A ‘5.“
€42.43: «.4.
.5. ..

9.1.1.

Hm}.

, . ‘ , ; .. I r. ,

.um.h...n9.un.1 . ‘ . . , . .. . V . .. V , .flbmrnm r wfltu:
A. , _ , V , . .. _ a 4%
_ _ , ._ . . . _ . ‘ @ X

#3 ,

I...

fire.“ .. .7..
Rmmm L”... g
. . .c.

.4

Jr 5
dwvw

3.
,1 x

 

lava-Eta... I6.

 

 

 

 

7 £55....
a ..

 

 

 

LIBRARY
Michigan State

 

.ty

n
D

Univer

This is to certify that the
dissertation entitled

THE REGULATION OF STAT5A EXPRESSION AND ITS
FUNCTION IN THE DEVELOPING MOUSE MAMMARY GLAND

presented by

Sarah Jane Santos

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Cell and Molecular Biology

 

 

 

 

Adm/L, g. 4m m3?

r Major Professor's Signature J

.;Z\ (t? (7/7/11? £14.) ’51 Qg’) 0%
J

 

Date

MSU is an affinnative-action, equal-opportunity employer

. -.-4-.-0-0-1-0-I-n-I-t-o-u-u—-a»

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/07 p:/ClRC/DateDue.indd-p.1

 

 

 

THE REGULATION OF STATSA EXPRESSION AND ITS
FUNCTION IN THE DEVELOPING MOUSE MAMMARY GLAND

By

Sarah Jane Santos

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Cell and Molecular Biology Program

2007

ABSTRACT

THE REGULATION OF STATSA EXPRESSION AND ITS
FUNCTION IN THE DEVELOPING MOUSE MAMMARY GLAND

By

Sarah Jane Santos

Signal transducer and activator of transcription (Stat)5a is a critical regulator of
proliferation, differentiation and survival in mammary epithelial cells, and has been
implicated in human breast cancer. While the pathways leading to Stat5a activation have
been clearly elucidated, the mechanisms which control its expression are not well
understood. To investigate potential regulators of Stat5a expression, an
immunohistochemical analysis of Stat5a levels throughout mammary gland development
was performed. The pattern of Stat5a expression suggested that it was regulated by the
ovarian hormones, estrogen (E) and progesterone (P). This was confirmed through
hormone ablation and reconstitution studies, where ovariectomy led to a severe decrease
in Stat5a levels and treatment with E+P was necessary to restore its expression. Further
studies demonstrated that Stat5a is expressed predominantly in estrogen receptor and
progesterone receptor positive cells, suggesting that E and P directly induce the
expression of StatSa through their respective receptors.

Although Stat5a was found to be present and activated in the pubertal mammary
gland, its functions outside of pregnancy and lactation are unknown. To investigate the
role of Stat5a in early mammary gland development, a Stat5a knockout model was used.

Aduclt nulliparous Stat5a-/- mice displayed defects in primary branching and side-

branching, as well as a diminished proliferative response to E+P treatment.

Having identified a role for Stat5a in E+P stimulated proliferation, the underlying
mechanism by which it was acting was studied. Double labeling experiments indicated
that Stat5a positive cells rarely proliferate, suggesting its actions are via a paracrine
mechanism. Receptor activator of NF-KB ligand (RANKL), a secreted factor that can
induce mammary epithelial cell proliferation, was demonstrated to be a downstream
mediator of Stat5a. In addition, the nuclear localization and thus function of Id2, a
critical RANKL target, was defective in Stat5a-/- mammary cells.

In summary, these studies reveal three novel findings concerning Stat5a in the
mouse mammary gland. First, they demonstrate that Stat5a expression is controlled by
E+P in the mammary gland. Secondly, a role for Stat5a in primary branching and side-
branching in the virgin gland is shown. Finally, these studies establish a potential

paracrine mechanism by which Stat5a induces proliferation via RANKL.

ACKNOWLEDGEMENTS

I would like to thank everyone who was involved with this dissertation.
Especially, I would like to thank Dr. Susan Conrad, who has been a great advisor and a
great scientist from whom to gain research experience from. I have genuinely enjoyed
my time as Dr. Conrad’s graduate student, and I deeply appreciate all of her guidance
throughout my research project. Also, thanks to the rest of my committee, Drs. Walter
Esselman, Kathy Gallo, Bob Hausinger and Richard Miksicek for all of their help and
support pertaining to my studies. The advice and guidance I received from Dr. Sandra
Haslam was also extremely helpful in completing this dissertation.

I would like to thank everyone that was involved with the animal work carried out
for this thesis, with special thanks to Beth Stames and Tuan Nguyen. Beth was
extremely helpfiil with processing mammary gland tissue, while Tuan carried out all of
the genotyping. In addition, Mark Aupperlee was an enormous support by teaching me
many of the techniques used my research, and I obtained valuable information about
mouse mammary gland biology from his studies.

Thanks to all faculty and staff in the Breast Cancer and Environment Research
Center. The friendliness and helpfulness of everyone there has made my time as a
graduate student very enjoyable. Jeff Leipprandt, Anastasia Kariagina, and Kristen
Bullard have all helped out with work for this thesis, and their participation is greatly
appreciated. A lot of technical help was also obtained from individuals at the MSU core

facilities. I would like to especially thank Dr. Annette Thelen for her assistance with the

iv

Real Time PCR experiments, and Dr. Melinda Frame for her help with the confocal
microscopy.

All of my friends and family have been extremely supportive during my years at
Michigan State University. To all the members of the Conrad lab, thank you for your
companionship and good times in the lab. Shibani and Emily, we have been side by side
throughout most our studies, and even though we are not in the same lab anymore, your
continued friendship and support have meant a lot to me. I would like to also thank the
members of the Haslam lab for accepting me as a part of their group. To Gabby and
May, thanks for the constant supply of conversation, and for all the great times had
outside of the lab, it’s been fun! Mom and Dad, your support has been there from the
beginning, and I could not have done it without you. I especially want to acknowledge
my husband Douglas, for being extremely encouraging and patient throughout my

endeavors in graduate school. I could not have done this without your love and support.

TABLE OF CONTENTS

LIST OF FIGURES viii

CHAPTER 1

Literature Review

Anatomy of the mammary gland ................................................................... 2
Mammary gland development ...................................................................... 4
Systemic mammogens: steroid hormones .......................................................... 9
Systemic mammogens: peptide hormones ....................................................... 13
Locally acting mammogens ........................................................................ 15
Signal transducer and activator of transcription (Stat) ......................................... 19
Stat structure ......................................................................................... 20
Stat pathway and regulation ........................................................................ 21
Stat function: normal and disregulated ........................................................... 26
Stat5 expression in the mammary gland ......................................................... 27
Stat5 function in the mammary gland ............................................................ 29
StatS regulated genes ............................................................................... 33

Stat5 and breast cancer ............................................................................. 34
Conclusion ........................................................................................... 35
References ............................................................................................ 37

CHAPTER 2

Estrogen and Progesterone are Critical Regulators of Stat5a Expression in the Mouse
Mammary Gland

Abstract ............................................................................................... 53
Introduction .......................................................................................... 54
Materials and Methods .............................................................................. 56
Results ................................................................................................ 60
Discussion ............................................................................................ 79
References ............................................................................................ 85
CHAPTER 3

Examining the Function of Stat5a in the Virgin Mammary Gland and its Role in E+P
Induced Proliferation

Abstract ............................................................................................... 90
Introduction .......................................................................................... 91
Materials and Methods .............................................................................. 93

vi

Results ................................................................................................ 97

Discussion .......................................................................................... 1 10
References .......................................................................................... 1 17
Concluding Remarks .............................................................................. 120
APPENDIX

Regulation of Stat5a Expression by R5020 in Primary Mammary Epithelial Cells. ..... 122

vii

LIST OF FIGURES

CHAPTER 1

Figure 1.1: Cross section of a mammary gland duct .......................................

Figure 1.2: Schematic presentation of the different stages of mammary gland

development

Figure 1.3: Basic overview of the Jak/Stat signaling pathway ............................

CHAPTER 2

Figure 2.1: Immunofluorescent staining of Stat5a during mouse mammary gland. . ..

development

Figure 2.2: Stat5a expression in hormone treated mice ....................................
Figure 2.3: Cellular localization of Stat5a in bromocriptine treated mice ...............
Figure 2.4: Colocalization of Stat5a with PRA or PRB ...................................
Figure 2.5: Colocalization of Stat5a with ERa .............................................

Figure 2.6: Irnmunodetection of Stat5a and BrdU in mammary glands of hormone......

treated mice

Figure 2.7: Irnmunodetection of StatSa and WAP or Stat5a and RANKL ..............

CHAPTER 3

Figure 3.1: Whole mount analysis of pubertal and adult virgin mice ....................
Figure 3.2: Morphological response of mammary glands to hormone treatment. . . ..
Figure 3.3: Proliferative response of mammary glands to hormone treatment ..........
Figure 3.4: RANKL expression in response to hormone treatment ......................

Figure 3.5: Progesterone receptor expression in response to hormone treatment. . .. .

viii

..... 3

.6

...22

...62

....65

....68

..... 71

....73

76

....78

....99

101

..104

...107

109

Figure 3.6: ld2 localization in response to hormone treatment ........................... 111

Figure 3.7: Proposed mechanism of Stat5a mediated proliferation in response ........115
to E+P

APPENDIX

Figure 4.1: Stat5a mRN A expression in response to R5020 ................................ 127

ix

CHAPTER 1

LITERATURE REVIEW

LITERATURE REVIEW

Anatomy of the mammary gland

The mammary gland presents a unique and fascinating system to study because
the majority of its development and differentiation occurs postnatally. Unraveling the
basic principles of mammary development is not only interesting, but is also central to
our understanding of breast cancer. According to the American Cancer Society, 40,910
breast cancer related deaths will occur this year. A comprehensive knowledge of normal
mammogenesis will aid our understanding of how mammary cells become deregulated in
cancer, and may ultimately lead to developing new therapeutics.

The mammary gland is an evolutionarily young organ that secretes milk to sustain
newborn offspring. In most mammals, the milk is delivered through a complex ductal
system to the nipple. In contrast, monotremes have mammary glands without a central
ductal system or nipple, and instead secrete milk into a milk patch (1). The discussion in
this dissertation will focus on the former glandular system. Historically, the mouse has
been favored as a model of both mammary gland development and breast cancer biology.
In contrast to humans which have a single pair of glands, mice have five pairs of
mammary glands which form along the entire trunk (2). Murine glands contain much
less fibrous tissue (3), and do not produce structures to the degree of complexity seen in
the human gland (2). Despite these differences, the mouse can still serve as an important
model for the investigation of basic mammary gland biology.

The mammary gland is composed of two distinct compartments, each containing

multiple cell types (Fig. 1.1). The epithelium makes up the secretory component of the

Fibroblast

 

Figure 1.1. Cross section of a mammary gland duct. Secretory lmninal epithelial cells
surround the lumen of the duct, and are apically located to a single layer of contractile
myoepithelial cells. Myoepithelial cells are surrounded by a basement membrane. In the
stroma, fibroblasts surround the basement membrane, and adipocytes fill the remainder of
the fat pad. Adaptation of figure is used with permission from Alexis Drolet.

organ, while the supporting stroma surrounds the epithelium (4). The two major
epithelial cell types are luminal and myoepithelial. The cuboidal luminal cells line the
ducts and alveoli, and are polarized and secretory in nature. Myoepithelial cells, also
referred to as basal cells, are elongated in shape and form a continuous layer surrounding
the outside of luminal cells. As implied by their name, myoepithelial cells possess
contractile properties that aid in the delivery of milk (5). The myoepithelial cells are
separated from the stroma by a layer of basement membrane. The predominant stromal
cell is the adipocyte, and for this reason the mammary stroma is also referred to as the fat
pad. Fibroblasts, endothelial cells, immune cells and neurons are also present in the
stroma. The development of a fully functional mammary gland requires an intricate
network of signaling between the epithelium and stroma. This communication process is
initiated in the embryo and continues through two major stages of development that occur

during puberty and pregnancy.

Mammary gland development

The mammary gland is derived from the epithelial cell lineage, and is produced as
a result of an invagination of the dermis. The first evidence of development in most
mammals is the milk line, an elevated ridge of ectoderm. The milk line then separates
into raised, lens-shaped aggregates of epithelial cells at specific locations where
mammary glands will form. In primates, these ectoderrnal placodes are produced in the
thoracic region (6), whereas rodents develop them along the entire trunk. In the mouse
embryo, this process can be observed as early as late embryonic day 10 (E10) to early

E11 (7). The formation of the mammary placodes is not through the proliferation of

cells, but rather through the migration of epidermal cells (7, 8). Within one day, the
placodes develop into mammary buds and are visible as elevated knob-like structures (9).
By the following day, the mammary buds begin to invaginate into the dermis, and by
E14.5, can no longer be detected externally (9). At approximately E15, cell proliferation
occurs at the tip of the invaginated bud to form a duct, called the primary sprout. Each
sprout forms a lumen, which opens on the surface of the skin, where the nipple forms.
By E185, the sprout has elongated and bifurcated to produce approximately 6-8 short
ducts, which comprise the rudimentary gland observed at partuition (9). This stage of
development is hormone independent, and occurs primarily in response to signaling
between the epithelium and mesenchyme. Several regulators of embryonic mammary
gland (anlage) development have been identified, and it is interesting to note that many of
these also have roles in breast cancer. Fibroblast grth factor (FGF)10, neuregulin
(Nrg)3, parathyroid hormone related peptide (PTHrP) and Wnts have all been shown to
be critical signalers in the formation of the mammary anlage (10).

The mammary anlage contains a population of stem cells with the capacity to give
rise to all cell types of the mature mammary gland. In fact, mammary buds can be
isolated and transplanted into cleared fat pads of recipient mice, to form fully developed
mammary glands (11). This is an important technique that is used to examine the effects
of deleting specific genes in mice when germ line deletion causes lethality.

After birth, the rudimentary gland grows isometrically with the rest of the body.
The first stage of dynamic mammary development does not occur until puberty, when

epithelial cell proliferation is initiated by the surge in ovarian hormones (see Figure 1.2).

PRB-PUBERTAL PUBERTAL ADULT PREGNANT LACTATING

mammary fat pad TEBs side-branches alveoli milk filled alveoli

 

t.

nipple primary branches

Figure 1.2. Schematic presentation of the different stages of mammary gland
development. A rudimentary ductal system is present within the mammary fat pad at
birth, and grows at the same rate as the animal until the onset of puberty. During puberty,
the production of E and P by the ovaries promotes ductal elongation, and conspicuous
club-shaped structures (terminal end buds (TEBs)), where the majority of epithelial cell
proliferation occurs, appear at the duct ends. In the adult virgin, the entire fat pad is filled
with a regularly spaced system of primary duets, with side-branches that form and regress
to some degree in each estrous cycle. The increase in hormones during pregnancy
(estrogen, progesterone, prolactin, and placental lactogens) increase cell proliferation and
induce the formation of alveoli. These alveoli expand and differentiate into milk-
secreting structures at the end of pregnancy. In the lactating gland, fully mature luminal
epithelial cells synthesize and secrete milk components into the alveolar lumina.

The production of estrogen (E) and progesterone (P) at puberty drives ductal elongation
into the fat pad. The majority of proliferation occurs in prominent club-shaped structures
located at the distal ends of ducts called terminal end buds (TEB)s (4). Two distinct cell
types define TEB anatomy, body cells and cap cells (12). Multiple layers of body cells,
which differentiate primarily into luminal cells, are enclosed by a single outer layer of
cap cells, which give rise to myoepithelial cells (12). The body cells closest to the cap
cell layer are highly proliferative and support the outward growth of the duct.
Meanwhile, the body cells furthest from the cap cell layer undergo apoptosis to support
the formation of a hollow lumen in the developing duct (13). Bifurcation and trifurcation
of the TEBs causes dichotomous primary branching of the epithelium to produce an
arborized ductal tree. When the perimeter of the fat pad is reached, TEBs regress and the
majority of proliferation ceases at this stage until pregnancy (13).

The remaining proliferation observed in the adult virgin gland is manifested in the
development of side-branches. These small structures branch off laterally from
established primary ducts. Side-branches grow and regress intermittently in response to
the rising and falling hormone levels during the estrous cycle (14). However, the side-
branches never fiilly regress, resulting in ever increasing mammary gland complexity as
the animal matures (14). In the mature mammary gland, primary ducts may be composed
of several layers of luminal epithelial cells, which become fewer as the distance from the
nipple increases. Most terminal ducts and side-branches have only one layer of luminal
epithelial cells (15).

The second major stage of postnatal mammary gland development occurs during

pregnancy and results in a fully functional gland. An increase in ovarian hormone levels,

particularly P, and the addition of placental hormones results in further ductal side-
branching, and alveolar units subsequently form at the ends of these branches. Alveoli
serve as the major milk-producing components of the gland. They are comprised of fully
differentiated luminal epithelial cells surrounding a central cavity. Alveolar cells are
polarized so that milk constituents are secreted from their apical membrane into the
lumen. There are two phases of lactogenesis. The first occurs in mid to late pregnancy
and is characterized by the expression of milk proteins. Lipid droplets are visible in the
cytoplasm of luminal cells during this phase. In the second phase, which occurs near
parturition, milk protein expression increases even further, and lipid droplets and proteins
are secreted into the alveolar lumen. In addition to lipids and lactose, the major protein
constituents of milk are casein, lactalbumin and whey acidic protein (WAP). In response
to suckling, oxytocin is released and stimulates the contraction of myoepithelial cells
surrounding alveoli and ducts, resulting in milk release. Cessation of lactation due to
weaning induces a massive remodeling of the mammary gland called involution. During
this period, alveolar cells undergo apoptosis and regress (16), resulting in a gland that
closely resembles that of mature, virgin animals. The ability of the mammary gland to
undergo the process of creating milk-producing alveoli with each successive pregnancy
underlines the existence of mammary stem or progenitor cells. Further analysis is
necessary to fully understand the roles and regulation of mammary stem cells in normal

and malignant tissue development.

Systemic mammogens: steroid hormones

Several signaling networks are required for the development of a functional
mammary gland, and systemic endocrine factors play a paramount role in this process. E
and P are the principle steroid hormone regulators of post-pubertal mammogenesis. The
control of mammary development by these ovarian hormones was initially revealed
through endocrine ablation studies over 100 years ago, when Halban demonstrated that
ovariectomy induced mammary regression (17).

E acts via binding to its two receptors, ERa and ERB. Like other members of the
steroid receptor super-family, when bound by their ligand, these receptors act as
transcription factors in the nucleus, initiating the expression of specific responsive genes
(18). In addition to this classical ER genomic action, a smaller amount of ER resides in
the cell membrane or cytoplasm, where it can initiate more rapid responses through direct
interactions with a variety of signaling pathways (19). EROL, but not ERB, plays a
primary role in the outgrth of mammary ducts during puberty (20). ERa-/— mice do
not produce TEBs, and the gland fails to develop past the prepubertal stage. Early
transplant experiments using ERa-/- tissue to investigate if stromal or epithelial ERoc was
required for mammary gland development resulted in contradictory results. One study
concluded that only stromal ERa was necessary for ductal outgrowth. This was shown
by transplanting recombined mammary stromal and epithelial tissue from neonatal wild-
type and ERa-/- glands under the renal capsule of nude mice (21). A separate study
demonstrated that both epithelial and stromal ERa were required for complete mammary
gland development in adult mice when mammary epithelial cells were isolated from adult

ERa-/- or wild-type mice and injected into epithelial-free mammary fat pads (22). These

data suggest that mammary development in neonatal and adult mammary epithelial tissue
is carried out via different ER mechanisms. However, all of these experiments were
complicated by the fact that they used hypomorphic knockout mammary glands (23).
Although full length ERa was not functional in these mice, alternative splicing gave rise
to a small amount of truncated ERa product which retained some transactivation
potential (24, 25). It has been recently shown using a new ERa-/- model, which has no
detectable EROL transcript, that epithelial ERa, but not stromal ERa, is essential for
ductal outgrth in puberty (26). Much less is understood about ERB fimction in the

mammary gland. Unlike ERa, ERB is not necessary for normal pubertal development,
but instead appears to play a role in differentiation during pregnancy (27).

