANKL is a paracrine mediator of increased D1 nuclear localization in PRA- cells in
both BALB/c and C57BL/6 glands.

Based on gene deletion studies in the C57BL/6 X129SV mixed genetic
background, D1 is considered to be required for alveologenesis during pregnancy (41-
43). However, P is also essential for alveologenesis (28). Since we found that the
C57BL/6 gland is less responsive to P and expresses high levels of nuclear D1, we sought
to determine the relative importance of P and D1 for alveologenesis in BALB/c vs.
C57BL/6 glands during pregnancy. To address this, we generated BALB/c D1" ' mice by
backcrossing C57BL/6 X 129SV D14 ° mice into the BALB/c genetic background. In the
pregnant BALB/c D1” ' gland, there was extensive alveolar development, although not as
extensive as in the wild-type BALB/c gland (Fig. 4.6C). Despite extensive alveolar
development, BALB/c D1 "' mice were lactation impaired and failed to nurse their pups.
C57BL/6 X 129SV D1 'l' mice also exhibit impaired lactation, but alveolar development

is also significantly impaired relative to BALB/c D1" ' (41-43).

Id2 localization is regulated differently in BALB/c versus C57BL/6 mammary gland

In response to RANKL signaling, Id2 is translocated to the nucleus (44). Nuclear

localization of Id2 is required for mammary gland proliferation (44). Mice

overexpressing D1, but deficient in Id2 exhibit a defect in mammary epithelial cell

159

Figure 4.6. Hormonal regulation of cyclin D1 in the BALB/c vs. C57BL/6 mammary
gland. Dual immunofluorescent detection of PRA and cyclin D1 was performed on
mammary gland sections for adult BALB/c and C57BL/6 OVX mice treated for 10 d with
C, P, or E+P. A. P or E+P treatment increased nuclear expression of cyclin D1 (green
nuclei) in the BALB/c mammary gland, whereas nuclear expression of cyclin D1 was
elevated in all C57BL/6 treatment groups. Nuclei were counterstained with DAPI (blue
nuclei). Scale bar = 25 pm. 8. Quantitation of percentage cyclin D1+ PRA- and
PRA+D1+ luminal epithelial cell nuclei. Total nuclear cyclin D1+ cells in 10 d P and 10
d E+P-treated BALB/c glands is significantly greater than in 10 d C glands (*, P<0.05).
Total nuclear cyclin D1+ cells in 10 d E+P-treated C57BL/6 glands is significantly
greater than in 10 d C or P-treated glands (#, P<0.05). The values represent the mean i
SEM from three mice per group with a minimum of 1000 cells per mouse analyzed. C.
Representative whole mounts from adult and 1 d postpartum (1d pp) BALB/c Cyclin D1 ‘
l' and 1 d postpartum wild-type BALB/c mice. Note significant alveolar development in 1
d postpartum BALB/c Cyclin D1 'I' gland.

160

A BALB/c C57BL/6

OVX
10d C

OVX
10d P

 

v, 70' D PRA-Cyclin D1 + #
B 60‘ l PRA+Cyclin D1 +

*

 

 

BALB/c C57BL/6
C D1 -/- virgin D1 -/- 1d pp WT 1d pp

:: ' . .
A " "‘3‘ , . .
.1. 1L - ~

  

161

proliferation (45). We hypothesized that reduced RANKL levels would lead to reduced
Id2 nuclear localization in the C57BL/6 mammary gland, and that the activation of Id2 by
RANKL was also required for proliferation.

In the BALB/c gland, P or E+P treatment for 5 or 10 days decreased cytoplasmic
localization and correspondingly increased nuclear localization of Id2 compared to OVX
controls (Fig 4.7A). In the C57BL/6 gland, only treatment with E+P decreased
cytoplasmic and increased nuclear localization of Id2 (Fig 4.7A). E+P treatment also

increased the level of nuclear Id2 to a greater extent in the BALB/c gland (Fig 4.78).

Differences in ERa expression between C57BL/6 and BALB/c

The above results indicate that the proliferative effect of E was more pronounced
in C57BL/6 OVX mice, as evidenced by sustained proliferation and enlargement of duct
ends. This led us to examine ERa expression in the two strains. There were 10% more
ERa+ cells (p < 0.05) in the C57BL/6 gland in ovary intact animals (Fig. 4.8A). As
previously reported for the BALB/c gland (1), ER colocalized with PRA in the C57BL/6
gland (Fig 4.88). However, the level of ERa expression per cell, as determined by the
intensity of immunofluorescent staining with anti-ERa antibody, was 1.7-fold higher in
the C57BL/6 mammary gland (Fig. 4.8C). ERa levels were decreased by treatment with
E by itself or with E+P in both strains. However, ERa levels were higher in C57BL/6

glands after B or E+P treatment.