Like E, P’s action is also mediated through its intracellular receptor, PR. There
are two major PR isoforms, PRA and PRB, which are produced by a single gene through
differential transcription from two initiation sites, and translation initiation at two
alternative AUG initiation codons (28). These receptors exhibit different temporal and
spatial patterns of expression in the mouse mammary gland (29). PRA is the
predominant isoform present in the virgin gland, where it is expressed in a subset of
epithelial cells located in a punctate pattern throughout the ducts. In the pregnant gland,
PRA continues to be expressed in both ductal and alveolar cells, but the overall
percentage of PRA positive cells and the amount of PRA protein per cell decreases as the
pregnancy proceeds. In contrast, PRB is not detectable in the virgin gland, and is
primarily confined to a subset of alveolar cells during pregnancy. Both isoforms become
undetectable during lactation. Expression of both receptors reappears in the post-

involuted gland, where PRA levels are similar to those seen in the late pregnant gland,

10

but PRB levels are lower. The two PR isoforms are rarely expressed in the same cell in
the mouse mammary gland (29). It is interesting to note that the expression pattern of PR
in humans and rats differs from the mouse in two ways. First, PRB is present in the
nulliparous human and rat mammary gland, and secondly, PRA and PRB are often
coexpressed in the same mammary epithelial cell in these species (30).

The role of P in mammary development was not examined in detail until recently,
when studies using PR gene-deleted mice (total PR-/-, PRA-/- only or PRB-/- only) (31-
33) and transgenic mice (PRA or PRB transgenes) (34, 35) were carried out. Whole
mount analysis revealed that the ductal structure of the adult PR-/- mammary gland was
similar to that of agematched wild-type mice. However, PR-/- mammary glands failed to
respond to exogenous E+P treatment, displaying a lack of side-branching and alveolar
development. Transplantation experiments have determined that the role of PR in
alveologenesis during pregnancy or in response to E+P is intrinsic to the epithelium (36).

Ovariectomized PRA-/— mice showed normal side-branching and alveologenesis
in response to E+P treatment, indicating that PRB is sufficient for this process (32).
PRB-/- mice failed to develop side-branches or alveoli during pregnancy or when treated
with E+P, (33) further demonstrating that it is the critical isoform for these
developmental processes. Adult glands from PRA transgenic mice displayed an
increased amount of side-branching, sometimes resulting in glands resembling those in
early pregnant animals (34). In contrast, robust alveolar development with only limited
side-branching, and an inhibition of ductal elongation was seen in glands from PRB

transgenic mice (35). Thus, PR is not critical for ductal outgrowth during puberty, but

11

PRB is required for alveologenesis during pregnancy, and PRA may play a role in side-
branching.

The glucocoriticoid receptor (GR) also participates in mammary development.
Deletion of the GR gene results in embryonic lethality, so normal mammary development
cannot be examined. To circumvent this problem, GR-I— outgrowths from embryonic
mammary buds were transplanted into cleared fat pads of wild-type hosts (37). The
resulting mammary glands displayed abnormal development during puberty with
distended ductal lumina and multiple layers of ductal epithelial cells instead of the
standard single layer. Another study demonstrated that mice expressing 3 GR that lacks
DNA binding ability had decreased side-branching in the adult virgin mammary gland
(38). In vitro experiments have established that glucocorticoids are lactogenic factors
(39). However, the conditional deletion of GR during pregnancy did not affect milk
production, but instead showed a reduction in epithelial cell proliferation (40).

The l,25-(OH)2D3 (vitamin D3) receptor (VDR) is another member of the
nuclear hormone receptor superfamily (41, 42), which participates in mammary
development (43). Loss of the VDR has been shown to result in defective regulation of
ductal outgrowth in the mouse mammary gland. After treatment to normalize fertility,
calcium levels and E levels, VDR -/- mice exhibited accelerated ductal elongation and an
increased number of TEBs during puberty (44). In addition, treatment of these mice with
ovarian hormones in viva revealed an increased proliferative response. Vitamin D3

appears to antagonize the proliferative signal from E+P in the pubertal mammary gland.

12

Systemic mammogens: peptide hormones

In the early twentieth century, hypophysectomy experiments revealed that factors
other than ovarian hormones were necessary for mammary gland development (45). In
1928, Stricker and Grueter showed that milk. secretion could be induced in virgin rabbits
by injecting pituitary extracts from lactating animals (46). The pituitary derived factor
responsible for the maturation of the mammary gland was isolated four years later and
named prolactin (PRL) for its milk inducing attributes (47). PRL is primarily produced
and secreted by specialized cells in the anterior pituitary gland called lactotrophs. E, and
to a lesser extent P, induce the hypothalamus to secrete galanin (48), which in turn
induces the lactotrophs to secrete PRL (49). During lactation, the suckling stimulus
drives secretion of PRL from the pituitary (50). The hormone is also synthesized to a
lesser extent locally in the mammary gland (51).

Unlike the steroid hormones E and P, PRL is a peptide hormone which acts
through a transmembrane receptor belonging to the cytokine receptor superfamily (52).
The mammary PRL receptor (PRLR) signals via the Janus kinase (J ak)2 protein to
activate the signal transducer and activator of transcription (Stat)5a, mitogen-activated
protein (MAP) kinase, and other signaling pathways. Several studies have used PRLR
gene knockout mice to determine the role of PRL in the mammary gland. Adult virgin
PRLR-/- glands exhibited decreased side-branching. However, transplantation studies
showed that this defect was not intrinsic to the epithelium, but rather was due to the
inability of PRL to stimulate P secretion from the ovaries (53-55). Since PRLR-/- mice
are infertile, PRLR+/- mice were used to examine mammogenesis during pregnancy. Up

to mid-pregnancy, mammary development was indistinguishable from wild-type mice.

13

However, a greater amount of alveolar development was noticeable in the wild-type
females after day 15 of pregnancy, and failed to produce milk after parturition (54).
Similar results were obtained when PRLR-/- epithelium was engrafted to wild-type
stroma (55). These results demonstrate that PRLR-/- and PRB-/- mammary glands have
similar developmental phenotypes. Thus, PRL is an important mitogen and
differentiation factor in the pregnant mammary gland.

In addition to PRL, other PRL-like hormones are produced by the placenta during
pregnacy. In mice and rats, identified PRL-related hormones now number more than a
dozen. The first two of these hormones were initially revealed by searches for placental
proteins in rodents that bind with high affinity to the PRL receptor and mimic the action
of PRL (56, 57). These two hormones have been designated as "placental lactogens"
(placental lactogen I (PL-I) and (PL-11)) reflecting their origin and roles as inducers of
milk production. At mid-gestation, pituitary derived PRL becomes less necessary for
mammopoiesis, as the placental lactogens increase in amount and take over as the major
stimulatory factors (58, 59). After partuition, when PL-I and PL-II are absent, PRL
becomes necessary for the maintenance of alveoli and production of milk. When the
suckling stimulus is removed at weaning, and pressure from milk build up increases, the
mammary gland undergoes involution (60).

Growth hormone (GH) is another factor secreted by the anterior pituitary gland
that regulates mammary development. Mammary glands of mice lacking the GH
receptor (GHR) gene exhibited delayed ductal outgrowth and restricted side-branching at
puberty (61). Transplantation of GHR-/- mammary epithelium into cleared fat pads of

wild-type mice showed that stromal GHR was sufficient for normal ductal outgrowth

14

(61). It is well accepted that GH and E are the principle hormones driving pubertal
mammary development (62). More recent data suggests that GH also plays a role in both
alveolar development and lactation. In PRLR+/- mice, treatment with GH during
pregnancy was able to restore alveolar development (63). Other studies have shown that
GH synergizes with PRL for normal milk production in rats (64, 65), and GH is able to

stimulate milk protein expression in primary mouse mammary cells (66).

Locally acting mammogens

Early models of systemic E action in the mammary gland were based on the
correlation of its ability to induce epithelial cell proliferation and the presence of ER in
the epithelium. This led to the hypothesis that E induces proliferation directly in the ER
expressing epithelial cells. However, the discovery that the majority of proliferating cells
in the developing mammary epithelium do not contain steroid hormone receptors (67-69)

complicated this initial model. Instead, the steroid receptor-positive cells are often in

close proximity to proliferating cells which incorporate 3H-thymidine (67). It is now
known that locally produced grth factors mediate the proliferative effects of the
mammogenic steroid hormones. Some growth factors are direct transcriptional targets of
hormone receptors, and the germline deletion of the genes encoding these growth factors
or their receptors results in mammary phenotypes similar to those in knockout models of
the steroid hormone receptors which control their expression.

The first example of a paracrine factor mediating the effect of a systemic
mammogen was revealed by the finding that insulin-like grth factor 1 (IGF-l) could

reproduce the effects of GH in promoting mammary gland development, and that GH

15

acted on the stromal compartment to increase IGF -1 mRNA levels (70—72). IGF-1-/- and
GHR-l— mammary glands exhibit similar defects in pubertal ductal outgrowth, suggesting
that they act in a common pathway (61, 73). Treatment with IGF-l + E was able to
rescue the defects seen in IGF-1-/- mice, while GH + E treatment could not (73). This
confirmed that IGF-l is downstream of GH. Although GH or IGF-l alone could induce
some mammary development in ovariectomized, hypophysectomized, pubertal animals,
the addition of E was required to achieve full mammary development, suggesting a
synergism of these two signaling pathways (74, 75). It was later demonstrated that mice
with a liver-specific deletion of the IGF-l gene had normal mammary IGF-l transcript
levels and normal mammary development at puberty despite having significantly reduced
serum IGF-l levels (76). This finding demonstrates that only locally produced IGF-l is
necessary for ductal outgrowth.

PRL induced proliferation is also accomplished via paracrine signaling in the
mammary gland. One mediator of this PRL activity is IGF-2. The first evidence that
IGF-2 was involved in PRL’s effects came from studies where ectopic expression of IGF-
2 was shown to restore alveologenesis in pregnant mammary glands derived from PRLR-
/- epithelial cells transplanted into wild-type stroma (55). Consistent with this notion was
the observation that transplanted [FG-2-l- epithelial cells exhibit a similar defect in
alveolar development as PRLR-/- cells (55). Finally, PRL treatment was able to induce
lGF-2 mRNA expression in primary mammary epithelial cells, and IGF-2 treatment of
primary cells resulted in an increase in cyclin D1 protein (55). Taken together, these
observations suggested that PRLR is required for the synthesis of IGF-2 by mammary

epithelial cells and that once produced, IGF-2 can act in a paracrine manner to induce

16

cyclin D1 expression. This pathway is similar to that of the GHR, the closest relative of
the PRLR, and its paracrine mediator IGF-l, except that PRL effects are intrinsic to the
epithelium.

Epidermal growth factor receptor (EGFR) is a critical downstream mediator of E
induced ductal elongation and branching in the pubertal mammary gland. The EGFR is a
member of the ErbB/typel family of receptor tyrosine kinases, and forms homodirners or
heterodimers with the other family members, ErbB2, ErbB3 and ErbB4 (77). Multiple
ligands bind to the EGFR, including but not exclusively EGF, transforming growth
factor-0t (TGF-a), and amphiregulin (AR) (78). The function of EGFR in
mammogenesis was demonstrated using two mouse models, one with targeted expression
of a dominant negative EGF R in the mammary gland (79) and a second in which neonatal
EGFR-/- mammary glands were transplanted into wild-type hosts (80). Both of these
models exhibited defective mammary ductal development at puberty. The transplant
experiments demonstrated that stromal, but not epithelial EGFR was essential for
pubertal ductal outgrowth (80). Further experiments have shown that ErbB2 also
contributes to pubertal ductal outgrowth, but unlike EGFR, ErbB2 acts primarily in the
epithelium (81).

E treatment stimulates tyrosine phosphorylation and activation of both EGFR and
ErbB2 in ovariectomized animals at puberty (82). This is likely due to the fact that
expression of AR, the chief EGFR ligand during puberty, is regulated directly by E (83).
AR mRNA is expressed at higher levels than other EGFR ligands during puberty and is
synthesized by ductal and TEB epithelial cells (84). AR-/- mice exhibit blunted ductal

outgrowth at puberty, similar to EROt-/- mice, and implanted AR-secreting pellets

17

promote ductal outgrth (84). Taken together, these data suggest a model in which E
acting through epithelial ER, induces AR synthesis and secretion, and the secreted AR
then acts on stromal EGFR and epithelial ErBZ to induce ductal outgrowth. The
downstream responses to AR action in stromal cells remain to be explored. In the
simplest scenario, stromal cells stimulated by AR could emit a proliferative signal that is
received by the neighboring epithelial cells. Several growth factors, such as hepatocyte
growth factor (HGF), IGF -1, and FGFlO, are good candidates because they are expressed
in the mammary stroma at the time of ductal elongation, whereas their respective
receptors are found in the epithelium (85).

The proliferative action of P through PR in mammary epithelial cells is also
paracrine in nature. This was first revealed by the observation that wild-type epithelium,
within close proximity of PR-/- epithelium, could mediate alveolar formation in the
knockout tissue (36). Wnts were proposed as likely mediators of P-induced side-
branching after ectopic expression of Wntl was shown to overcome the defect in PR-/-
glands (86). However, Wntl is not normally expressed in the mammary gland. Instead,
Wnt4, which is expressed in the early to mid pregnant gland, was demonstrated to be
critical for side-branching and was induced by P in vivo (86). To date, studies have only
implicated Wnts in the side-branching which mostly occurs during pregnancy.

Another paracrine mediator of hormone induced mammary development is the
TNF family member, receptor activator of NF-kB ligand (RANKL), also known as
OPGL, GDP, and TRANCE (87). Although RANKL is primarily known for its functions
in bone remodeling and immunity, it has been shown recently that RANKL is also a key

regulator of proliferation during mammary gland development (87). Pubertal and

18

nulliparous adult mice lacking RANKL or its receptor, RANK, show normal mammary
gland morphology (88). However, during pregnancy, RANKL-/- mice display impaired
alveolar formation, and mammary tissue transplant experiments showed that the effect of
RANKL was intrinsic to the epithelial compartment (88). Overexpression of RANK in
the mammary gland resulted in an increase in proliferation and a decrease in
differentiation during pregnancy (89). Thus, RANKL is a critical mitogen in the
pregnant mammary gland.

RANKL expression is controlled by a number of factors. P, PRL and PTHrP, all
of which are upregulated during pregnancy, have all been shown to induce the expression
of RANKL in the mammary gland (88). This is consistent with the fact that RANKL
expression is not detectable by Western blotting in the nulliparous adult gland, but is
increasingly expressed as pregnancy progresses (88). In contrast, its receptor RANK is

constitutively expressed in the mammary gland throughout development (8 8).

Signal transducer and activator of transcription (Stat)

The signal transducer and activator of transcription (Stat) signaling pathway is
critical for normal mammary gland development. This pathway transmits extracellular
signals received by transmembrane receptors to the nucleus, resulting in specific genetic
responses. In their inactivated form, latent Stats are located in the cytoplasm. Following
receptor activation, Stats translocate to the nucleus and directly induce transcription of
responsive genes. Hence they act as signal transducers in the cytoplasm and transcription
factors in the nucleus. This pathway allows for an efficient transcriptional response to

external signals without the need for secondary messengers.

l9

Stat structure

Stats are evolutionarily conserved, and are found in eurkaryotes ranging from
slime molds to humans (90). In mammals, seven Stat proteins (Statl, Stat2, Stat3, Stat4,
Stat5a, StatSb, and Stat6) are transcribed from separate genes. These proteins range in
size from 750 and 850 amino acids (91). To date, no crystal structure of a whole Stat
molecule has been solved. Most of what is known about the three-dimensional structure
stems from crystallographic studies of specific domains from Stats 1, 3 and 4 (91).

Stat proteins are defined by the presence of five conserved domains. The amino-
terrninal half of the protein consists of two relatively under-characterized domains. The
first domain consists of ~125 amino acids, and is reported to aid in DNA binding and to
regulate nuclear translocation (92, 93). There is also a coiled-coil domain (amino acids
~135 to ~315) comprised of a four-helix bundle, which functions in protein-protein
interactions (90). The domains that compose the carboxy-terminus are better
characterized. The DNA-binding domain (DBD) (amino acids ~320 to ~480) binds to
members of the gamma activating sites (GAS) family of enhancers, and also appears to
regulate nuclear export (94, 95). The structure of the Statl or Stat3 DBD bound to DNA
consists of several beta sheets, and is similar in configuration to domains of other
transcription factors such as p53 and NF-KB (96). Adjacent to the DBD is a linker
domain (amino acids ~480 to ~575), which is important in the stability of the Stat-DNA
interaction (97). A Src homology 2 (SH2) domain (amino acids «575 to ~680) is the
most highly conserved motif and is critical for both the recruitment of Stats to receptors

and Stat dimerization (92). Finally, the carboxy-terminus contains a transcriptional

20

activation domain (TAD), which varies considerably in both length and sequence,

conferring specificity to the Stat family members (98).

Stat pathway and regulation

A large variety of cytokine and growth factor (polypeptide ligands) can induce
Stat activation. When these ligands bind their cognate receptor, they induce the
dimerization or oligomerization of the receptors. This leads to the apposition of the
associated J aks, which are non-covalently bound to the cytOplasmic tail of receptors (99)
(see Figure 1.3). Once within close proximity, transphosphorylation of the Jaks occurs
on tyrosine residues. The activated J aks subsequently phosphorylate tyrosines within the
cytoplasmic domains of the cytokine-receptor, providing docking sites for the SH2
domains in Stat proteins. The Stats are then recruited to the receptor complex,
whereupon they are phosphorylated on a single tyrosine residue near their carboxy
terminus by the Jaks (100) . Alternatively, Stats can be directly phosphorylated and
activated by receptor tyrosine kinases such as the EGFR, colony stimulating factor-l
receptor (CSF-lR), and platelet derived growth factor receptor (PDGF-R) (92). The non
receptor tyrosine kinase Src can also phosphorylate Stat proteins (101). Regardless of
how they are phosphorylated, Stat dimerization then occurs through reciprocal
phosphotyrosine—SH2 interactions, and released from the receptor complex (102).
Phosphorylated Stat monomers have never been detected, and it is speculated that this is
because two Stats are activated simultaneously within such close proximity that

dimerization occurs instantaneously.

21

—_—__ __,,__f_,__,_____,=__h ,4__:__, _:_

 

 

 

Figure 1.3. Basic overview of the Jak/Stat signaling pathway. Upon ligand binding,
cytokine receptors dimerize, bringing their associated J aks within close proximity to each
other. Once activated by transphosphorylation, the Jaks phosphorylate tyrosines on the
receptor cytoplasmic tail. Stat proteins are then recruited to the phospho-tyrosines via
their SH2 domains. Jaks phosphorylate specific tyrosine residues within recruited Stat
proteins, and phosphorylated Stats dimerize and release from the receptor complex. Stat
dimers translocate to the nucleus and bind to members of the GAS family of enhancers to
induce transcription of Stat regulated genes.

22

Once dimerized, nuclear localization signals are revealed, and Stats quickly
translocate to the nucleus (91). Due to the large size (~180 kDa) of these complexes, Stat
dimers require the assistance of an (rt-importin protein to enter the nucleus through the
nuclear pore complex (103). When Stats are bound by the importin inside the nucleus,
their DNA-binding site is masked (104, 105). However, competition with specific DNA
sequences dislodges the importin, since these sequences have a higher binding affinity for
Stat DBDs (104).

The major effects of activated Stats are through their ability to induce
transcription of target genes. Phosphorylated Stat proteins bind to gene promoters
containing GAS enhancer elements (TTCNNNGAA) (94). The specificity for gene
expression induced by the different Stat proteins is attained through their varying binding
affinities for target DNA sites, and variations in the nucleotide sequence (106). Stats
bind to the DNA directly, and associate with other transcription factors and co-activators
to induce gene expression (91). In particular, Stats rely on their carboxy-terminal TAD to
bind to co-activators. The TADS in Statl, Stat2, Stat3, Stat5 and Stat6 are all known to
interact with histone acetyltranferases (HATS) (90), and the p300/CBP HAT appears to
be highly recruited to the Stat proteins (107). In addition to HATS, interactions with
other proteins can occur at the same time to produce synergistic activation of gene
expression. A variety of such interactions have been described. Stat3 has been reported
to bind to c-jun, GR, and androgen receptor (108-110), Stat6 can bind to
CCAAT/enhancer binding protein (C/EBP) (111), Stat5 can bind to GR and PR (112),
and Statl can bind to Spl (113). Interactions between Stat dimers can also occur at

special tandem binding sites on the DNA, allowing multiple dimers to bind, resulting in

23

maximal transcription activation. To date, there is no record of interactions between
dimers of different Stat proteins (i.e. Stat3 and Stat5).

In addition to tyrosine phosphorylation, serine phosphorylation, arginine
methylation and acetylation of stats has been reported, however details about these
pathways are limited (91). The best described is a serine phosphorylation within the
carboxy-terminus, which has been shown to enhance the transcriptional activity of most
Stat proteins (114). Some co-activators are known to bind more readily when Stats are
phosphorylated on this serine residue in addition to the tyrosine residue in the TAD
(114). However, this does not hold true for all Stat proteins, Since mutation of the
conserved serine in Stat5 did not affect transcriptional activation of a PRL-responsive
promoter in COS cells (115). The most Simple explanation for this is that
phosphorylation of the serine residue in Stat5 proteins may not affect transcription.
Alternatively, the requirement for a phosphorylated serine could be promoter or cell type-
specific. There is a great deal of speculation about the kinases responsible for serine
phosphorylation of Stats, and several candidates within the p38, ERK, PKC, mTOR and
INK pathways have been implicated (114).

The length and intensity of a cytokine response is determined by the balance of
activation and inactivation of the Stat pathway, and ultimately by the number of active
Stat complexes in the nucleus. Stats are exported back to the cytoplasm in a process that
appears to be mediated by the Crml/Ran-GTPase (95), and this export depends on
residues in coiled-coil domain and DBD (95, 116). Dephosphorylation of Stats in the
nucleus initiates the nuclear export pathway. TC45 is one nuclear tyrosine phosphatase

linked to the dephosphorylation of Statl and Stat3 (117). Based on studies using

24

staurosporine to inhibit tyrosine phosphorylation in fibroblasts, it takes approximately 20
minutes for an individual Statl molecule to go through the activation and deactivation
cycle (118).

Besides dephosphorylation, negative regulation of Stat activity in the nucleus is
controlled by at least two other mechanisms. The first is through the actions of a family
of proteins called proteins that inhibit activated Stats (PIAS). These proteins interact
only with tyrosine-phosphorylated Stats, and have been shown to block DNA binding of
Stats, as well as inhibit their transcriptional activity by sumoylation (119). The second
mechanism is via a set of naturally occurring truncated Stats which act as dominant
negative forms of the proteins (91). Shortened forms Statl, Stat3 and Stat5 have all been
found (108, 120, 121). These proteins inhibit the Stat pathway because they contain a
truncated carboxy-terminus that lacks a fully functional TAD. Thus, truncated Stats can
compete with full length Stats for DNA binding and inhibit the activation of gene
expression.

In addition to the factors that deactivate Stat proteins in the nucleus, negative
regulators of the J ak/Stat pathway also exist in the cytoplasm. The supressors of cytokine
Signaling (SOCS) proteins are a family of molecules that act as negative regulators by
binding to receptor and/or Jak catalytic sites to block further phosphorylation of Stat
proteins. SOCS proteins can also induce the tumover of Jaks and some receptors via the
proteasome degradation pathway by recruiting ubiquitin ligases to the activated receptor
complex (122). To date, SOCS are the only known inducible inhibitors of the Jak/Stat
pathway. The expression of SOCS mRNA is directly induced by Stat transcription

factors, thus providing a negative feedback loop (123). A final mechanism of Stat

25

attenuation is via the SH2-containing phosphatase (SHP) proteins. These proteins are
constitutively expressed, and can attenuate Stat signaling by dephosphorylating both J aks

and receptors.

Stat function: normal and disregulated

Stat proteins were first discovered during the analysis of interferon signaling
pathways (124, 125). It is now known that Stats are activated by a large number of
cytokines, and participate in many of the cellular processes, such as proliferation,
differentiation, apoptosis, and cell survival, that are induced by cytokine, growth factor,
and hormone signaling (126). One of the main physiological roles of Stat proteins is to
regulate the immune response to infection. Stats 1, 2, 3, 4 and 6 have all been implicated
in host defense, and the deletion of these genes in mice leads to a higher sensitivity to
infections (91). Many different aspects of the immune response are regulated through the
Stat pathway, including interleukin signaling, differentiation of T cells, and lymphocyte
survival (91). Stats 3 and 5 have also been implicated in mammary gland development,
and a detailed description of these functions is presented in a later section.

Signaling pathways that regulate proliferation and survival are frequently
deregulated in cancer cells, and the Jak/Stat pathway is no exception. Increased
expression of Statl has been observed in many human neoplasias (127). However,
contradictory to this, Statl is also considered a potential tumor suppressor, since it
promotes growth arrest and apoptosis (127). Thus, Statl may play multiple roles in the
process of malignant transformation, and these roles are not yet clearly understood. In

contrast, Stat3 and Stat5 are considered potential oncogenes, since they can induce

26

expression of c-Myc, cyclin D1, and bcl-xl, promoting cell-cycle progression, cellular
transformation, and preventing apoptosis (126). Constitutively activated Stats have been
observed in a number of human cancer cell lines and primary tumors. ( 126). A
persistently active form of Stat3 appears to be required for the survival of many head and
neck and multiple myeloma cancer cells, since the introduction of a dominant-negative
Stat3 into these cells often induces apoptosis (91). The activation of Stat3 in these cells
has been shown to be the result of excess production of EGF R ligand, increased amounts

of normal EGFR, or mutations that increase EGF R activity (128).

Stat5 expression in the mammary gland

Two members of the Stat family, Stat3 and Stat5, have been well documented as
regulators of mammary gland development. While both proteins are critical for the
normal function of the gland, they are believed to play opposing roles (129). Stat3 has
been demonstrated to induce mammary involution by promoting apoptosis (130, 131),
whereas Stat5 appears to inhibit this process (132). In addition, Stat5 is necessary for
robust mammary development during pregnancy, and is vital for lactation. This review
will focus on the functions of Stat5 in the murine mammary gland.

The Stat5 gene was originally cloned from lactating sheep mammary glands (133)
and the protein was identified as a “mammary gland factor” (MGF) that is stimulated by
PRL to initiate the expression of milk proteins (134). There are two closely related
isoforms of Stat5, a and b, each encoded by a separate gene. In the mouse, the genes are
located within a 110 kb stretch on chromosome 11 in a head to head orientation (135).

StatSa and Stat5b show 96% similarity at the amino acid level. They differ mainly within

27

the carboxy-terminus, where the last 8 amino acids of Stat5b are completely different
from Stat5a, and Stat5a is 12 amino acids longer (136). When activated by
phosphorylation, the two isoforms can homo- or hetero-dimerize. Stat5a and Stat5b carry
out distinct functions in vivo. For example, Stat5b is selectively important for OH
Signaling in the liver (137, 138), while Stat5a is critical for the mammary gland
development induced by PRL (138, 139). However, these differences may not be the
result of differences in the proteins themselves, but rather due to the differential
expression of each isotype. For example, Stat5b is the predominant isoform in the liver
(140), while Stat5a is more highly expressed in mammary tissue (138).

The expression of Stat5a and Stat5b (collectively referred to as Stat5 from here
on) is developmentally regulated in the mouse mammary gland. Northern analysis of
whole mammary gland homogenates showed that Stat5 mRNA was only slightly
detectable in the virgin and early pregnant gland, increased dramatically during
pregnancy, and remained high in lactating glands (141). A similar expression pattern was
found in the rat when mRNA levels of the Stat5a isoform were measured (142). Protein
expression of both Stat5a and Stat5b isoforms was measured by Western blotting of
whole mammary gland homogenates, and exhibited a developmental pattern similar to
the mRNA. While total Stat5 protein levels increased during pregnancy and lactation, the
phosphorylation of both isoforms increased even more significantly (143). Both the
protein and phosphorylation levels of Stat5 dropped dramatically after weaning. One
limitation of these studies is that they examined protein expression in the entire
mammary gland, and the relative levels in the epithelium and stroma cannot be

determined from the results. More recently, an immunohistochemical study reported that

28

phosphorylated Stat5 was present in adult virgin mouse and human epithelial cells (144),
but other developmental stages were not examined.

Stat5 activation in the mammary gland can result from signaling through the
GHR, PRLR or EGF R. PRL activates Stat5 only in epithelial cells due to the absence of
PRLR in the stroma, while GHR and EGFR activate Stat5 primarily in the stromal
compartment (61). Pituitary factors are the predominant activators of Stat5 in the non-
pregnant gland, as demonstrated by the lack of phospho-StatS staining following
hypophysectomy (144). The finding that PRLR-/- mice exhibit an 80% reduction in the
level of phosphorylated Stat5 in the mammary gland indicated that PRL is the primary
pituitary factor that induces Stat5 activation (144). J ak2 was shown to mediate the
activation of Stat5 by PRLR in response to PRL treatment (145), and in agreement with
this, PRLR-/-, Jak2-/- and Stat5-/- mammary glands all exhibit similar defects alveolar

development (55, 146, 147).

Stat5 function in the mammary gland

The use of knockout models has been critical to understanding the physiology of
Stat5 in the mammary gland. Two approaches have been taken to produce Stat5 null
mice. The first knockout models were made by the gerrnline deletion of either the entire
110kb Stat5 locus, or of the Stat5a and Stat5b genes independently (referred to as Stat5-l-
, Stat5a-/- and Stat5b-/- mice from here on) (138, 139, 147). Additionally, two mammary
specific knockouts of the entire Stat5 locus were produced to avoid the reproductive
defects seen in Stat5-/- mice, and to examine defects during later development (148).

The conditional knockouts were generated by creating a mouse line which contained the

29

StatSa and Stat5b genes flanked by loxP recombination sites. These mice were then bred
with two transgenic strains, each of which contained the gene for cre recombinase under
the control of a mammary Specific promoter. Expression of cre recombinase in one
mouse line was controlled by the MMTV promoter, which is active in the mammary
gland epithelium throughout development. The second mouse line expressed the cre
recombinase transgene under the control of the WAP promoter, which is activated by
Stat5 in the late pregnant and lactating mammary gland. Thus, in the second system,
Stat5 was deleted only in cells that had already become fully differentiated.

Gerrnline deletion of both Stat5 isoforms resulted in incomplete alveolar
development, and a complete absence of lactation (138, 147), which was similar to the
mammary phenotypes seen in Jak2-/- (146) and PRLR-/- mice. When only the Stat5a
isoform was deleted, mammary development was comparable to the total Stat5 knockout
(138, 139). In contrast, deletion of Stat5b was reported have either no effect or an
extremely blunted effect on mammary development when compared to Stat5a knockouts
(138, 139). This difference is likely due to the disparity in expression levels between the
Stat isoforms in the mammary gland (138). In concordance with this model, Stat5b was
able to compensate for the loss of Stat5a after multiple pregnancies in Stat5a-/- mice, and
an increase in Stat5b levels was observed in these mice (149).

Another important phenotype of Stat5-/- mice to note is their infertility. This
defect was determined to be the result of non-functioning corpora lutea, the PRL
stimulated exocrine glands in the ovary that secrete P ( 138). PRL has a critical role in
ovarian function, as indicated by the fact that both PRL-/- mice (150) and PRLR-l- mice

are infertile (53), as well as the role of PRL in activating Stat5. It is interesting to note

30

that both Stat5a -/- and Stat5b -/- mice are fertile, indicating the functional redundancy of
the Stat5 isoforms in the ovary. However, it is not known if the corpora lutea are
maximally functional in these animals. To circumvent the problem of infertility in Stat5
knockout mice, mammary gland transplantation was used to examine mammogenesis
during pregnancy.

Mammary glands comprised of Stat5-/- epithelium grown in wild-type stroma
exhibited a lack of functional alveoli at parturition (147). Further investigation
determined that this phenotype was associated with a defect in proliferation in response
to E+P treatment. Wild-type mice were transplanted with Stat5-/- epithelium, and
mammary glands were allowed to develop for 6 weeks. The mice were then treated with
an acute 2 day dose of E + P to mimic early pregnancy, and proliferation was assessed by
the percentage of cells that incorporated BrdU during a short labeling period prior to
sacrifice. The results showed that there were approximately 50% fewer 5-Bromo-2’-
Deoxyuridine (BrdU) positive cells in Stat5-/- epithelium compared to wild-type glands
(147). Similarly, MMTV-ore recombinase Stat5-/— mice Showed an 80% decrease in
mammary epithelial cells positive for the proliferation marker, phosphorylated histone 3
(H3P) in response to the same 2 day hormone treatment ( 148). Together, these findings
demonstrate that a major function of Stat5 in the mammary gland is to mediate the
proliferation of epithelial cells in response to ovarian hormones.

Stat5 has also been described as a regulator of mammary cell differentiation, and
this role appears to be especially critical in the pregnant and lactating gland. Evidence
for this function comes from the observation that epithelial cells in Stat5-/- mammary

glands do not express markers of fully differentiated cells during pregnancy, and instead

31

maintain virgin-like features (147). The expression of the Na-K-Cl cotransporter
(NKCCl) is normally present in the virgin mammary epithelium, but at much lower
levels in alveoli during pregnancy. When the alveolar—like structures in pregnant Stat5-l-
mammary glands were examined, expression of NKCCI was maintained at the level seen
in virgin glands. Further investigation found that the sodium phosphate cotransporter
Npt2b, a protein that is normally expressed at high levels in the lactating gland, was not
detectable in Stat5-/- mammary cells at parturition. In agreement with these findings,
expression of markers of fully differentiated secretory cells, WAP, B-casein, and or-
lactalbumin, was decreased in Stat5a—/— glands at parturition.

Genetic disruption of Stat5 in the mammary gland has also uncovered a role for
the protein in regulating cell survival. The loss of Stat5 late in pregnancy in WAP-ore
recombinase Stat5-/- mice resulted in premature mammary cell death via apoptosis.
When expression of the cre recombinase protein was examined during pregnancy, the
number of positive cells continually decreased, and very few expressing cells remained
by day 18.5 of pregnancy. This indicated that cells producing cre recombinase, and thus
lacking Stat5 expression, were not able to survive in the pregnant gland. Terminal
uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed that ~5
times more apoptotic cells were present in the WAP-cre-recombinase Stat5-/- mammary
glands at day 15.5 of pregnancy compared to control glands. In summary, the results
obtained from the Stat5 knockout studies have demonstrated that Stat5 controls the
formation of functionally differentiated alveoli during pregnancy through the regulation

of proliferation, differentiation and survival of mammary epithelial cells.

32

Stat5 regulated genes

A number of genes are transcriptionally regulated by Stat5 and, as predicted by its
roles in the mammary gland and other tissues, many of these genes contribute to cell
proliferation, survival, and differentiation. Gene array experiments have contributed to
defining which genes are induced by Stat5 in the mammary gland. The induced
dimerization of Stat5-gyrase fusion proteins in KIMZ mammary epithelial cells elicited
the upregulation of numerous genes (132). Of the most highly induced genes, many
encoded epithelial differentiation markers and anti-apoptotic proteins, such as or-casein
and prosaposin, respectively.

Many of the genes involved in differentiation that are directly induced by Stat5 in
the mammary gland encode milk constituents such as WAP, B-casein and (rt-lactalbumin.
However, Stat5 can also induce the expression of other transcription factors, which in
turn can regulate expression of milk genes. Expression of Etsl was found to be directly
regulated by Stat5 (132), and Etsl has been shown to induce the WAP gene (151). EIfS,
another member of the Ets family of transcription factors, is hypothesized to be induced
by Stat5, and it also regulates milk production in the mammary gland (152).

Stat5 can also activate transcription of several genes implicated in proliferation
and survival. Expression of RANKL is induced by PRLR signaling through the
activation of Stat5 in mammary epithelial cells (153). Multiple pathways may be
regulated by RANKL in mammary cells, and the exact mechanisms by which it induces
proliferation are not yet clear. Stat5 can also activate transcription of the cyclin D1 gene
directly at GAS elements within the promoter, and Stat5 has been observed to bind to the

cyclin D1 gene promoter in the MCF-7 and HC-ll mammary epithelial cell lines in

33

response to hypoxic conditions (154). Finally, expression of the anti-apoptotic protein
Bcl-xl has been shown to be induced by Stat5 in several systems, but it is not known

whether this occurs in mammary epithelial cells (155).

Stat5 and breast cancer

In addition to its role in normal mammary gland development, Stat5 has also been
implicated in mammary oncogenesis. Transgenic animals that express either wild-type or
a constitutively active form of Stat5 in the mammary gland develop mammary tumors at
an incidence rate of up to 22% within 8-12 months (156). In addition, the deletion of
Stat5 impairs tumor development in mice with mammary specific over-expression of
TGF-a or SV40 large-T antigen (157, 158). Finally, PRL is a reported tumor promoter in
rodents (159), and this may be via the activation of Stat5.

Stat5 may also play a role in human breast cancer as well, although the data are
somewhat paradoxical. An early study examined Stat5 activity in extracts from a small
number of invasive carcinomas, benign lesions and normal tissue by electrophoretic
mobility shift assay (EMSA) (160). The majority of invasive carcinomas exhibited
increased Stat5 activity compared to the other two sample types, suggesting that Stat5
may aid in the progression of tumors. Although this study presented evidence for
changes in Stat5 activity in breast cancer, the conclusions that could be drawn were
limited by a lack of normalization to total Stat5 protein levels, and a small sample size.

Data obtained in a number of more recent studies using larger sample Sizes
somewhat oppose the early findings. When nuclear-localized, tyrosine phosphorylated

Stat5 was scored in tumor samples from more than 1000 patients, tumors with active

34

Stat5 corresponded to a reduced risk of tumor recurrence and patient morbidity (161).
The prognosis based on Stat5 activity was independent of many other predictors of
survival including ER and PR status, Her2/neu status, tumor size, and histological grade.
The link between Stat5 activity and patient survival may be due to its ability to suppress
tumorinvasiveness. Activation of Stat5 via PRL in both T-47D and ZR-75-l breast
cancer cell lines leads to increased levels of E-cadherin, an adhesion molecule known to
suppress cell invasion (162).