162

 

A C P E+P
I.-
III

BALB/c C57BL/6
c P E+P c P E+P

w .—__ —.-. 1...,

 

 

 

ld2

 

Figure 4.7. Hormonal regulation of ld2 in the BALB/c vs. C57BL/6 mammary
gland. A. Immunofluorescent detection of Id2 in mammary gland sections from adult
BALB/c and C57BL/6 OVX mice treated for 5 d with C, P, or E+P. Nuclear localization
of Id2 (green nuclei, white arrowheads) was increased by 5 d P and E+P treatment in the
BALB/c and 5d E+P treatment in the C57BL/6 mammary gland. Scale bar = 25 pm. 8.
Immunoblot analysis of Id2 in mammary glands from adult BALB/c and C57BL/6 OVX
mice treated for 5 d with C, P, or E+P. Nuclear extracts from mammary glands were
subjected to SDS-PAGE and Id2 detected in a western blot as described in Materials and
Methods. Nuclear Id2 was detected as a single 15 kDa species and increased in
abundance following E+P treatment in the BALB/c and C57BL/6 mammary gland.

163

Figure 4.8. Expression and hormonal regulation of ERa in BALB/c and C57BL/6
mammary glands. Immunofluorescent detection of ERol was performed on mammary
gland sections from adult BALB/c and C57BL/6 intact or OVX mice treated for 5 d with
C, E, P, or E+P. A. Quantitation of the percentage ERoc positive luminal epithelial cells
in intact C57BL/6 and BALB/c mammary gland. The values represent the mean :1: SEM
from three mice per group with a minimum of 1000 cells per mouse analyzed. The
percent ERa+ cells in the BALB/c gland was significantly lower than in the C57BL/6
gland C“, P < 0.05). 8. Representative images of PRA (green nuclei), ERa (red nuclei),
and ERor and PRA colocalization (yellow nuclei) in the C57BL/6 mammary gland. Scale
bar = 25 pm. C. Quantitation of ERa, immunofluorescence staining intensity. Values
represent the mean pixel intensity in ERa-positive cells i SEM from three mice per
group with a minimum of 1000 cells per mouse analyzed. Control treated C57BL/6
intact and OVX mice have more ERor than BALB/c mice (*, P < 0.05; ANOVA). D.
Representative ERor immunofluorescence staining of mammary glands from adult
BALB/c and C57BL/6 intact mice. Scale bar = 25 pm.

164

A 70% EIBALB/c B
60% ICS7BL/6
*

50%
40%1 FL
30%

20%

Percent ERol positive cells

10% merge

 

 

 

0

 

 

Adult intact

O

1000

(O
O
O

D BALB/c
I C57BL/6

O) \I (D
O O O
O O O

11

Relative Fluorescence Intensity
N (p J} 0'!
O O O O
O O O O

—L
O
O

 

Intact 5d C 5d P 5d E+P
BALB/c C573L/6

U

intact

 

165

Estrogen, Stat5a expression, and RANKL induction in BALB/c and C57BL/6

mammary glands

As shown above, C57BL/6 mice required E in addition to P for the induction of
RANKL and Id2 nuclear localization. In contrast, P alone was sufficient for these effects
in the BALB/c gland, although E enhanced the effects of P. Signal transducer and
activator of transcription 5a (Stat5a) has been shown to increase expression of RANKL
(46). In BALB/c mice, E+P treatment induces expression of activated nuclear Stat5a (47).
Because both E and P were required to induce RANKL in C57BL/6 mice, we
hypothesized that E played a critical role in RANKL induction through activation of
Stat5a. We found that E treatment increased nuclear Stat5a in the C57BL/6 gland (Fig.
4.9A) and there was an overall increase in Stat5a expression after E+P treatment (Fig
4.98). Additionally, E+P treatment induced RANKL colocalization with Stat5a (data not

shown).

166

l‘

A c E
P E+P
B
'A'

450

 

El BALB/C
I CS7BL/6 *

150
100

01
O

 

 

Figure 4.9. Hormonal regulation of Stat5a in BALB/c and C57BL/6 mammary
glands. A. Immunofluorescence detection of Stat5a in mammary gland sections from
adult BALB/c and C57BL/6 OVX rrrice treated for 3 d with C, E, P, or E+P.
Representative images showing Stat5a expression (teal) in the 3d C, E, P, and E+P-
treated C57BL/6 mammary gland; nuclei were counterstained with DAPI (dark blue).
Scale bar 2 25 pm. 8. Quantitation of Stat5a immunofluorescence staining intensity.
Values represent the mean i SEM pixel intensity in nuclear Stat5a-positive cells from
three mice per group with a minimum of 1000 cells per mouse analyzed. Stat5a intensity
is increased in BALB/c and C57BL/6 mammary glands by 3d E+P treatment relative to
OVX controls C“, P < 0.05). StatSa intensity is increased in C57BL/6 mammary glands by
3d E treatment relative to OVX controls (#, P < 0.05).