Although it seems somewhat contradictory, Stat5 appears to play a positive role in
tumor formation and an inhibitory role in tumor progression. The ability of Stat5 to
induce proliferation may explain why constitutively active forms of the protein can lead
to tumor formation. Additionally, increased Stat5 activity could lead to a larger pool of
epithelial cells that can become transformed. However, once tumors have formed, the
presence of active Stat5 could prohibit metastasis through inducing differentiation of

tumor cells, potentially explaining the increase in patient survival.

Conclusion

Mammary gland development is a dynamic process that requires a network of
several signaling pathways. Numerous studies have established the signaling molecule,
Stat5a as one of the critical regulators of pregnancy-induced mammary gland
development. Since its discovery as a key mediator of PRL signaling, considerable
progress has been made in defining the mechanisms leading to Stat5a activity. However,
several aspects of Stat5a in the mammary gland are still relatively unknown. The

underlying mechanisms that regulate Stat5a expression in the mammary gland are not

35

known. In addition, the function of Stat5a outside of pregnancy has not been thoroughly
explored. Finally, the mechanisms by which Stat5a controls proliferation in the
mammary gland have not been elucidated. The research presented in this dissertation
addresses these aspects, and provides new insight into both the regulation and function of

Stat5a in the mouse mammary gland.

36

10.

ll.

12.

13.

REFERENCES

Griffiths M 1978 Biology of the Monotremes. Academic Press, New York

Cardiff RD, Wellings SR 1999 The comparative pathology of human and mouse
mammary glands. J Mammary Gland Biol Neoplasia 4:105-22

Hovey RC, McFadden TB, Akers RM 1999 Regulation of mammary gland
growth and morphogenesis by the mammary fat pad: a species comparison. J
Mammary Gland Biol Neoplasia 4:53-68

Daniel CW, Silberstein G 1987 Postnatal development of the rodent mammary
gland. In: Neville MC, Daniel CW (eds) The Mammary Gland: Development,
Regulation and Function. Plenum Press, New York; pp 3-36

Richardson KC 1949 The effector contractile tissues of the mammary gland. J
Endocrinol 6:Suppl, xxv

Howard BA, Gusterson BA 2000 Human breast development. J Mammary
Gland Biol Neoplasia 5:119-37

Balinsky B1 1950 On the prenatal growth of the mammary gland rudiment in the
mouse. J Anat 842227-35

Propper AY 1978 Wandering epithelial cells in the rabbit embryo milk line. A
preliminary scanning electron microscope study. Dev Biol 67:225-31

Veltmaat J M, Mailleux AA, Thiery JP, Bellusci S 2003 Mouse embryonic
mammogenesis as a model for the molecular regulation of pattern formation.
Differentiation 71 :1-17

Howard B, Ashworth A 2006 Signalling pathways implicated in early mammary
gland morphogenesis and breast cancer. PLoS Genet 2:e112

Deome KB, Faulkin LJ, J r., Bern HA, Blair PB 1959 Development of
mammary tumors from hyperplastic alveolar nodules transplanted into gland—free
mammary fat pads of female C3H mice. Cancer Res 19:515-20

Williams JM, Daniel CW 1983 Mammary ductal elongation: differentiation of
myoepithelium and basal lamina during branching morphogenesis. Dev Biol
97:274-90

Humphreys RC, Krajewska M, Krnacik S, Jaeger R, Weiher H, Krajewski S,
Reed JC, Rosen J M 1996 Apoptosis in the terminal endbud of the murine

37

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

mammary gland: a mechanism of ductal morphogenesis. Development 122:4013-
22

Vonderhaar BK 1988 Regulation of development of the normal mammary gland
by hormones and grth factors. Cancer Treat Res 40:251-66

Sekhri KK, Pitelka DR, DeOme KB 1967 Studies of mouse mammary glands. I.
Cytomorphology of the normal mammary gland. J Natl Cancer Inst 392459-90

Strange R, Li F, Saurer S, Burkhardt A, Friis RR 1992 Apoptotic cell death
and tissue remodelling during mouse mammary gland involution. Development
115:49-58

Halban J 1900 Die innere Sekretion von Ovarium und Placenta und ihre
Bedeutung fuer die Function der Milchdruese. Mschr Geburtsh Gynaek2496—5 O3

Tsai MJ, O'Malley BW 1994 Molecular mechanisms of action of steroid/thyroid
receptor superfamily members. Annu Rev Biochem 632451-86

Cato AC, N estl A, Mink S 2002 Rapid actions of steroid receptors in cellular
signaling pathways. Sci STKE 20021RE9

Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned
and where will they lead us? Endocr Rev 202358-417

Cunha GR, Young P, Hom YK, Cooke PS, Taylor JA, Lubahn DB 1997
Elucidation of a role for stromal steroid hormone receptors in mammary gland

grth and development using tissue recombinants. J Mammary Gland Biol
Neoplasia 2:393-402

Mueller SO, Clark JA, Myers PH, Korach KS 2002 Mammary gland
development in adult mice requires epithelial and stromal estrogen receptor alpha.
Endocrinology 143:2357-65

Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993
Alteration of reproductive function but not prenatal sexual development after

insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci U
S A 90:11162-6

Couse JF, Curtis SW, Washburn T F, Lindzey J, Golding TS, Lubahn DB,
Smithies O, Korach KS 1995 Analysis of transcription and estrogen insensitivity

in the female mouse after targeted disruption of the estrogen receptor gene. Mol
Endocrinol 9: 1441-54

38

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Kos M, Denger S, Reid G, Korach KS, Gannon F 2002 Down but not out? A
novel protein isoform of the estrogen receptor alpha is expressed in the estrogen
receptor alpha knockout mouse. J Mol Endocrinol 29:281-6

Mallepell S, Krust A, Chambon P, Brisken C 2006 Paracrine Signaling through
the epithelial estrogen receptor alpha is required for proliferation and
morphogenesis in the mammary gland. Proc Natl Acad Sci U S A 103:2196-201

Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, Warner M,
Gustafsson JA 2002 Involvement of estrogen receptor beta in terminal
differentiation of mammary gland epithelium. Proc Natl Acad Sci U S A

99: 15578-83

Conneely OM, Mulac-Jericevic B, Lydon JP 2003 Progesterone-dependent
regulation of female reproductive activity by two distinct progesterone receptor
isoforms. Steroids 68:771-8

Aupperlee MD, Smith KT, Kariagina A, Haslam SZ 2005 Progesterone
receptor isoforms A and B: temporal and Spatial differences in expression during
murine mammary gland development. Endocrinology 146:3577-88

Kariagina A, Aupperlee MD, Haslam SZ 2007 Progesterone receptor isoforms
and proliferation in the rat mammary gland during development. Endocrinology
148:2723-36

Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA,
J r., Shyamala G, Conneely OM, O'Malley BW 1995 Mice lacking progesterone
receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266-78

Mulac—Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM 2000
Subgroup of reproductive functions of progesterone mediated by progesterone
receptor—B isoform. Science 289: 175 1-4

Mulac—Jericevic B, Lydon JP, DeMayo FJ, Conneely OM 2003 Defective
mammary gland morphogenesis in mice lacking the progesterone receptor B
isoform. Proc Natl Acad Sci U S A 100:9744-9

Shyamala G, Yang X, Silberstein G, Barcellos-Hoff MH, Dale E 1998
Transgenic mice carrying an imbalance in the native ratio of A to B forms of
progesterone receptor exhibit developmental abnormalities in mammary glands.
Proc Natl Acad Sci U S A 95:696-701

Shyamala G, Yang X, Cardiff RD, Dale E 2000 Impact of progesterone

receptor on cell-fate decisions during mammary gland development. Proc Natl
Acad Sci U S A 97:3044-9

39

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

Brisken C, Park S, Vass T, Lydon JP, O'Malley BW, Weinberg RA 1998 A
paracrine role for the epithelial progesterone receptor in mammary gland
development. Proc Natl Acad Sci U S A 95:5076-81

Kingsley-Kallescn M, Mukhopadhyay SS, Wyszomierski SL, Schanler S,
Schutz G, Rosen J M 2002 The mineralocorticoid receptor may compensate for
the loss of the glucocorticoid receptor at Specific stages of mammary gland
development. Mol Endocrinol 16:2008-18

Reichardt HM, Horsch K, Grone HJ, Kolbus A, Beug H, Hynes N, Schutz G
2001 Mammary gland development and lactation are controlled by different
glucocorticoid receptor activities. Eur J Endocrinol 145:519-27

Ganguly R, Ganguly N, Mehta NM, Banerjee MR 1980 Absolute requirement
of glucocorticoid for expression of the casein gene in the presence of prolactin.
Proc Natl Acad Sci U S A 77:6003-6

Wintermantel TM, Bock D, Fleig V, Greiner EF, Schutz G 2005 The epithelial
glucocorticoid receptor is required for the normal timing of cell proliferation
during mammary lobuloalveolar development but is dispensable for milk

production. Mol Endocrinol 19:340-9

McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, O'Malley BW 1987
Molecular cloning of complementary DNA encoding the avian receptor for
vitamin D. Science 235:1214-7

Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler
MR, Pike JW, Shine J, O'Malley BW 1988 Cloning and expression of full-
length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci U S A
85:3294-8

Welsh J, Wietzke JA, Zinser GM, Smyczek S, Romu S, Tribble E, Welsh JC,
Byrne B, Narvaez CJ 2002 Impact of the Vitamin D3 receptor on growth-

regulatory pathways in mammary gland and breast cancer. J Steroid Biochem Mol
Biol 83:85-92

Zinser G, Packman K, Welsh J 2002 Vitamin D(3) receptor ablation alters
mammary gland morphogenesis. Development 129:3067-76

Reece RP, Turner, C.W., Hill RT. 1936 Mammary gland development in the
hypophysectomized albino rat. Proc Soc Exp Biol Med 34:204-217

Stricker PR, Grueter, R. 1928 Action du lobe anterieur de. l’hypophyse sur la
montee laiteuse. Comptes Rendus des Seances Société biologie 99:1978-1980

40

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

Riddle O, Bates, RW. and Dykshorn, SW. 1933 The preparation, identification
and assay of prolactin-a hormone of the anterior pituitary. Am. J. Physiol.
105:191-216

Rossmanith WG, Marks BL, Clifton DK, Steiner RA 1996 Induction of
galanin mRNA in GnRH neurons by estradiol and its facilitation by progesterone.
J Neuroendocrinol 8: 185-91

Ormandy CJ, Lee CS, Ormandy HF, Fantl V, Shine J, Peters G, Sutherland
RL 1998 Amplification, expression, and steroid regulation of the preprogalanin
gene in human breast cancer. Cancer Res 58:1353-7

Grosvenor CE, Whitworth N 1974 Evidence for a steady rate of secretion of
prolactin following suckling in the rat. J Dairy Sci 57:900-4

Reynolds C, Montone KT, Powell CM, Tomaszewski JE, Clevenger CV 1997
Expression of prolactin and its receptor in human breast carcinoma.
Endocrinology 138:5555-60

Bazan J F 1989 A novel family of grth factor receptors: a common binding
domain in the grth hormone, prolactin, erythropoietin and IL-6 receptors, and
the p75 IL-2 receptor beta-chain. Biochem Biophys Res Commun 164:788-95

Ormandy CJ, Binart N, Kelly PA 1997 Mammary gland development in
prolactin receptor knockout mice. J Mammary Gland Biol Neoplasia 2:355-64

Naylor MJ, Lockefeer JA, Horseman ND, Ormandy CJ 2003 Prolactin
regulates mammary epithelial cell proliferation via autocrine/paracrine
mechanism. Endocrine 20:1 1 1—4

Brisken C, Kaur S, Chavarria TE, Binart N, Sutherland RL, Weinberg RA,
Kelly PA, Ormandy CJ 1999 Prolactin controls mammary gland development
via direct and indirect mechanisms. Dev Biol 210:96-106

Talamantes F, Jr. 1975 Comparative study of the occurrence of placental
prolactin among mammals. Gen Comp Endocrinol 27:115-21

Sakai S, Kohmoto K 1976 Binding of mouse choriomammotropin to prolactin
receptors in the mouse mammary gland. Endocrinol Jpn 23:499-503

Collip J, Seyle H, Thompson D 1933 Further observations on the effect of
hypophysectomy on lactation. Proc Soc Exp Biol Med 30:913

Thordarson G, Talamantes F 1987 Role of the placenta in mammary gland
development and function. In: Neville MC, Daniel CW (eds) The Mammary

41

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

Gland: Development, Regulation, and Function. Plenum Press, New York; pp
459-498

Wilde CJ, Knight CH, Flint DJ 1999 Control of milk secretion and apoptosis
during mammary involution. J Mammary Gland Biol Neoplasia 42129-36

Gallego MI, Binart N, Robinson GW, Okagaki R, Coschigano KT, Perry J,
Kopchick JJ, Oka T, Kelly PA, Hennighausen L 2001 Prolactin, growth
hormone, and epidermal growth factor activate Stat5 in different compartments of

mammary tissue and exert different and overlapping developmental effects. Dev
Biol 229: 163-75

Hovey RC, Trott J F, Vonderhaar BK 2002 Establishing a framework for the
functional mammary gland: from endocrinology to morphology. J Mammary
Gland Biol Neoplasia 7: 1 7-38

Allan GJ, Tonner E, Barber MC, Travers MT, Shand JH, Vernon RG, Kelly
PA, Binart N, Flint DJ 2002 Growth hormone, acting in part through the insulin-
like grth factor axis, rescues developmental, but not metabolic, activity in the

mammary gland of mice expressing a single allele of the prolactin receptor.
Endocrinology 143 :43 10-9

Caron RW, J ahn GA, Deis RP 1994 Lactogenic actions of different growth
hormone preparations in pregnant and lactating rats. J Endocrinol 142:535-45

Flint DJ, Gardner M 1994 Evidence that growth hormone stimulates milk
synthesis by direct action on the mammary gland and that prolactin exerts effects
on milk secretion by maintenance of mammary deoxyribonucleic acid content and
tight junction status. Endocrinology 135:11 19-24

Ways J, Markoff E, Ogren L, Talamantes F 1979 Lactogenic response of
mouse mammary explants from different days of pregnancy to placental lactogen
and pituitary prolactin. In Vitro 15:891-4

Clarke RB, Howell A, Potten CS, Anderson E 1997 Dissociation between
steroid receptor expression and cell proliferation in the human breast. Cancer Res
57:4987-91

Russo J, A0 X, Grill C, Russo IH 1999 Pattern of distribution of cells positive
for estrogen receptor alpha and progesterone receptor in relation to proliferating
cells in the mammary gland. Breast Cancer Res Treat 53:217-27

Zeps N, Bentel JM, Papadimitriou JM, D'Antuono MF, Dawkins HJ 1998

Estrogen receptor-negative epithelial cells in mouse mammary gland development
and growth. Differentiation 62:221-6

42

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

Ruan W, Newman CB, Kleinberg DL 1992 Intact and amino-terminally
Shortened forms of insulin-like growth factor I induce mammary gland
differentiation and development. Proc Natl Acad Sci U S A 89:10872-6

Kleinberg DL, Ruan W, Catanese V, Newman CB, Feldman M 1990 Non-
lactogenic effects of growth hormone on grth and insulin-like growth factor-I
messenger ribonucleic acid of rat mammary gland. Endocrinology 126:3274-6

Walden PD, Ruan W, Feldman M, Kleinberg DL 1998 Evidence that the
mammary fat pad mediates the action of grth hormone in mammary gland
development. Endocrinology 139:659-62

Ruan W, Kleinberg DL 1999 Insulin-like grth factor I is essential for terminal

end bud formation and ductal morphogenesis during mammary development.
Endocrinology 140:5075-81

Ruan W, Catanese V, Wieczorek R, Feldman M, Kleinberg DL 1995 Estradiol
enhances the stimulatory effect of insulin-like grth factor-I (IGF-I) on
mammary development and grth hormone-induced IGF-I messenger
ribonucleic acid. Endocrinology 136: 1296-302

Kleinberg DL 1998 Role of IGF-I in normal mammary development. Breast
Cancer Res Treat 47:201-8

Richards RG, Klotz DM, Walker MP, Diaugustine RP 2004 Mammary gland
branching morphogenesis is diminished in mice with a deficiency of insulin-like

growth factor-I (IGF-I), but not in mice with a liver-specific deletion of IGF-I.
Endocrinology 145 :3 1 06-1 0

Earp HS, Dawson TL, Li X, Yu H 1995 Heterodimerization and functional
interaction between EGF receptor family members: a new signaling paradigm
with implications for breast cancer research. Breast Cancer Res Treat 35:115-32

Pinkas-Kramarski R, Alroy I, Yarden Y 1997 ErbB receptors and EGF-like
ligands: cell lineage determination and oncogenesis through combinatorial
Signaling. J Mammary Gland Biol Neoplasia 2:97-107

Xie W, Paterson AJ, Chin E, Nabell LM, Kudlow JE 1997 Targeted expression
of a dominant negative epidermal growth factor receptor in the mammary gland of

transgenic mice inhibits pubertal mammary duct development. Mol Endocrinol
1 1 : 1 766-8 1

Wiesen JF, Young P, Werb Z, Cunha GR 1999 Signaling through the stromal

epidermal growth factor receptor is necessary for mammary ductal development.
Development 126:335-44

43

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

Howlin J, McBryan J, Martin F 2006 Pubertal mammary gland development:
insights from mouse models. J Mammary Gland Biol Neoplasia 11:283-97

Sebastian J, Richards RG, Walker MP, Wiesen JF, Werb Z, Derynck R,
Horn YK, Cunha GR, DiAngustine RP 1998 Activation and function of the
epidermal grth factor receptor and erbB-2 during mammary gland
morphogenesis. Cell Growth Differ 9:777-85

Martinez-Lacaci I, Saceda M, Plowman GD, Johnson GR, Normanno N,
Salomon DS, Dickson RB 1995 Estrogen and phorbol esters regulate
amphiregulin expression by two separate mechanisms in human breast cancer cell
lines. Endocrinology 136:3983-92

Luetteke NC, Qiu TH, Fenton SE, Troyer KL, Riedel RF, Chang A, Lee DC
1999 Targeted inactivation of the EGF and amphiregulin genes reveals distinct
roles for EGF receptor ligands in mouse mammary gland development.
Development 126:2739-50

Imagawa W, Pedchenko VK, Helber J, Zhang H 2002 Hormone/growth factor
interactions mediating epithelial/stromal communication in mammary gland
development and carcinogenesis. J Steroid Biochem Mol Biol 80:213-30

Brisken C, Heineman A, Chavarria T, Elenbaas B, Tan J, Dey SK,
McMahon JA, McMahon AP, Weinberg RA 2000 Essential function of Wnt-4

in mammary gland development downstream of progesterone signaling. Genes
Dev 14:650-4

Boyce BF, Xing L 2007 Biology of RANK, RANKL, and osteoprotegerin.
Arthritis Res Ther 9 Suppl 1:S1

Fata JE, Kong YY, Li J, Sasaki T, llrie-Sasaki J, Moorehead RA, Elliott R,
Scully S, Voura EB, Lacey DL, Boyle WJ, Khokha R, Penninger JM 2000
The osteoclast differentiation factor osteoprotegerin-ligand is essential for
mammary gland development. Cell 103:41-50

Gonzalez-Suarez E, Branstetter D, Armstrong A, Dinh H, Blumberg H,
Dougall WC 2007 RANK overexpression in transgenic mice with mouse
mammary tumor virus promoter-controlled RANK increases proliferation and
impairs alveolar differentiation in the mammary epithelia and disrupts lumen
formation in cultured epithelial acini. Mol Cell Biol 27: 1442-54

Horvath CM 2000 STAT proteins and transcriptional responses to extracellular
signals. Trends Biochem Sci 25:496-502

Levy DE, Darnell JE, Jr. 2002 Stats: transcriptional control and biological
impact. Nat Rev Mol Cell Biol 3:651—62

44

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW 2002 Signaling
through the J AK/STAT pathway, recent advances and future challenges. Gene
285:1-24

Vinkemeier U, Moarefi I, Darnell JE, J r., Kuriyan J 1998 Structure of the
amino-terminal protein interaction domain of STAT-4. Science 279:1048-52

Decker T, Kovarik P, Meinke A 1997 GAS elements: a few nucleotides with a
major impact on cytokine-induced gene expression. J Interferon Cytokine Res
17:121-34

McBride KM, McDonald C, Reich NC 2000 Nuclear export signal located
within theDNA-binding domain of the STATltranscription factor. EMBO J
19:6196-206

Chen X, Vinkemeier U, Zhao Y, Jeruzalnri D, Darnell JE, J r., Kuriyan J
1998 Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to
DNA. Cell 93:827-39

Yang E, Henriksen MA, Schaefer O, Zakharova N, Darnell JE, Jr. 2002
Dissociation time from DNA determines transcriptional function in a STATl
linker mutant. J Biol Chem 277:13455-62

Schindler CW 2002 Series introduction. J AK-STAT signaling in human disease.
J Clin Invest 109:1133-7

Leonard WJ 2001 Role of J ak kinases and STATS in cytokine signal
transduction. Int J Hematol 73 :27 1 -7

Darnell JE, Jr. 1997 STATS and gene regulation. Science 277: 1630-5

Olayioye MA, Beuvink I, Horsch K, Daly JM, Hynes NE 1999 ErbB receptor-
induced activation of stat transcription factors is mediated by Src tyrosine kinases.
J Biol Chem 274:17209-18

Greenlund AC, Morales MO, Viviano BL, Yan H, Krolewski J, Schreiber RD
1995 Stat recruitment by tyrosine-phosphorylated cytokine receptors: an ordered
reversible affinity-driven process. Immunity 2:677-87

Mattaj IW, Englmeier L 1998 Nucleocytoplasmic transport: the soluble phase.
Annu Rev Biochem 67:265-306

McBride KM, Banninger G, McDonald C, Reich NC 2002 Regulated nuclear

import of the STAT] transcription factor by direct binding of importin-alpha.
Embo J 21:1754-63

45

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

Fagerlund R, Melen K, Kinnunen L, Julkunen I 2002 Arginine/lysine-rich
nuclear localization signals mediate interactions between dimeric STATS and
importin alpha 5. J Biol Chem 277:30072-8

Ehret GB, Reichenbach P, Schindler U, Horvath CM, Fritz S, Nabholz M,
Bucher P 2001 DNA binding Specificity of different STAT proteins. Comparison
of in Vitro Specificity with natural target sites. J Biol Chem 276:6675-88

Paulson M, Pisharody S, Pan L, Guadagno S, Mui AL, Levy DE 1999 Stat
protein transactivation domains recruit p300/CBP through widely divergent
sequences. J Biol Chem 274:25343-9

Schaefer TS, Sanders LK, Nathans D 1995 Cooperative transcriptional activity
of Jun and Stat3 beta, a short form of Stat3. Proc Natl Acad Sci U S A 92:9097-
101

Zhang Z, Jones S, Hagood J S, Fuentes NL, Fuller GM 1997 STAT3 acts as a
co-activator of glucocorticoid receptor signaling. J Biol Chem 272:30607-10

Ueda T, Bruchovsky N, Sadar MD 2002 Activation of the androgen receptor N-
terrninal domain by interleukin-6 via MAPK and STAT3 signal transduction
pathways. J Biol Chem 277:7076-85

Delphin S, Stavnezer J 1995 Characterization of an interleukin 4 (IL-4)
responsive region in the immunoglobulin heavy chain germline epsilon promoter:
regulation by NF-IL-4, a C/EBP family member and NF-kappa B/p50. J Exp Med
181 : 1 8 1-92

Stoecklin E, Wissler M, Schaetzle D, Pfitzner E, Groner B 1999 Interactions in
the transcriptional regulation exerted by Stat5 and by members of the steroid
hormone receptor family. J Steroid Biochem Mol Biol 69:195-204

Look DC, Pelletier MR, Tidwell RM, Roswit WT, Holtzman MJ 1995 Statl
depends on transcriptional synergy with Spl. J Biol Chem 270:30264-7

Decker T, Kovarik P 2000 Serine phosphorylation of STATS. Oncogene
19:2628-37

Yamashita H, Xu J, Erwin RA, Farrar WL, Kirken RA, Rui H 1998
Differential control of the phosphorylation state of proline-juxtaposed serine

residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells. J Biol
Chem 273:30218-24

46

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

Begitt A, Meyer T, van Rossum M, Vinkemeier U 2000 Nucleocytoplasmic
translocation of Statl is regulated by a leucine-rich export Signal in the coiled-coil
domain. Proc Natl Acad Sci U S A 97: 10418-23

ten Hoeve J, de Jesus Ibarra—Sanchez M, Fu Y, Zhu W, Tremblay M, David
M, Shuai K 2002 Identification of a nuclear Statl protein tyrosine phosphatase.
Mol Cell Biol 22:5662-8

Haspel RL, Darnell JE, Jr. 1999 A nuclear protein tyrosine phosphatase is
required for the inactivation of Statl. Proc Natl Acad Sci U S A 96:10188-93

Shuai K 2000 Modulation of STAT signaling by STAT-interacting proteins.
Oncogene 19:2638-44

Schindler C, Fu XY, Improta T, Aebersold R, Darnell J E, Jr. 1992 Proteins of
transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins
that are activated by interferon alpha. Proc Natl Acad Sci U S A 89:7836-9

Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN 1996 Naturally
occurring dominant negative variants of Stat5. Mol Cell Biol 16:6141-8

Wormald S, Hilton DJ 2004 Inhibitors of cytokine signal transduction. J Biol
Chem 279:821-4

Krebs DL, Hilton DJ 2001 SOCS proteins: negative regulators of cytokine
signaling. Stem Cells 19:378-87

Fu XY, Kessler DS, Veals SA, Levy DE, Darnell JE, Jr. 1990 ISGF 3, the
transcriptional activator induced by interferon alpha, consists of multiple
interacting polypeptide chains. Proc Natl Acad Sci U S A 87:8555-9

Kessler DS, Veals SA, Fu XY, Levy DE 1990 Interferon-alpha regulates nuclear
translocation and DNA-binding affinity of ISGF 3, a multimeric transcriptional
activator. Genes Dev 4: 1 753-65

Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo
A 2003 STAT proteins: from normal control of cellular events to tumorigenesis. J
Cell Physiol 197:157-68

Adamkova L, Souckova K, Kovarik J 2007 Transcription protein STATI:
biology and relation to cancer. F olia Biol (Praha) 53: 1-6

Song JI, Grandis JR 2000 STAT signaling in head and neck cancer. Oncogene
19:2489-95

47

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

Hennighausen L, Robinson GW, Wagner KU, Liu X 1997 Developing a
mammary gland is a stat affair. J Mammary Gland Biol Neoplasia 22365-72

Kritikou EA, Sharkey A, Abell K, Came PJ, Anderson E, Clarkson RW,
Watson CJ 2003 A dual, non-redundant, role for LIF as a regulator of
development and STAT3-mediated cell death in mammary gland. Development
130:3459-68

Chapman RS, Lourenco PC, Tonner E, Flint DJ, Selbert S, Takeda K, Akira
S, Clarke AR, Watson CJ 1999 Suppression of epithelial apoptosis and delayed
mammary gland involution in mice with a conditional knockout of Stat3. Genes
Dev 13:2604-16

Clarkson RW, Boland MP, Kritikou EA, Lee JM, Freeman TC, Tiffen PG,
Watson CJ 2006 The genes induced by signal transducer and activators of
transcription (STAT)3 and STAT5 in mammary epithelial cells define the roles of
these STATS in mammary development. Mol Endocrinol 20:675-85

Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF) is a
novel member of the cytokine regulated transcription factor gene family and
confers the prolactin response. EMBO J 13:2182-91

Wakao H, Gouilleux F, Groner B 1995 Mammary gland factor (MGF) is a
novel member of the cytokine regulated transcription factor gene family and
confers the prolactin response. EMBO J 14:854-5

Miyoshi K, Cui Y, Riedlinger G, Robinson P, Lehoczky J, Zon L, Oka T,
Dewar K, Hennighausen L 2001 Structure of the mouse Stat 3/5 locus: evolution
from Drosophila to zebrafish to mouse. Genomics 71:150-5

Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning
and expression of Stat5 and an additional homologue (Stat5b) involved in

prolactin Signal transduction in mouse mammary tissue. Proc Natl Acad Sci U S
A 92:8831-5

Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ,
Davey HW 1997 Requirement of STATSb for sexual dimorphism of body grth
rates and liver gene expression. Proc Natl Acad Sci U S A 94:7239-44

Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D,
Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b proteins have

essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841-
50

48

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A,
Hennighausen L 1997 Stat5a is mandatory for adult mammary gland
development and lactogenesis. Genes Dev 11:179-86

Standke GJ, Meier VS, Groner B 1994 Mammary gland factor activated by
prolactin on mammary epithelial cells and acute-phase response factor activated

by interleukin-6 in liver cells share DNA binding and transactivation potential.
Mol Endocrinol 8:469-77

Philp JA, Burdon TG, Watson CJ 1996 Differential activation of STATS 3 and
5 during mammary gland development. FEBS Lett 396:77-80

Kazansky AV, Raught B, Lindsey SM, Wang YF, Rosen JM 1995 Regulation
of mammary gland factor/Stat5a during mammary gland development. Mol
Endocrinol 9: 1 598-609

Liu X, Robinson GW, Hennighausen L 1996 Activation of StatSa and Stat5b by
tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol
Endocrinol 10:1496-506

Nevalainen MT, Xie J, Bubendorf L, Wagner KU, Rui H 2002 Basal
activation of transcription factor Si gnal transducer and activator of transcription

(Stat5) in nonpregnant mouse and human breast epithelium. Mol Endocrinol
1 6: 1 1 08-24

Hynes NE, Cella N, Wartmann M 1997 Prolactin mediated intracellular
signaling in mammary epithelial cells. J Mammary Gland Biol Neoplasia 2: 19-27

Wagner KU, Krempler A, Triplett AA, Qi Y, George NM, Zhu J, Rui H 2004
Impaired alveologenesis and maintenance of secretory mammary epithelial cells
in J ak2 conditional knockout mice. Mol Cell Biol 24:5510-20

Miyoshi K, Shillingford JM, Smith GH, Grimm SL, Wagner KU, Oka T,
Rosen JM, Robinson GW, Hennighausen L 2001 Signal transducer and
activator of transcription (Stat) 5 controls the proliferation and differentiation of
mammary alveolar epithelium. J Cell Biol 155:531-42

Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW,
Hennighausen L 2004 Inactivation of Stat5 in mouse mammary epithelium
during pregnancy reveals distinct functions in cell proliferation, survival, and
differentiation. Mol Cell Biol 24:8037-47

Liu X, Gallego MI, Smith CH, Robinson GW, Hennighausen L 1998

Functional rescue of Stat5a-null mammary tissue through the activation of
compensating signals including Stat5b. Cell Growth Differ 9:795-803

49

150.

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M, Nakashima K,
Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective mammopoiesis,
but normal hematopoiesis, in mice with a targeted disruption of the prolactin
gene. EMBO J 16:6926-35

McKnight RA, Spencer M, Dittmer J, Brady JN, Wall RJ, Hennighausen L
1995 An Ets Site in the whey acidic protein gene promoter mediates
transcriptional activation in the mammary gland of pregnant mice but is
dispensable during lactation. Mol Endocrinol 9:7 17-24

Hennighausen L, Robinson GW 2005 Information networks in the mammary
gland. Nat Rev Mol Cell Biol 6:715-25

Srivastava S, Matsuda M, Hou Z, Bailey JP, Kitazawa R, Herbst MP,
Horseman ND 2003 Receptor activator of NF-kappaB ligand induction via J ak2
and Stat5a in mammary epithelial cells. J Biol Chem 278:46171-8

Joung YH, Lim EJ, Lee MY, Park JH, Ye SK, Park EU, Kim SY, Zhang Z,
Lee K], Park DK, Park T, Moon WK, Yang YM 2005 Hypoxia activates the

cyclin D1 promoter via the J ak2/STAT5b pathway in breast cancer cells. Exp Mol
Med 372353-64

Bowman T, Garcia R, Turkson J, Jove R 2000 STATS in oncogenesis.
Oncogene 19:2474-88

Iavnilovitch E, Cardiff RD, Groner B, Barash I 2004 Deregulation of Stat5
expression and activation causes mammary tumors in transgenic mice. Int J
Cancer 112:607-19

Humphreys RC, Hennighausen L 1999 Signal transducer and activator of
transcription 5a influences mammary epithelial cell survival and tumori genesis.
Cell Growth Differ 10:685-94

Ren S, Cai HR, Li M, Furth PA 2002 Loss of Stat5a delays mammary cancer
progression in a mouse model. Oncogene 21:4335-9

Clevenger CV, Furth PA, Hankinson SE, Schuler LA 2003 The role of
prolactin in mammary carcinoma. Endocr Rev 24: 1-27

Watson CJ 200] Stat transcription factors in mammary gland development and
tumorigenesis. J Mammary Gland Biol Neoplasia 6:115-27

Nevalainen MT, Xie J, Torhorst J, Bubendorf L, Haas P, Kononen J, Sauter

G, Rui H 2004 Signal transducer and activator of transcription-5 activation and
breast cancer prognosis. J Clin Oncol 22:2053-60

50

162. Sultan AS, Xie J, LeBaron MJ, Ealley EL, Nevalainen MT, Rui H 2005 Stat5
promotes homotypic adhesion and inhibits invasive characteristics of human
breast cancer cells. Oncogene 24:746-60

51

CHAPTER TWO

ESTROGEN AND PROGESTERONE ARE CRITICAL REGULATORS OF
STATSA EXPRESSION IN THE MOUSE MAMMARY GLAND

Note: The contents of this chapter have been published in Santos S.J., Haslam S.Z., and
Conrad SE. Estrogen and progesterone are critical regulators of Stat5a expression in the
mouse mammary gland. Endocrinology. (2007) Sept 20. Published online ahead of print.

52

ABSTRACT

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of
mammary gland development. Several pathways for activating Stat5a have been
identified, but little is known about the mechanisms that regulate its expression in this
tissue. In this report, we used immunofluorescent staining to examine Stat5a expression
in mammary epithelial cells during normal development and in response to treatment
with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very
low levels in the pre-pubertal gland, and was highly induced in a subset of luminal
epithelial cells during puberty. The percentage of positive cells increased in adult virgin,
pregnant, and lactating animals, dropped dramatically during involution and then
increased again post-weaning. Ovariectomy ablated Stat5a expression in virgin animals,
and treatment with both E and P was necessary to restore it. Double labeling experiments
in animals treated with E+P for three days demonstrated that Stat5a was localized
exclusively to cells containing both estrogen and progesterone receptors. Together, these
results identify a novel role for E and P in inducing Stat5a expression in the virgin
mammary gland, and suggest that these hormones act at the cellular level through their

cognate receptors.

53

INTRODUCTION

Signal transducer and activator of transcription (Stat)5 plays an important role in
mammary gland development. Two isoforms of Stat5, a and b, are produced by separate
genes (1), and act as signaling mediators involved in numerous cellular functions
including proliferation, differentiation and survival (2-4). They are members of the Stat
family of proteins, which are latent transcription factors that localize to the cytoplasm
until activated by a variety of cytokines, grth factors and hormones (5-7). Binding of
these ligands to their cognate receptors activates either an intrinsic receptor kinase
domain or an associated J ak kinase, which then recruits and phosphorylates Stat proteins
(8, 9). Phosphorylated Stat proteins translocate to the nucleus where they bind DNA and
activate responsive genes.

The majority of studies of Stat5 in the mammary gland have focused on its role in
pregnancy and lactation. When both a and b isoforms were deleted, Stat5-/- mice
exhibited reduced alveolar expansion during pregnancy and a lactational defect (10). A
similar phenotype was seen when only the Stat5a isoform was deleted, but Stat5b
deficient mice had a much less severe mammary gland defect (10-12). This indicates a
specific requirement for Stat5a during mammary gland development, which is consistent
with the fact that it is the predominant isoform expressed in this tissue (10).

In terms of Specific functions, Stat5a has been reported to play important roles in
mammary cell differentiation, proliferation and survival. In the final stages of mammary

cell differentiation, it activates expression of genes encoding milk constituents such as or-

lactalbumin, [3-casein and whey acidic protein (WAP) (10, 13). Further supporting a role

54

for Stat5a in differentiation, recent studies have demonstrated that after parturition,
Stat5-/- epithelial cells lack a specific marker of alveolar cells (Npt2b), while retaining a
marker of virgin-like ductal cells (NKCCI) (14, 15). Stat5a may also play a role in the
proliferative response to estrogen (E) and progesterone (P) during pregnancy, since BrdU
incorporation was significantly decreased in Stat5-/- mouse mammary epithelial cells in
response to E+P treatment (14, 15). Finally, conditionally deleting Stat5 (a and b)
during pregnancy induced premature cell death, indicating that it is critical for cell
survival at this developmental stage (14).

Developmental studies using Northern and Western blotting of whole murine
mammary gland homogenates demonstrated that Stat5a is present in both the immature
and mature virgin, increases during pregnancy, and reaches a maximum level during late
pregnancy and lactation (16, 17). However, expression in the stroma] and epithelial
compartments could not be discriminated using these approaches, and the increase in
Stat5a might therefore reflect increased epithelial cell number rather than increased
expression per epithelial cell. In addition, Western blotting does not permit one to
localize expression to specific epithelial structures such as ducts, end buds and alveoli. A
more recent study used immunohistochemistry to examine total Stat5 expression in
mouse mammary epithelium. It was found that Stat5 was expressed in the adult virgin as
well as the pregnant gland, but immature animals were not examined (18). Surprisingly,
the majority of Stat5 was activated, even in virgin animals. In the mammary gland, Stat5
can be activated by growth hormone, EGF, or prolactin (PRL) (19, 20). In lactating
animals, Stat5a induces expression of milk protein genes, largely in response to PRL, and

Stat5a activation in virgin animals also seems to be predominantly due to PRL (18).

55

Although the pathways leading to Stat5a activation have been extensively studied,
little information is available regarding the regulation of its expression in mammary cells.
Progestin treatment was reported to induce Stat5a transcription in the human breast
cancer cell line, T47D. There are two forms of the progesterone receptor (PR), PRA and
PRB (21), and Stat5a induction occurred only in cells expressing the PRB isoform (22).
However, the factors regulating Stat5a expression in the normal mammary gland have not
been identified. In the current work, we have utilized immunofluorescence to study
Stat5a expression in mouse mammary epithelial cells during normal development and in
response to hormone treatments. The results demonstrate that Stat5a expression increases
dramatically at puberty and after ovariectomy in response to the ovarian hormones E+P.
Furthermore, Stat5a extensively co-localizes with both estrogen and progesterone
receptors (ER and PR), suggesting that it is a target of these receptors and may mediate

some of the effects of E and P in the mammary gland.