167

DISCUSSION

Progesterone, in conjunction with E and other hormones and growth factors, is
required for the extensive sidebranching and alveologenesis that occurs during
pregnancy. Previous studies indicate that P promotes ductal sidebranching and
alveologenesis through the induction of PRB (l, 19), increased RANKL expression (29),
and increased expression of nuclear cyclin D1 (1, 19, 29). We observed a significant
delay in ductal sidebranching and alveologenesis during pregnancy in C57BL/6 mice
compared to BALB/c mice. To understand the basis for this difference, we have
compared hormonal regulation of PRB, RANKL, Id2 and cyclin D1 in C57BL/6 vs.
BALB/c mammary glands.

We found that the C57BL/6 gland was less responsive to P than the BALB/c
gland, as evidenced by the lack of P-induced PRB and RANKL expression, reduced
Id2nuclear translocation, and reduced proliferation. However, RANKL expression, Id2
nuclear translocation, and proliferation could be induced by E+P, suggesting a greater
dependence on E for alveologenesis in the C57BL/6 mouse. In contrast, P by itself could
induce PRB and RANKL expression, proliferation, and alveologenesis in the BALB/c
gland, indicating a greater responsiveness to P.

Studies of C57BL/6 cyclin D1 'l' mice show cyclin D1 expression and its nuclear
localization to be critical for alveolar development (41-43). In the BALB/c gland, nuclear
cyclin D1 expression was dramatically decreased after ovariectomy, but could be
increased by P treatment. Nuclear cyclin D1 levels were increased predominantly in

PRA- cells, and were associated with P- or E+P-induced proliferation in PRA- cells.

168

Surprisingly, we found high levels of nuclear cyclin D1 even after OVX in the C57BL/6
gland. Notably, nuclear cyclin D1 was predominantly expressed in PRA+ cells. While
treatment with P did not affect nuclear cyclin D1 levels, treatment with E+P led to an
increase in nuclear cyclin D1 in PRA- cells; most proliferation induced by E+P also
occurred in PRA- cells. These results demonstrate very different inherent patterns of
nuclear cyclin D1 expression and regulation between the two strains, and further
demonstrate reduced sensitivity to P in the C57BL/6 gland.

Deletion of the cyclin D1 gene in the C57BL/6 genetic background results in a
lack of alveologenesis during pregnancy (42, 43). In contrast, BALB/c cyclin D1 'I' mice
exhibited extensive alveologenesis during pregnancy. Viewed in the context of the
reduced responsiveness to P that we found in C57BL/6 mice, these results suggest that
impaired alveologenesis in C57BL/6 cyclin D1 'l' mice is likely the result of inherent
strain-specific reduced responsiveness to P rather than the lack of cyclin D1 p_er_se.
However, both BALB/c and C57BL/6 cyclin D1 '/' mice were lactation-deficient, in
agreement with previous reports that cyclin D1 plays a specific role in lactational
differentiation in addition to its cell cycle regulatory function (41-43).

The proliferative effect of E was more pronounced in C57BL/6 than in BALB/c
mice, as evidenced by sustained E-induced enlargement of and proliferation in the distal
tips of ducts. Furthermore, E+P-treated C57BL/6 glands exhibited enlarged distal tips
similar to those seen with E alone, indicating increased responsiveness to E and reduced
responsiveness to P. We found 10% more ERa+ cells and higher levels of ERa per cell in
C57BL/6 glands, consistent with their greater sensitivity to E. Similar to our results,

Montero Girard et al (33) also found that virgin C57BL/6 mice exhibited a hypoplastic

169

ductal phenotype and reduced morphological response to P compared to BALB/c mice.
However, opposite to our findings, they reported that the percentages of ERa+ and PRA+
cells were greater in BALB/c glands compared to C57/BL/6 glands. One possible
explanation for the difference in results lies in the methods used for immunodetection. In
the Montero Girard et a1. (2007) study, no antigen retrieval was used prior to antibody
staining. Using the same antibodies, we and others have found poor detection of ERa or
PRA without antigen retrieval (48)(Aupperlee & Haslam unpublished observations).
Thus, it is possible that the different results obtained for the percentages of ERa+ and
PRA+ cells might be attributed to differences in detection with or without antigen
retrieval.