MATERIALS AND METHODS

Animal Experiments:

18 week old virgin, female BALB/c mice from our own colony were bilaterally
ovariectomized one week prior to hormone treatment. Animals were injected
subcutaneously in the dorsum of the neck with saline, 17B-estradiol (Sigma, Saint Louis,
MO) (1 pg), progesterone (Sigma) (1 mg), or the combination of both hormones every 24

h for either 3 or 10 consecutive days. Prolactin (Sigma) was injected subcutaneously

56

every 12 h (1 ug/ g body weight per day). For bromocriptine (BC) experiments, 0.5 mg of
BC (Sigma) was suspended in 0.1 ml sesame oil (Sigma) and administered
subcutaneously every 24 h for 3 days. This amount of BC is reported to be adequate for a
sustained suppression of serum prolactin levels for 24 h (23). Animals were injected
intraperitoneally with 70 ug/g body weight of 5-bromo-2-deoxyuridine (BrdU) (Sigma)
two h before they were euthanized. All animal experimentation was conducted in
accordance with accepted standards of humane animal care and approved by the All

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

Antibodies:

Rabbit polyclonal ct-Stat5a (cat# sc-1081), goat polyclonal a-phospho Stat5 (cat#
sc-11761) and rabbit polyclonal (X-WAP (cat# sc-25526) antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal (rt-Stat5a (cat# 611834)
antibody was obtained from BD Biosciences (San Jose, CA). Goat polyclonal a-RANKL
(AF462) antibody was obtained from R&D Systems (Minneapolis, MN). Mouse
monoclonal a-smooth muscle actin antibody (clone 14A) was from Sigma. Mouse
monoclonal a-human ERa antibody (NCL-ER-6F11) was obtained from Novocastra
Laboratories (Newcastle, UK). Rabbit polyclonal (rt-PR, which detects only PRA (cat#
A0098) was obtained from Dako (Carpinteria, CA). Rabbit polyclonal a-PRB was
produced commercially using a peptide corresponding to 15 amino acids unique to mouse
progesterone receptor B by Affinity Bioreagents (Golden, CO) and has been verified to

detect only PRB (24). Mouse monoclonal a-BrdU antibody was provided in a kit from

57

Amersharn Biosciences (Piscataway, NJ). All secondary antibodies were conjugated to

Alexa fluor dyes and were obtained from Invitrogen (Carlsbad, CA).

Immunohistochemistry:

Inguinal mammary glands were fixed in 10% phosphate-buffered formalin
overnight at 4 C, dehydrated, cleared and embedded in paraffin. Tissue was sectioned to
5 pm, mounted on 3-aminopropyl triethoxysilane-coated coverslips, and allowed to dry
for 24 h at room temperature. The tissue was then dewaxed and rehydrated through a
descending gradient of ethanol. Sections were autoclaved (121 C at 15 psi) for 20 min in
0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. Specific blocking and
antibody incubation protocols are detailed below. Each incubation step was followed by
two 5 min washes in phosphate buffered saline (PBS).

For Stat5a labeling, sections were blocked for 30 min in 2% bovine serum
albumin in PBS, pH 7.3 (2% PBSA). Samples were first incubated with rabbit or-StatSa
antibody (in 2% PBSA, overnight at 4 C), then incubated with goat or-rabbit antibody
conjugated to Alexa 488 (green) (in PBS, 30 min). For double labeling experiments,
sections were first blocked with goat (rt-mouse IgG Fab fragments (Jackson
ImmunoResearch Laboratories, West Grove, PA) [1:100 1% PBSA, 1 h], then blocked
with normal goat serum (NGS) (Vector Laboratories, Burlingame, CA ) (1 :1 in PBS, 30
min). The tissue was incubated with either mouse a-BrdU for 1 h, or mouse oc-SMA (in

PBS), a-ERO. (in PBS-0.5% Triton X-100) or or-StatSa antibody (in PBS) overnight at 4

C. This was followed by incubation. with goat a-mouse Alexa 488 for Stat5a-PRA

58

double labeling or Alexa 546 (red) for all other staining (in PBS, 30 min). Samples were
blocked with 2% PBSA for 30 min, incubated with one of the following: rabbit or-Stat5a,
or-WAP, or-PRA or or-PRB antibody in 2% PBSA overnight at 4 C. This was followed
by incubation with goat or-rabbit Alexa 546 for Stat5a-PRA labeling or Alexa 488 for all
other staining (in PBS, 30 min). For Stat5a-RANKL double staining: sections were
incubated with normal rabbit serum (1:1 in PBS, 30 min), a-RANKL antibody (in PBS
0.5% Triton X-100, overnight at 4 C), rabbit or-goat Alexa 488 (30 min), goat a-mouse
IgG Fab (1:100 in 1% PBSA, 1 h), NGS (1 :1 in PBS, 30 min), mouse a-StatSa (overnight
at 4 C), and goat oc-mouse Alexa 546 (30 min). For Stat5a and SMA, WAP, RANKL,
ERa, PRA or BrdU staining, nuclei were stained with 4',6-diamidino-2-phenylindole
(DAPI) (Sigma) and pictures were taken on a Nikon Eclipse fluorescent microscope.
Intensity measurements were made using MetaMorph 6 Software. For Stat5a and PRB
double staining, nuclei were counterstained using TOPRO-3 iodide (Molecular Probes),
and visualized using a Pascal laser scanning confocal microscope (Zeiss). For Stat5a
visualization following BC treatment, nuclei were counterstained using TOPRO-3 iodide
and visualized using a Flroiew laser scanning confocal microscope (Olympus).

To detect phosphorylated Stat5, antigen retrieval was accomplished as previously
described (20). After blocking in 10% normal rabbit serum (NRS) (Vector Laboratories)
in PBS for 30 min, the sections were incubated overnight a 4 C with goat or-phospho-
Stat5 antibody (in 10% NRS in PBS). The tissue was then incubated with rabbit or-goat
Alexa 488 antibody (in PBS, 30 min). Nuclei were stained with DAPI, and samples were

visualized using a Nikon Eclipse microscope and MetaMorph software.

59

RESULTS

Immunofluorescent localization and quantitation of Stat5a in epithelial cells during

mammary gland development

To examine Stat5a expression during mammary gland development, the level and
pattern of expression was examined by immunofluorescence in mammary tissue from
prepubertal (4 wk), pubertal (5 wk), adult (19 wk), pregnant (7 day), lactating (10 day),
and involuting (8 and 28 day) animals (Fig. 2.1A). In 4 wk old animals, a diffuse Signal
that was slightly above the secondary antibody alone control was observed. By 5 weeks
of age, intense staining was seen in a subpopulation of luminal epithelial cells that were
present throughout the ducts in a punctate pattern. An exception to this punctuate pattern
was noted in end buds, where only diffuse light staining was present. Most of the intense
staining in ductal cells was nuclear, suggesting that the protein is activated even at this
early age. In mature (19 wk) virgin animals, where the ducts have reached the limit of
the fat pad and end buds are no longer present, the punctate distribution of positive cells
persisted, and the staining remained predominantly nuclear.

Since Stat5a is required for lobuloalveolar development and lactation, its
expression was also examined in the pregnant, lactating and involuting mammary gland
(Fig. 2.1A). Intense nuclear Stat5a staining was observed in both ductal and alveolar
cells in the pregnant mammary gland, and an increase in cytoplasmic staining was also
observed in the Stat5a+ alveolar cells. In sections from lactating animals, the vast

majority of cells exhibited both intense nuclear and cytoplasmic Stat5a staining.

60

Figure 2.1. Immunofluorescent staining of Stat5a during mouse mammary gland
development. A. Sections from 4 wk old pre-pubertal, 5 wk old pubertal, 19 wk old
mature adult, 7 day pregnant, 10 day lactating, and 8 and 28 day involuting mammary
glands were stained with a-StatSa antibody (green) and nuclei were labeled with DAPI
(blue) as described in Materials and Methods. Control sections fiom 4 wk old glands
without primary antibody are also shown. Green arrowheads indicate Stat5a positive
cells and white arrowheads indicate Stat5a negative cells. B. Mammary glands from
mature (19 wk old) mice were stained for smooth muscle actin (red) and StatSa (green).
Scale bar for all images = 5m. C. Quantitation of nuclear Stat5a expression at different
stages of mouse mammary gland development. Immunofluorescent staining using an or-
Stat5a antibody was carried out as described in (A) on tissue sections fiom 4-, 5-, or 19-
week old virgin, 7 day pregnant (P), 10 day lactating (L), 8 or 28 day involuting (1) mice.
The values represent the mean i SEM from three animals per group, with a minimum of
1000 cells/mouse analyzed, and significance was determined by Student’s t test. No
nuclear staining was detected (ND) at 4 weeks of age or 8 days of involution. Images in
this dissertation are presented in color.

61

A pre-pubertal duct pre-pubertal duct pre-pubertal duct
+ primary antibody + primary antibody - primary antibody
- DAPI + DAPI - DAPI pubertal duct

.-

adult virgin duct pregnant alveoli

         
 

   

pubertal end bud

end bud (high mag.)

 

lactating 8 day' rm oluting 28 day involuting adult virgin duct

 

C 100 p < 0.005

% Stat5a+ nuclei

 

4wk 5wk19wk 7dP 7dP 10dL 8d128d1
duct alveoli

62

Expression was dramatically decreased in the 8 day post-lactational involuting gland,
where staining was less intense than in the virgin gland and resembled that seen in the
pre-pubertal gland. However, Stat5a staining was restored by 28 days after weaning, and
approached the level seen in the adult virgin gland. Double staining for Stat5a and the
myoepithelial cell marker, smooth muscle actin, demonstrated that Stat5a expression was
limited to luminal epithelial cells (Fig. 2.1B).

Quantitation of the results shown in figure 2.1A revealed that 37% of luminal
epithelial cells had intense nuclear Stat5a staining in 5 wk old animals, and this increased
to 46% in 19 wk old mice (Fig. 2.1C). Stat5a was expressed at similar levels in ducts in
the pregnant gland, where 45% of luminal epithelial cells were positive. Expression was
higher in the alveoli, where 67% of cells were positive. Expression was highest during
lactation, with 87% of cells staining positive. Stat5a staining was non-detectable at day 8

of involution, but was present 28 days after weaning, where 34% of cells were positive.

Regulation of Stat5a expression by ovarian hormones

The appearance of Stat5a between 4 and 5 weeks of age, when ovarian cycles
typically begin, and the further increase during pregnancy, suggested that its expression
might be regulated by the ovarian hormones E and P. To test this possibility, 18 week
old mice were ovariectomized and allowed to recover for 1 week. They were then
injected with E, P, E+P, or vehicle control once per day for 3 days, and their mammary
glands were examined for Stat5a expression. After ovariectomy, control-treated animals

had weak Stat5a staining (Fig. 2.2A), similar to that seen in the 4 wk old, intact pre-

63

Figure 2.2. Stat5a expression in hormone treated mice. A. 18 wk old virgin mice
were ovariectomized, then treated with vehicle control, E, P, PRL, E+P or PRL+P for 3
days as described in Materials and Methods. Representative photomicrographs of Stat5a
staining are shown. B. 3 day E+P treated mammary glands were stained with an or-
pY694 Stat5 antibody. All images Show Stat5a staining (green) in the left panel and the
merged image with DAPI- stained nuclei in the right panel. Scale bar for all images = 20
mm. C. Quantitation of Stat5a staining intensity in E+P treated mice. The total pixel
intensity of every cell (nucleus + cytoplasm) was measured from a single representative
structure from the 3 day control (white bars) and E+P treated (black bars) mice (each bar
= 1 cell). Images in this dissertation are presented in color.

64

Control

Estrogen + Progesterone

1.:

Prolactin

Pixel Intensity

Control

 

65

pubertal gland (see Fig. 2.1A). Ovariectomized animals treated with P alone were
indistinguishable from the control-treated mice. Estrogen treated animals had a slight
increase in nuclear Stat5a staining in a few cells (Fig. 2.2A). In contrast, robust Stat5a
expression was observed in both the nucleus and cytoplasm in E+P treated mice, with
41i1% of luminal epithelial cells being positive (Fig. 2.2A). Overall, both the
percentage and distribution of StatSa+ cells in the 3 day E+P treated animals were similar
to that observed in the mammary glands of intact, mature mice (see Fig. 2.1A), indicating
that expression of Stat5a in the mammary gland is largely dependent on ovarian
hormones.

Nevalainen et a]. previously used phospho-specific antibodies to demonstrate that
Stat5 is activated in adult virgin mouse mammary epithelial cells (18), and the nuclear
localization of Stat5a in the glands from E+P treated animals (Fig. 2.2A) suggested that
Stat5a is also phosphorylated and activated under these conditions. To determine if this
was the case, immunofluorescent staining was carried out with an antibody that detects
both a and b Stat5 isoforms phosphorylated on tyrosine 694 (pY694) (Fig. 2.23). When
scored, 47i7% of luminal epithelial cells from E+P treated animals were positive for
pY694, similar to the percentage of cells showing intense nuclear Stat5a staining. Since
the Stat5a observed after E+P treatment was predominantly nuclear, we considered the
possibility that some or all of the apparent increase in expression was actually due to
translocation of the protein into the nucleus. To address this issue, the staining intensities
of individual cells (cytoplasm + nucleus) in representative structures from the 3 day
control and E+P treated mice were quantitated and compared. Cells with a low level of

staining in the E+P gland had similar intensities to the cells in the control gland (Fig.

66

2.2A and C). However, cells with bright nuclear staining in the E+P gland had
approximately twice the intensity of the other cells (Fig. 2.2A and C). This increase in
intensity demonstrates that total cellular Stat5a protein levels are increased in a subset of
luminal epithelial cells in response to E+P treatment.

PRL treatment increased the amount of activated Stat5 in hypophysectomized
animals (18), and E can induce PRL secretion (25, 26). One potential role of E in these
experiments could therefore be to induce PRL, which would then lead to Stat5a nuclear
accumulation. To examine the possibility that PRL might increase expression as well as
activation of Stat5a, ovariectomized virgin animals were treated for 3 days with PRL or
PRL+P. Neither treatment increased StatSa expression significantly compared to the
control (Fig. 2.2A), indicating that E is not increasing Stat5a expression via PRL.
However, B may contribute to Stat5a activation and nuclear localization via PRL. The
results shown in Figure 2.2 demonstrated that E+P treatment increased Stat5a expression
in the mammary epithelium, and that the protein was phosphorylated and nuclear. Since
PRL is the major activator of Stat5a in the adult virgin gland (18), this suggested that
E+P treatment in the absence of PRL would lead to increased protein without nuclear
localization. To test this hypothesis, bromocriptine (BC) was utilized to inhibit the
secretion of PRL from the pituitary gland. Ovariectomized mice were treated with E+P
for 3 days as described above, and were additionally given BC or vehicle control.
Mammary glands were examined by confocal microscopy to assess the cellular
localization of Stat5a. As expected, strong nuclear Stat5a staining was observed in the
tissue from E+P+vehicle treated mice (Fig. 2.3). In contrast, mammary epithelial cells

from E+P+BC treated mice had predominantly cytoplasmic Stat5a staining (Fig. 2.3).

67

StatSa nuclei merge

U
CD
+
Q:
+
[L]

 

Figure 2.3. Cellular localization of Stat5a in bromocriptine treated mice. 18 wk old
virgin mice were ovariectomized, and treated with E+P + vehicle control (oil) or E+P +
bromocriptine (BC) for 3 days. Staining for Stat5a (green) and nuclei (red) was
visualized with an Olympus laser scanning confocal microscope. White arrowheads
indicate nuclear Stat5a staining and red arrowheads indicate cytoplasmic Stat5a staining.
Scale bar = 20 pm. Images in this dissertation are presented in color.

68

Based on these results, we propose a model where E+P induces the expression of Stat5a,

which can subsequently be activated by PRL, leading to its nuclear localization.
Colocalization of Stat5a with estrogen and progesterone receptors

The induction of Stat5a expression by E+P suggested that the gene might be a
target of the ER and/or PR. Previous studies in T47D human breast cancer cells have
indicated that transcription of the Stat5a gene is induced by progestin in cells expressing
PRB, but not PRA (22). However, PRA is the dominant isoform observed in virgin mice,
where it is present in ductal epithelial cells in a punctate pattern that is reminiscent of that
seen with Stat5a (27). In contrast, PRB is not detectable in adult virgin mice, increases
during pregnancy, and is predominately localized in alveoli. To determine if Stat5a
colocalizes with PRA or PRB in the mouse mammary gland, samples from the 3 and 10
day E+P treated animals were examined by double indirect immunofluorescent staining
using Stat5a and PR-isoform specific antibodies. After 3 days of E+P treatment, only
ducts were scored since no alveoli were present. In these ductal structures, 42% of
luminal cells were Stat5a+, 46% were PRA+ and 42% were both Stat5a and PRA positive
(Fig. 2.4). Thus, after 3 days of E+P treatment, virtually all cells expressing Stat5a
contained PRA, but there was a small subset (~9%) of PRA+ cells that did not express
Stat5a.

We previously observed that PRB is absent from ducts and is only detectable in
alveoli during pregnancy (27) or after 5 or more days of E+P treatment (28). To

determine if Stat5a colocalizes with PRB, we examined Stat5a/PR colocalization in

69

Figure 2.4. Colocalization of Stat5a with PRA or PRB. A. Staining for Stat5a and
PRA was carried out on sections from 3 and 10 day E+P treated ovariectomized mice,
and was visualized with a Nikon Eclipse fluorescent microscope. Staining for Stat5a and
PRB was carried out on sections from 10 day E+P treated mice only, and was visualized
with a Pascal laser scanning confocal microscope. Yellow arrowheads indicate Stat5a
and PRA or Stat5a and PRB colocalization (yellow/orange nuclei). White arrowhead
indicates Stat5a negative, PRA positive cell (red nucleus). Green arrowheads indicate
Stat5a positive, PRA or PRB negative cells (green nuclei). Scale bar = 20 pm. B.
Quantitation of Stat5a and PR colocalization in 3 and 10 day E+P treated animals. The
values represent the mean 1- SEM from three animals per group, with a minimum of 800
cells/mouse analyzed. The percentage of PRA positive cells and Stat5a/PRA positive
cells were both significantly lower in 10 day E+P alveolar structures than 3 day E+P
ducts (p < 0.001) as determined by Student’s t test. Images in this dissertation are
presented in color.

70

A 3 d Duct 10 d Alveoli 10 d Alveoli

      

p<0.001

0‘

— —
a

mi
p < 0.001

    

w I
% Positive Cells

 

 

0
‘3‘” 65"
£3 <33. a. 941? ‘33. $3" 879 039
X X X
‘3‘” ‘3‘!" 58
N N
é" a” e"
l___l ; l
3 day ducts 10 day alveoli

71

alveolar structures after 10 days of E+P treatment. As expected, PRB was not detected in
ducts (data not shown), but was present in 31% of alveolar cells (Fig. 2.4). PRA was
present in 18% of alveolar cells, compared to 47% of ductal cells from 3 day treated
animals. When StatSa expression was quantitated, 43% of alveolar cells were positive,
which is similar to the percentage seen in the 3 day treated samples. Scoring for both
Stat5a and PRA or PRB revealed that 35% of Stat5a expressing cells were PRAI, and
65% were PRB+ (Fig. 2.4). Since PRA and PRB are rarely expressed in the same cells in
the mouse mammary gland (27), we infer from these data that virtually all Stat5a+
alveolar cells are also PRI, with roughly one third containing PRA and the other two
thirds containing PRB.

Since both E and P were required to induce Stat5a expression in ovariectomized
animals, we examined whether ERor and Stat5a colocalize in ductal epithelial cells from
3 day E+P treated mammary glands. Double staining revealed that the two proteins were
highly colocalized, with 43% of cells being Stat5a+, 45% ERor+, and 42% both Stat5a and
ERor positive (Fig. 2.5). Thus, virtually all Stat5a+ cells in ducts contain EROt. Since
PRA also colocalizes with Stat5a in ducts, this suggests that a sub-population of ductal

cells contain ERor, PRA and Stat5a.

Colocalization of Stat5a and BrdU

Mouse mammary glands lacking both Stat5a and b (Stat5-/—) display a significant
reduction in epithelial cell proliferation in response to E+P treatment compared to wild-

type glands, suggesting that Stat5 is a positive regulator of proliferation in response to

72

CD

% Positive Cells

Stat5a+ ERa+ Stat5a+
ERor+

 

Figure 2.5. Colocalization of Stat5a with ERa. A. Staining for Stat5a and ERa was
carried out on sections from 3 day E+P treated ovariectomized mice. White arrowheads
indicate Stat5a and ERor positive cells. Scale bar = 20 um. B. Quantitation of Stat5a
and ERor colocalization in 3 day E+P treated animals. The values represent the mean 1
SEM from three animals per group, with a minimum of 800 cells/mouse analyzed.
Images in this dissertation are presented in color.

73

these hormones (14, 15). Since Stat5a is the predominant isoform in the mammary gland
and Stat5a-/- mice display a more pronounced mammary gland developmental defect
than Stat5b-/— mice, the effect on proliferation is likely due to the lack of Stat5a. To
determine if Stat5a+ cells proliferate in response to E+P, 3 and 10 day E+P treated
animals were injected with BrdU 2 hours before they were euthanized, and tissue sections
were double-labeled for BrdU and Stat5a. After 3 days of treatment, 38% of ductal
epithelial cells were Stat5a+, 21% were BrdU+, and 3% were both Stat5a and BrdU
positive (Fig. 2.6). Thus, only 9% of the cells expressing Stat5a were BrdU. A Similar
result was observed in the 10 day E+P treated mice, where 6% of ductal cells and 8% of
alveolar cells were BrdU, but only 2% and 4% of the Stat5a expressing cells were
BrdU+, respectively (Fig. 2.6). When BrdU incorporation was compared between the
three populations of cells (total, Stat5a+ and Stat5a), StatSa' cells were 3 to 6 times more
likely to incorporate BrdU than Stat5a+ cells (p < 0.05) after either 3 or 10 day E+P
treatments. Thus, although some Stat5a+ cells incorporate BrdU in response to hormone

treatment, they are underrepresented in the pool of proliferating cells.
Colocalization of Stat5a and WAP or RANKL

The observation that Stat5a is expressed at significant levels in the virgin gland
prompted the investigation of potential target genes at this developmental stage. Stat5a is
a well-established differentiation factor that induces the expression of milk protein genes
including WAP in the lactating mammary gland. Irnmunohistochemistry was used to

investigate whether this gene was also induced in Stat5a expressing cells in E+P treated

74

Figure 2.6. Immunodetection of Stat5a and BrdU in mammary glands of hormone
treated mice. Ovariectomized 19 wk old mice were treated with E+P for 3 or 10 days,
and animals were injected with BrdU 2 hours before being euthanized as described in
Materials and Methods. A. Representative structures from mammary glands showing
Stat5a (green), BrdU (red), DAPI stained nuclei (blue), and merged images. Green
arrowheads indicate Stat5a positive, BrdU negative cells. Red arrowheads indicate BrdU
positive, Stat5a negative cells. White arrowheads indicate BrdU positive, Stat5a positive
cells. Scale bar = 25 um. B. Quantitation of Stat5a and BrdU colocalization in 3 and 10
day E+P treated animals. The values represent the mean i SEM from three animals per
group, with a minimum of 800 cells/mouse analyzed. Images in this dissertation are
presented in color.

75

A 3 day duct 10 day duct

4 <
I ”a...‘ I y 7‘. .

   

- B 3 d 10 da
10 day alveoli |_L‘a |__—Xfi
50— d d ‘ ‘ '

 

 

 

ucts ucts alveoli
40—
fl
3
o 30-
.Z
a
_Sat5a B_dU a? 20“
°\ 10—
0-500 S etc o «to o
S b ‘ b b " b b
0963‘ 694°93‘Xsi a'i’e‘xsv‘
‘3‘» (3‘0 ‘D
9‘9 9‘99 9'58)

76

mice. In agreement with previous studies, WAP staining was present on the luminal
surface of alveoli when lactating mammary glands were examined (Fig. 2.7A), and a
large percentage of the alveolar cells were StatSa positive. AS expected, both WAP and
Stat5a were absent from mammary glands in control ovariectomized mice. Despite the
induction of nuclear Stat5a in mammary glands from 3 day E+P treated mice, no WAP
staining was detected. Thus, the presence of Stat5a is not sufficient to induce the
expression of the milk protein, WAP, in the virgin gland.

In addition to its role in differentiation, Stat5a may also be a proliferation factor in
the mammary gland. Since Stat5a+ cells are less likely to proliferate than their Stat5a'
neighbors (Fig. 2.6), it is likely that any proliferative activity of Stat5a is accomplished
via a paracrine mechanism. One candidate paracrine factor is the TNF family member,
receptor activator of NF-KB ligand (RANKL). RANKL is a secreted protein that is
essential for normal mammary gland development (29). PRL induces RANKL
expression in primary mammary epithelial cells, and experiments in heterologous cell
culture models suggest that this is through the J ak2/Stat5a pathway (30). To investigate
whether RANKL might be a Stat5a target in vivo, mammary gland sections from 3 day
control or E+P treated mice were stained for both Stat5a and RANKL. Mammary cells
from control mice lacked both Stat5a and RANKL staining (Fig. 2.7B), and both were
induced after 3 days of E+P treatment. In addition, there was a high level of
colocalization of the two proteins (Fig. 2.7B), supporting the hypothesis that they are

functionally linked.

77

Control E+P Lactatin

     

 

Figure 2.7. Immunodetection of Stat5a and WAP or Stat5a and RANKL. Tissue
sections from 3 day E+P and control treated ovariectomized mice were stained for Stat5a
and either WAP or RANKL. 10 day lactating mice were included as a control for WAP.
A. Representative structures showing merged images of Stat5a (red), WAP (green), and
DAPI stained nuclei (blue). B. Representative structures of merged images of StatSa
(red), RANKL (green), and DAPI stained nuclei (blue). Scale bar = 25 um. Images in
this dissertation are presented in color.

78

DISCUSSION

Despite the importance of Stat5a in mammary gland biology, very little is known
about the mechanisms that regulate its expression in this tissue. To address this issue, we
studied the pattern of Stat5a expression in mammary epithelial cells during normal
development and in response to treatment with the ovarian hormones E and P.
Immunofluorescent staining revealed that Stat5a was expressed at extremely low levels in
the pre-pubertal gland. Expression increased at puberty, with 37% of luminal cells in
ducts exhibiting bright nuclear staining. The percentage of Stat5a positive cells increased
significantly in adult virgin ducts, and increased further in alveolar cells during
pregnancy. It was at its highest level during lactation, where more than 80% of luminal
epithelial cells had both nuclear and cytoplasmic staining, decreased dramatically during
involution, and was then restored to near virgin levels. The increase in Stat5a expression
at puberty suggested that ovarian steroid hormones induce its expression. This was
confirmed by the finding that Stat5a levels dropped dramatically following ovariectomy,
and treatment with E+P was necessary to restore it. While pregnancy levels of hormones
were used in these studies, it is likely that lower concentrations are sufficient to support
Stat5a expression since it was present at high levels in the adult virgin gland. Activation
and expression of Stat5 are not affected by variations in circulating hormone levels
during the estrous cycle in mice (18), suggesting that relatively low levels of E and P are
required for the amount of Stat5a observed in the virgin gland.

To investigate the mechanism by which E and P induce Stat5a, we examined the

pattern of ERa, PR and Stat5a expression by immunohistochemistry. After 3 or 10 days

79

of E+P treatment, virtually all Stat5a+ cells also contained either PRA or PRB, which is
consistent with a model in which PR directly or indirectly activates the Stat5a gene.
Since the two PR isoforms are rarely coexpressed in the same cell in the mouse
mammary gland (27), these findings further suggest that either isoform can induce Stat5a
expression in this system. This differs from the situation in human T47D breast cancer
cells, where progestin induced Stat5a mRNA in cells expressing PRB, but not those
expressing PRA (22). One potential explanation for this difference is that we have
examined Stat5a protein instead of mRNA. Our experiments do not therefore establish if
the observed increase in Stat5a levels after E+P treatment is due to increased gene
transcription, mRNA stabilization, mRNA translation, protein stabilization, or a
combination of the above.

The fact that combined E+P treatment was required to induce Stat5a in
ovariectomized animals indicated an important role for E in regulating its expression.
Since PR levels decrease significantly following ovariectomy (28, 31), one role for E
could be to induce PR, which would then activate the Stat5a gene. EROL is present in the
almost all Stat5a”7PRA+ cells after 3 days of E+P treatment (Fig. 2.4), which is consistent
with this hypothesis. An alternative possibility is that E and P, acting through their
respective receptors, are independently required to induce Stat5a in PRA+ cells. The
situation in PRB+ cells is more complex, since the majorityof PRBI cells in the
mammary gland do not contain ERor (28). However, E is required for high levels of PRB
expression in 10 day E+P treated animals (28), and may therefore contribute to Stat5a

expression in these cells by indirectly regulating PRB levels.

80

We also considered a model in which E acts indirectly by increasing systemic
PRL. It is well established that PRL activates Stat5 via the PRLR/Jak pathway (8, 32,
33), and E induces PRL secretion from the pituitary gland (25, 26). To investigate if this
pathway also induces Stat5a expression, we examined glands from PRL and PRL+P
treated animals. Neither treatment reproduced the increase in Stat5a expression seen with
E+P. However, when PRL secretion was suppressed by bromocriptine in E+P treated
mice, StatSa was still induced, but was localized predominantly in the cytoplasm. This
leads us to propose a model in which E+P, acting through their receptors, induce Stat5a
expression in luminal epithelial cells in the virgin mammary gland. The protein is then
activated via the PRL/PRLR/Jak pathway, leading to its phosphorylation and nuclear
accumulation. This model is consistent with the results of Nevalainen et al., who
demonstrated that treatment with PRL 24 hours after hypophysectomy increased the level
of phosphorylated Stat5 in mammary epithelial cells (18), but did not affect the level of
total cellular Stat5. It is likely that ovarian function was maintained for 24 h after
hypophysectomy, and that Stat5a was present but inactive. It is interesting to note that
alternative mechanisms are likely to be responsible for the high levels of Stat5a
expression during lactation, since E, P and PR all decrease dramatically at parturition
(34-36).

The importance of Stat5a during pregnancy and lactation is well established (10,
11, 14, 15), and the presence of activated Stat5 in the adult virgin gland suggested that it
also has a fimction prior to pregnancy (18). The current observation that Stat5a appears in
epithelial cells at puberty, when the mammary gland is undergoing ductal development,

suggests that it regulates growth or differentiation in the virgin gland. The absence of

81

Stat5a in terminal end bud cells argues against a role in ductal elongation, and suggests a
possible role in differentiation or branching. The phenotype of Stat5a deficient mice
should help to provide insight into its function. Although no overt mammary phenotype
was initially noted in Stat5a-/- virgin mice (10, 11), one report using mammary
transplants indicated that Stat5a-/- epithelium had maintained terminal end buds and
reduced secondary branching at 8 weeks compared to wild-type epithelium (37). The
reduction in branching suggests that Stat5a is required for the differentiation of cells that
are programmed to become alveolar cells during pregnancy. Such a lack of alveolar
progenitor cells could then account for the stunted alveolar development seen in pregnant
Stat5a-/- mice. The alveolar-like structures that do exist in Stat5-/- mice do not express
the normal alveolar differentiation marker Npt2b, and retain a marker normally seen only
in ducts, NKCCl (14, 15), indicating that even those alveolar structures that do arise are
not fully differentiated. In addition to its potential role in differentiation in the virgin
gland, Stat5 has been reported to positively regulate proliferation in the pregnant or E+P
treated mammary gland (14, 15). The present results demonstrate that the majority of
cells proliferating in response to E+P do not express Stat5a, indicating that this isoform
could only stimulate proliferation through a paracrine mechanism.

In order to fully understand the role of Stat5a in the virgin mammary gland, it will
be important to identify its downstream targets. The WAP gene is induced by Stat5a
during lactation, but the protein not expressed at detectable levels in Stat5a+ cells in the
virgin gland. Thus, it is likely that novel Stat5a targets remain to be identified. One
potential candidate is the secreted cytokine RANKL. RANKL expression in mouse

mammary cells in vivo requires E, P, and PRL (Fig. 2.7 and (30)), and in vitro

82

experiments indicate that PRL is acting through Stat5a (30). Furthermore, Stat5a-l- and
RANKL-/- mice exhibit similar mammary gland phenotypes (10, 11, 29), suggesting that
the two genes may act in the same pathway. RANKL promotes cell cycle progression by
binding to its receptor, RANK, and initiating a signaling cascade leading to the activation
of the cyclin D1 gene (3 8). Our results demonstrate that RANKL colocalizes with Stat5a
in the mammary epithelium (Fig. 2.7), which is consistent with the hypothesis that Stat5a
induces RANKL, which then stimulates proliferation in adjacent, StatSa' cells. However,
it is also possible that Stat5a and RANKL are independently induced by E+P. A second
candidate Stat5a target in the virgin gland is Wnt-4. Wnt-4 is required for ductal Side
branching, and has been implicated as a mediator of the paracrine effects of P in
mammary gland development (39). Since Stat5a co-localizes with PR and is induced by
P, it could be an intermediate in a PR/StatSa/Wnt-4 pathway.

In summary, the experiments reported here identify a novel role for E and P as
critical regulators of Stat5a expression in the pubertal and adult virgin mammary gland,
and indicate that this regulation likely occurs at the cellular level through epithelial ERor
and PR. The newly synthesized Stat5a is then phosphorylated via PRL signaling, leading
to accumulation of the active transcription factor in the nucleus where it can regulate
genes involved in differentiation and/or proliferation. Our results further indicate that any
proliferative effects of Stat5a are through a paracrine mechanism, and suggest that
RANKL may mediate these or other effects. A detailed analysis of the developmental
phenotypes of Stat5a-/- mice, and identification of its target genes, will be required to

fully elucidate its functions in the virgin mammary gland.

83

ACKNOWLEDGEMENT

The authors thank Mark Aupperlee for his help providing materials and assistance
and Brenda Marrero for helping with the RANKL staining. This publication was made
possible by the Breast Cancer and the Environment Research Centers grant number 1-
U0] ES/CA 012800 01 from the National Institute of Environmental Health Sciences
(NIEHS), and the National Cancer Institute (NCI), National Institutes of Health (NIH),
Department of Health and Human Services (DHHS). Its contents are solely the
responsibility of the authors and do not necessarily represent the official views of the

NIEHS, NCI, or NIH.

84

10.

ll.

12.

REFERENCES

Lin X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning
and expression of Stat5 and an additional homologue (Stat5b) involved in
prolactin Signal transduction in mouse mammary tissue. Proc Natl Acad Sci U S
A 92:8831-5

Leonard WJ, O'Shea JJ 1998 Jaks and STATS; biological implications. Annu
Rev Irnmunol 162293-322

Akira S 1999 Functional roles of STAT family proteins: lessons from knockout
mice. Stem Cells 17:138-46

Buitenhuis M, Coffer PJ, Koenderman L 2004 Signal transducer and activator
of transcription 5 (STATS). Int J Biochem Cell Biol 36:2120-4

Darnell JE, Jr. 1997 STATS and gene regulation. Science 277:]630-5

Darnell JE, Jr., Kerr IM, Stark GR 1994 J ak-STAT pathways and
transcriptional activation in response to IFNS and other extracellular signaling
proteins. Science 264: 1415-21

Ihle JN 1996 STATS: signal transducers and activators of transcription. Cell
84:33 1-4

Ihle JN, Kerr IM 1995 J aks and Stats in signaling by the cytokine receptor
superfamily. Trends Genet 11:69-74

Schindler C, Darnell JE, Jr. 1995 Transcriptional responses to polypeptide
ligands: the J AK-STAT pathway. Annu Rev Biochem 642621-51

Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D,
Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b proteins have

essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841-
50

Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A,
Hennighausen L 1997 Stat5a is mandatory for adult mammary gland
development and lactogenesis. Genes Dev 11:179-86

Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ,

Davey HW 1997 Requirement of STATSb for sexual dimorphism of body growth
rates and liver gene expression. Proc Natl Acad Sci U S A 94:7239-44

85

13.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

Rosen JM, Wyszomierski SL, Hadsell D 1999 Regulation of milk protein gene
expression. Annu Rev Nntr 192407-36

Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW,
Hennighausen L 2004 Inactivation of Stat5 in mouse mammary epithelium
during pregnancy reveals distinct functions in cell proliferation, survival, and
differentiation. Mol Cell Biol 24:8037-47

Miyoshi K, Shillingford JM, Smith GH, Grimm SL, Wagner KU, Oka T,
Rosen JM, Robinson GW, Hennighausen L 2001 Signal transducer and
activator of transcription (Stat) 5 controls the proliferation and differentiation of
mammary alveolar epithelium. J Cell Biol 155:531-42

Kazansky AV, Raught B, Lindsey SM, Wang YF, Rosen JM 1995 Regulation
of mammary gland factor/Stat5a during mammary gland development. Mol
Endocrinol 9: 1598-609

Liu X, Robinson GW, Hennighausen L 1996 Activation of Stat5a and Stat5b by
tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol
Endocrinol 10:1496-506

Nevalainen MT, Xie J, Bubendorf L, Wagner KU, Rui H 2002 Basal
activation of transcription factor signal transducer and activator of transcription

(Stat5) in nonpregnant mouse and human breast epithelium. Mol Endocrinol
1 6: 1 1 08-24

Gallego MI, Binart N, Robinson GW, Okagaki R, Coschigano KT, Perry J,
Kopchick JJ, Oka T, Kelly PA, Hennighausen L 2001 Prolactin, growth
hormone, and epidermal growth factor activate Stat5 in different compartments of

mammary tissue and exert different and overlapping developmental effects. Dev
Biol 229:163-75

Jones FE, Welte T, Fu XY, Stern DF 1999 ErbB4 signaling in the mammary
gland is required for lobuloalveolar development and Stat5 activation during
lactation. J Cell Biol 147:77-88

Lydon JP, Sivaraman L, Conneely OM 2000 A reappraisal of progesterone
action in the mammary gland. J Mammary Gland Biol Neoplasia 5:325-38

Richer JK, Jacobsen BM, Manning NG, Abel MG, Wolf DM, Horwitz KB
2002 Differential gene regulation by the two progesterone receptor isoforms in
human breast cancer cells. J Biol Chem 277:5209-18

McMurray R, Keisler D, Kanuckel K, Izni S, Walker SE 1991 Prolactin

influences autoimmune disease activity in the female B/W mouse. J Immunol
147:3780-7

86

24.

25.

26.

27.

28.

29.

30.

3].

32.

33.

34.

35.