In C57BL/6 mice, E is also critically required in addition to P for alveologenesis. In
the C57BL/6 gland, induction of RANKL, a major P-induced paracrine factor promoting
alveologenesis (29), also required E in addition to P. In this regard, Stat5a has also been
shown to increase RANKL expression (46). Stat5a expression colocalizes with RANKL
and it has been suggested that colocalization of the two proteins may be functionally
linked (47). E+P treatment also increases Stat5a activation. However, co-treatment of
E+P with bromocryptine, which blocks prolactin secretion, only increases cytoplasmic
StatSa indicating that prolactin is required for activated nuclear Stat5a (47). Since E
increases prolactin levels, we hypothesize that one way that E contributes to
alveologenesis in C57BL/6 mice is through increased prolactin secretion, leading to
activation of Stat5a and Stat5a-dependent induction of RANKL. In support of this

hypothesis, we found that E and E+P increased nuclear Stat5a levels in the C57BL/6

170

gland. Further studies are warranted into the detailed mechanism(s) by which E
contributes to ductal sidebranching and alveologenesis in the C57BL/6 mouse.

The phenotypic differences between BALB/c and C57BL/6 cyclin D1 'l' mice
highlight the importance of inherent strain-specific differences when interpreting the
results of genetic manipulations. In this regard, the inherent reduced responsiveness to P
of the C57BL/6 strain indicates that this may not be the best strain for studying P-
mediated responses, such as sidebranching and alveologenesis. Additionally the variable
genetic contribution of mixed genetic backgrounds (i.e. C57BL/6 X 129SV) often used
for gene deletion studies may yield inconsistent outcomes, thus confounding the
interpretation of a phenotype. However, this problem can be overcome by backcrossing
genetically modified mice into a pure genetic background with a well-defined wildtype
phenotype.

In summary, we have demonstrated that the BALB/c and C57BL/6 mouse strains
differ significantly in their proliferative and morphological responses to estrogen and
progesterone. We have identified differences in the progesterone-mediated regulation of
several factors: PRB, cyclin D1, RANKL, and Id2. These differences shed light on the
basis for the reduced response to P in the C57BL/6 mouse strain. The fact that two
genetic backgrounds in the same species differ so significantly in E and P responses
indicates that caution is required in generalizing mechanisms of hormone action on the
basis of studies in a single mouse strain. These results also suggest that in human
populations with heterogeneous genetic backgrounds, individuals may respond
differentially to the same hormone through inherent differences in their regulation of

downstream signaling pathways. For example, the association of combined estrogen +

171

progestin hormonal therapy with increased breast cancer risk may be associated with a
genetic background in human populations that reflects increased sensitivity to P. These
differences may apply to hormonal regulation in both the normal breast and in breast
cancers. The differences in expression level and regulation that we have observed for
elements in the progesterone-regulated proliferative pathways of mouse mammary
epithelial cells suggest their potential utility as biomarkers for the assessment of P-
sensitivity in the human breast, as well as their potential as novel biomarkers for breast

cancer susceptibility.

172

ACKNOWLEDGEMENTS

This work was supported by the Breast Cancer and the Environment Research Centers
Grant U01 ES/CA 012800 from the National Institute of Environment Health Science
(NIEHS) and the National Cancer Institute (NCI), National Institutes of Health (NIH),
Department of Health and Human Services. Its contents are solely the responsibility of

the authors and do not necessarily represent the official views of the NIEHS or NCI,

NIH.

173

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CONCLUDING REMARKS

Progesterone is an important mitogen in the mammary gland. It is critical for
normal lobuloalveolar development and has also been implicated in the etiology of breast
cancer. Progesterone acts through binding to the progesterone receptor (PR), which
exists as two isoforms, PRA and PRB. In this thesis, I set out to examine the
developmental regulation of expression and localization of PRA and PRB in the mouse
mammary gland, to determine the hormonal regulation of PRA and PRB in viva in the
mouse mammary gland, and to examine the mechanism of progesterone action in mouse
strains with different genetic backgrounds.

While PR expression in the mouse mammary gland has been previously
examined, the developmental regulation of PRA and PRB remained unknown. In chapter
2, I demonstrated that protein expression of PRA and PRB in the developing BALB/c
mammary gland is temporally and spatially separated. PRA is the primary PR isoform
expressed in the virgin mammary gland, whereas PRB is the primary isoform expressed
upon pregnancy. In the virgin mammary gland PRB is not detected, and thus cells
containing PR express only PRA. In the pregnant mammary gland when both PRA and
PRB are expressed, PRA and PRB expression generally do not colocalize. These results
in the mouse contrasted with published reports of complete PRA and PRB co-expression
in the human premenopausal breast, and suggested that the separation of PRA and PRB
expression during mouse mammary gland development offered a unique opportunity to
examine the role of PRA during ductal development and sidebranching and the role of

PRB during alveologenesis. Initial analysis during puberty and pregnancy suggested that

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PRA primarily mediates proliferation via a paracrine mechanism, whereas during
pregnancy PRB may mediate proliferation via a direct mechanism.

The results presented in chapter 2 demonstrated that PRA level decreases during
pregnancy, whereas PRB level increases. The regulation of PRA and PRB protein
expression in vivo is poorly understood, but it has generally been suggested that estrogen
upregulates PR and progesterone downregulates PR. In chapter 3, I demonstrated that in
the BALB/c mammary gland PRA level is increased by estrogen and decreased by
progesterone. In contrast, PRB level is increased by progesterone and is further increased
by estrogen + progesterone. The results presented in chapter 3 also reveal potential
functional roles for PRA and PRB in the mammary gland. The increase in PRB
expression is associated with alveolar formation, consistent with a role of PRB in
alveologenesis. PRA expression is associated with ductal sidebranching, suggesting that
PRA-mediated proliferation plays a role in the formation of sidebranches. Additionally,
PRA-mediated proliferation during sidebranching occurs primarily in PRA negative cells,
whereas PRB-mediated proliferation during alveologenesis is present in both PRB
positive and negative cells, confirming the conclusion from chapter 2 that PRA and PRB
mediate proliferation via different mechanisms.

The results from chapter 2 and chapter 3 suggested that PRA mediates
proliferation by a paracrine mechanism. In order to further explore the role of PRA in
mediating progesterone-induced proliferation, two adult strains of mice, BALB/c and
C57BL/6 were analyzed for their responses to progesterone. In chapter 4 of this
dissertation, I presented data showing that C57BL/6 mice have reduced sidebranching

prior to pregnancy and a delay in alveologenesis during pregnancy compared to BALB/c

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mice. 1 showed that the lack of sidebranching is due to adult C57BL/6 mice having a
reduced morphological and proliferative response to progesterone. When I examined
potential downstream signals of progesterone, I found reduced progesterone-induced
PRB expression in the C57BL/6 mammary gland. In chapter 4, I also presented results
that identified a paracrine mechanism for progesterone action in BALB/c mammary
glands. RANKL expression increases in PRA positive cells in response to progesterone,
which was associated with increased nuclear localization of Id2 and nuclear localization
of cyclin D1 in PRA negative cells. The reduced sensitivity of the C57BL/6 mammary
gland to progesterone correlated with reduced progesterone-induced PRB and RANKL
expression, reduced nuclear localization of Id2, and significantly different regulation of
cyclin D1 expression. These results are the first to provide mechanistic insight into the
difference in hormonal responsiveness between mouse strains.

In conclusion, the findings presented in this dissertation provide novel insight into
the developmental and hormonal regulation of PRA and PRB, and thus provide a
framework for further study of PRA and PRB function. Currently, PRA and PRB
expression in the human breast has only been examined in premenopausal women. The
temporal and spatial separation of PRA and PRB expression during mouse mammary
gland development highlights the importance of similar developmental studies in the
human breast. Additionally, PRA and PRB are differentially regulated by hormones in
the mouse. Future studies are planned to examine PRA and PRB expression in breast
tissue from postmenopausal women receiving hormone replacement therapy with
estrogen alone or with estrogen + progesterone. Thus, it remains important to translate

these findings in the mouse into a greater understanding of progesterone action in the

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human breast. It is expected that there will be changes in PR isoform expression
associated with certain developmental states in the human breast, such as puberty and
pregnancy, and that these alterations will be associated with increased proliferation.

The results presented in this dissertation also significantly advance our
understanding of progesterone mechanism of action in genetically unaltered mice and
highlight the importance of genetic background in determining hormone responsiveness.
The identification of markers of progesterone action that are differently regulated in two
different mouse strains suggests that similar markers of progesterone action need to be
found in the human breast. Due to the genetic heterogeneity present in human
populations, it is likely that a range of responsiveness to progesterone exists. In order to
better understand the role of progesterone in the normal human breast and in the etiology
of breast cancer, it is important to consider the role of genetic background in influencing
progesterone responsiveness. The contribution of progesterone to the etiology of breast

cancer may be determined through greater insight into progesterone responsiveness.

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