Kariagina A, Aupperlee M, Haslam S 2007 Progesterone Receptor Isofonns
And Proliferation In The Rat Mammary Gland During Development.
Endocrinology

Ajika K, Krnlich L, Fawcett CP, McCann SM 1972 Effects of estrogen on
plasma and pituitary gonadotrOpins and prolactin, and on hypothalamic releasing
and inhibiting factors. Neuroendocrinology 9:304-15

Chen CL, Meites J 1970 Effects of estrogen and progesterone on serum and
pituitary prolactin levels in ovariectomized rats. Endocrinology 86:503-5

Aupperlee MD, Smith KT, Kariagina A, Haslam SZ 2005 Progesterone
receptor isoforms A and B: temporal and spatial differences in expression during
murine mammary gland development. Endocrinology 146:3577-88

Aupperlee MD, Haslam SZ 2007 Differential hormonal regulation and function
of PR isoforms in normal adult mouse mammary gland. Endocrinology

Fata JE, Kong YY, Li J, Sasaki T, Irie—Sasaki J, Moorehead RA, Elliott R,
Scully S, Voura EB, Lacey DL, Boyle WJ, Khokha R, Penninger JM 2000
The osteoclast differentiation factor osteoprotegerin-ligand is essential for
mammary gland development. Cell 103241-50

Srivastava S, Matsuda M, Hon Z, Bailey JP, Kitazawa R, Herbst MP,
Horseman ND 2003 Receptor activator of NF-kappaB ligand induction via J ak2
and Stat5a in mammary epithelial cells. J Biol Chem 278:46171-8

Shyamala G, Barcellos-Hoff MH, Toft D, Yang X 1997 In Situ localization of
progesterone receptors in normal mouse mammary glands: absence of receptors in

the connective and adipose stroma and a heterogeneous distribution in the
epithelium. J Steroid Biochem Mol Biol 63:251-9

Ihle JN, Stravapodis D, Parganas E, Thierfelder W, Feng J, Wang D,
Teglund S 1998 The roles of J aks and Stats in cytokine signaling. Cancer J Sci
Am 4 Suppl 1:S84-91

Groner B, Gouilleux F 1995 Prolactin-mediated gene activation in mammary
epithelial cells. Curr Opin Genet Dev 5:587-94

Mizognchi Y, Yamaguchi H, Aoki F, Enami J, Sakai S 1997 Corticosterone is
required for the prolactin receptor gene expression in the late pregnant mouse
mammary gland. Mol Cell Endocrinol 132:177-83

Tucker HA 1979 Endocrinology of lactation. Semin Perinatol 32199-223

87

36.

37.

38.

39.

Haslam SZ, Shyamala G 1979 Progesterone receptors in normal mammary
glands of mice: characterization and relationship to development. Endocrinology
1 05 :786-95

Liu X, Gallego MI, Smith CH, Robinson GW, Hennighausen L 1998
Functional rescue of Stat5a-null mammary tissue through the activation of
compensating signals including Stat5b. Cell Growth Differ 9:795-803

Kim NS, Kim HJ, Koo BK, Kwon MC, Kim YW, Cho Y, Yokota Y,
Penninger JM, Kong YY 2006 Receptor activator of NF-kappaB ligand
regulates the proliferation of mammary epithelial cells via Id2. Mol Cell Biol

26: 1002-13

Brisken C, Heineman A, Chavarria T, Elenbaas B, Tan J, Dey SK,
McMahon JA, McMahon AP, Weinberg RA 2000 Essential function of Wnt-4

in mammary gland development downstream of progesterone signaling. Genes
Dev 14:650-4

88

CHAPTER 3

EXAMINING THE FUNCTION OF STATSA IN THE VIRGIN MAMMARY
GLAND AND ITS ROLE IN E+P INDUCED PROLIFERATION

89

ABSTRACT

Signal transducer and activator of transcription (Stat)5a is a critical regulator of
mammary gland deveIOpment. The majority of Stat5a studies has focused on its roles in
the pregnant and lactating mammary gland. However, the expression of active Stat5a in
mammary epithelial cells was demonstrated as early as 5 wks of age, suggesting a
function in early mammary gland development. To date, very little is known about
StatSa’s role in the mammary gland of nulliparous mice. In the present study, early
mammary gland development was examined using Stat5a knockout mice. Virgin Stat5a-
/- mice exhibited defective primary branching and side-branching, providing evidence
that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed a
defective proliferative response to E+P treatment. The pathway by which Stat5a induces
proliferation was subsequently examined. It was previously demonstrated that Stat5a
positive cells rarely proliferate, suggesting that it induces proliferation through a
paracrine mechanism. One potential mediator, receptor activator of NF-KB ligand
(RANKL), was examined. RANKL is a secreted factor that can induce mammary
epithelial cell proliferation. StatSa-/- mammary glands were defective in upregulating the
expression of RANKL in response to E+P treatment. In addition, the nuclear localization
of a critical RANKL target, Id2, was diminished in Stat5a-/- mammary cells. Thus, the
activation of the RANKL signaling pathway is one mechanism by which Stat5a can

induce proliferation.

90

INTRODUCTION

The mouse mammary gland undergoes the majority of its development after birth.
This postnatal program of mammogenesis occurs in two distinct stages. The first is
initiated by the surge of ovarian hormones at puberty and results in a ductal tree
extending to the edges of the mammary fat pad. The second major stage of mammary
development is seen during pregnancy, when massive proliferation of the epithelial
compartment results in additional side-branches and the formation of alveoli at the ends
of those structures. A combination of several Signaling pathways acting together is
necessary to produce a fully functional mammary gland.

Signal transducer and activator of transcription (Stat)5a is one of the critical
regulators of normal mammogenesis. The Stat proteins are a family of transcription
factors which transduce extracellular cytokine and grth factor signals to the nucleus (1,
2). Two Stat5 isoforms exist (a and b, collectively referred to as Stat5), which share
~96% identity at the amino acid level (3). However, Stat5a has emerged as the chief
isoform involved in mammary development, likely because it is expressed at much higher
levels in the mammary gland than Stat5b (4). The vast majority of Stat5 studies have
examined its fimctions during pregnancy. Inactivation of Stat5 in mouse lines by
germline deletion has provided much of the information on its function.

Stat5a knockout mice (Stat5a-/-) exhibit reduced alveolar development at
parturition and fail to lactate (4, 5). Transplanted Stat5 knockout (Stat5-/-) epithelium
into wild-type stroma incorporates less BrdU in response to an acute E+P treatment (6).

Thus, Stat5 regulated proliferation occurs through a mechanism autonomous to the

91

epithelial cells. When Stat5 was conditionally deleted specifically in the mammary gland
during pregnancy, fewer phosphorylated histone 3 (a marker of cell proliferation) positive
cells were present compared to control glands, again demonstrating a function in
proliferation (7). Consistent with these results, increased proliferation and a
predisposition to mammary tumors was seen in transgenic animals expressing a
constitutively active form of Stat5 [comprised of the amino acid sequences from position
1 to 750 of the Stat5 molecule, the TAD (positions 677—847) of Stat6, and the kinase
domain (positions 757—1 129) of J ak2] in the mammary gland (8).

Although Stat5 has been implicated in mammary epithelial cell proliferation, very
little is known about the underlying mechanism by which it acts. Data shown in chapter
2 demonstrate that Stat5a positive cells rarely proliferate in response to E+P compared to
adjacent Stat5a negative cells, strongly suggesting that a paracrine molecule may mediate
Stat5a’s effect in neighboring cells. To gain additional information about the role of
Stat5a outside of pregnancy and the pathways by which it controls epithelial cell
proliferation, a more detailed examination of mammary gland morphology in pubertal
and mature virgin, as well as in hormone treated Stat5a-/- mice is presented here. These
studies establish a role for Stat5a in normal primary branching and side-branching in the
nulliparous gland, and a critical role in the proliferative response to E+P. Finally, we
Show a loss of Stat5a leads to reduced RANKL levels following E+P treatment, and

propose that this protein is an important mediator of Stat5a regulated proliferation.

92

MATERIALS AND METHODS

Animals:

Mammary glands were obtained from pubertal (6 wk old) and adult ( 18 wk old)
Stat5a-/-, Stat5a+/+ and Stat5a+/- female mice in the C57B16/129-svj mixed background.
For hormone treatments, 18 wk old virgin mice were ovariectomized (OVX) and 1 wk
after OVX animals were injected every 24 h for five days with E+P (1 pg + 1 mg,
respectively, per injection) administered subcutaneously into the dorsum of the neck.
Animals were injected intraperitoneally with 70 jig/g body weight of 5-bromo-2’-
deoxyuridine (BrdU) (Sigma, St Louis, MO) two hours before they were euthanized. All
animal experimentation was conducted in accordance with accepted standards of humane
animal care and approved by the All University Committee on Animal Use and Care at
Michigan State University.

Tail DNA was prepared as described (9). Genotyping of mice was performed by
PCR. For PCR analysis, the wild-type StatSa allele was detected by using primer F9 (5'-
AAG GGA CAG GAA GAG AGA AGG-3'), and primer R1 (5'-CCC ATA CAA CAC
TTG CAT CT-3') located at either Side of the hygromycin resistance gene insertion site.
This primer pair amplifies a fragment of 274 bp from wild-type and heterozygous mice
but not from Stat5a knockout mice. DNA was also amplified by using primers R1 and
TKp; the latter is located within the hygromycin resistance vector used to disrupt the
Stat5a locus (5'-GCA AAA CCA CAC TGC TCG AC-3') to detect the Stat5a knockout

allele. In this case, a 110-bp fragment was detected in mice heterozygous or homozygous

93

for the Stat5a knockout allele, while no signal was detected in wild-type mice.
Genotyping was confirmed by immunohistochemical analysis for Stat5a as described in

Materials and Methods from chapter 2.

Whole mount analysis:

To perform whole mount analyses, the left inguinal mammary gland (gland # 4)
was used. The excised glands were fixed in 10% phosphate-buffered formalin overnight
at 4 C for 24 h, hydrated, stained overnight at 4 C in Carmine alum, dehydrated, cleared

in xylene, and stored in methyl salicylate until ready for analyses.

Immunohistochemistry:

Inguinal mammary glands were fixed in 10% phosphate-buffered formalin
overnight at 4 C, dehydrated, cleared and embedded in paraffin. Tissue was sectioned to
5 pm, mounted on 3-aminopropyl triethoxysilane-coated coverslips, and allowed to dry
for 24 h at room temperature. The tissue was then dewaxed and rehydrated through a
descending gradient of ethanol. Sections were autoclaved (12] C at 15 psi) for 20 min in
0.0] M sodium citrate buffer (pH 6.0) for antigen retrieval. Specific blocking and
antibody incubation protocols are detailed below. Each incubation step was followed by
two 5 min washes in PBS.

For RANKL, Id2, and PRA labeling, sections were blocked for 30 min in 2%

bovine serum albumin in PBS, pH 7.3 (2% PBSA). Samples were first incubated with

94

goat or-RANKL antibody (R&D Systems, Minneapolis, MI), rabbit or-Id2 antibody (Santa
Cruz, Santa Cruz, CA), or rabbit polyclonal or-PR antibody, which detects only PRA
(Dako Carpinteria, CA) in 2% PBSA overnight at 4 C, then incubated with rabbit a-goat
(for RANKL staining) or goat a-rabbit antibody conjugated to Alexa 488 (green)(for Id2)
or Alexa 546 (red)(for PRA) (Invitrogen, Carlsbad, CA) in PBS for 30 min. Nuclei were
stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma, Saint Louis, MO) and
pictures were taken on a Nikon Eclipse fluorescent microscope (Tokyo, Japan) with
MetaMorph software (Molecular Devices Corp., Downington, PA). For Id2 staining,
nuclei were counterstained using TOPRO-3 iodide (Invitrogen), and visualized using a
Pascal laser scanning confocal microscope (Zeiss, Oberkochen, Germany).

For BrdU labeling, sections were first blocked with goat a-mouse IgG Fab
fragments (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:100 in 1%
PBSA for 1 h, then blocked with normal goat serum (NGS) (Vector Laboratories,
Burlingame, CA ) diluted 1:] in PBS for 30 min. The tissue was incubated with mouse
or-BrdU antibody (provided as a kit from GE Healthcare, Piscataway, NJ) for l h. This
was followed by 30 min with a biotinylated goat (rt-mouse antibody (Dako, Carpinteria,
CA) (1:400) and ABC reagent (Vector Laboratories). Irnmunoperoxidase localization of
antibody staining was achieved using 3'-3'-diarninobenzidene. The sections were
counterstained with hematoxylin and visualized using a Nikon Eclipse 50i microscope

and a Qimaging color camera with ImageQuant software (GE Healthcare).

95

Branch Quantitation and Statistical Analysis:

Primary branch point quantitation was achieved by examining one area of the #4
mammary gland whole mounts from 18 wk old mice. Using PowerPoint software, a
rectangle of identical size corresponding to ~15% of the mammary gland area was
aligned on photographs of each gland with similar lymph node placement. Primary ducts
within the rectangle were traced, and branch points counted. The pixel area of each gland
quantitated, as determined by the perimeter of duct ends, was calculated using Image]
software (NH-I, Bethesda, MA), and the number of branch points per gland was
normalized to this area. This process was carried out for each mammary gland by three
individuals, and the final data represents the average of these findings.

Sections examined for BrdU or PRA by immunoperoxidase and
immunofluorescence methods were quantitated for the number of BrdU or PRA-positive
cells with the aid of a light microscope (immunoperoxidase) or from captured images
(immunofluorescence). Five mice per treatment were analyzed. A minimum of 1200
total cells and two independent sections per mouse were analyzed. Results are expressed
as mean i SEM, and differences are considered significant at P < 0.05 using Student’s t

ICSI.

Immunoblot Analysis:

Whole mammary glands were obtained from 5 day E+P treated mice. The glands

were minced and homogenized in a buffer of 50 mM Tris HCl pH 7.2, 6 mM MgC12, 1

96

mM EDTA, 10% Sucrose (w/v) (1 m1/O.2 g mammary tissue) containing complete mini
protease inhibitor cocktail tablet (Roche, Indianapolis, IN) and phosphatase inhibitor
cocktails I and II (Sigma, St. Louis, MO) using a Polytron homogenizer. Homogenates
were centrifirged at 14,000 x g for 30 min and supematants were used for immunoblots.
Mammary gland extract (20 pg) was mixed with 1/ 10 volume of 10x SDS sample buffer
with reducing agent and boiled for 5 min at 100 C. Protein samples were resolved on 12%
polyacrylamide gels under denaturing conditions, and transferred onto Polyvinylidene
difluoride membranes (Perkin Elmer, Walthan, MA). Membranes were blocked in 5%
milk in phosphate-buffered saline with 0.5% Tween 20 overnight at 4 C and incubated
with goat monoclonal or-RANKL (R&D Systems)(dilution 1:1000) overnight at 4 C. The
secondary antibody was horseradish peroxidase-labeled rabbit a-goat (dilution 122000)
(Biorad Laboratories, Hercules, CA). After 1 h incubation with secondary antibody,
membranes were washed, incubated with Super Signal West Pico chemiluminescent

substrate (Pierce Biotechnology, Rockford, IL), and exposed to x-ray film.

RESULTS

Mammary gland morphology in pubertal and adult nulliparous mice

Previous studies of Stat5 in the mammary gland have focused heavily on its role

in pregnancy related mammary development. The few reports concerning the

morphology of virgin Stat5-/- and Stat5a-/- glands have been contradictory. While one

study described a lack of side-branching 8 wks after Stat5a-/- epithelium was transplanted

97

into wild-type stroma (10), others reported no defects in intact, adult Stat5a-/- mice (4),
or in outgrowths from transplanted Stat5-/- epithelium (6). However, the results
presented in chapter 2 demonstrated that Stat5a is present and activated in mouse
mammary epithelial cells as early as 5 wks of age, suggesting that it plays a role in the
development of the virgin gland. To investigate potential functions of StatSa in the non-
pregnant mammary gland, whole mounts from 6 wk and 18 wk old Stat5a-/-, Stat5a+/-
and Stat5a+/+ mice were compared. We observed that the ductal outgrowth in the 6 wk
old pubertal glands had progressed to the same distance beyond the lymph node in all of
the mice, regardless of Stat5a genotype (Fig. 3.1A), indicating that Stat5a is not
necessary for ductal elongation. Although there was some heterogeneity observed
between animals of the same genotype, the Stat5a-/- glands appeared to contain fewer
ducts and TEBS, and looked less organized overall. Mammary glands from mature 18 wk
old Stat5a-/- mice also had varying degrees of ductal morphology, but overall appeared
less developed and organized than age-matched wild-type glands, with a clear paucity of
Side-branching (Fig. 3.18). This finding supports earlier reports that Stat5a deletion in
transplanted virgin mammary gland epithelium resulted in less side-branching (7), and
emphasizes the specific requirement for the Stat5a isoform in early development. The
heterozygote glands displayed a morphology intermediate between the Stat5a-/- and
Stat5a+/+ mammary glands. The presence of this dosage effect demonstrates that
maximum expression of the protein is required for normal mammary gland development.
In addition to the lack of side-branches, the 18 wk old Stat5a-/- glands appeared
to contain fewer primary ducts. To test if the difference was Significant, primary branch

points were quantitated from similar regions of 18 wk old Stat5a-/-, Stat5a +/- and Stat5a

98

 

 

0.0025
0.0020

 

 

0.0015-
0.0010-
0.0005-

# of primary branches
normalized to area

 

0.0000:

 

Figure 3.1. Whole mount analysis of pubertal and adult virgin mice. The fourth
inguinal mammary glands were removed from Stat5a+/+, Stat+/- and Stat-/- mice and
prepared as whole mounts. A. Mammary glands fiom 6 wk old pubertal mice and B. 18
wk old mature mice. Scale bar for all figures = 2 mm. C. Analysis of primary branch
points in 18 wk old Stat5a+/+, Stat5a +/- and Stat5a -/- mammary glands. Quantitation
was carried out as described in Materials and Methods for 3-9 animals per genotype, and
is shown as the average of three individual analyses i- tlre standard deviation.

99

+/+ mammary glands as described in Materials and Methods. This analysis indicated that
Stat5a-/- glands contain significantly fewer (~32%) primary branch points than wild-type
glands (Fig. 3.1C). Stat5a+/- glands exhibited an intermediate phenotype, with ~21%
fewer branch points than wild-type glands and ~14% more than knockout glands,

although neither of these differences were statistically significant.
Morphological response to hormone treatment

To examine the role of Stat5a in mammary gland development, the response to
E+P treatment was examined. 18 wk old virgin Stat5a-/-, Stat5a+/— and Stat5a+/+ mice
were ovariectomized and allowed to recover for one week. Animals were then treated
with a combination of E and P for five days. Previous studies have used two day
treatments to examine the effects of Stat5 deletion on proliferation. However, a treatment
period of two days is not sufficient to elicit a discemable morphological response. In
contrast, five days of E+P treatment generates a visible response that includes enlarged
tips at the ends of ducts and side-branches as a precursor to alveolar formation. As
expected, mammary gland whole mounts from five day E+P treated wild-type mice
displayed enlarged tips at the ends of their numerous side-branches and ducts (Fig. 3.2).
Stat5a+/- and Stat5a-/- mice also exhibited enlarged tips at the ends of their side-branches
and ducts. However, similar to the intact virgin mice, dramatically fewer branches were
observed in the Stat5a-/- glands compared to the Stat5a+/+ glands. Again, the severity of
this defect was variable between individual Stat5a-/- animals. The lack of branches from

which alveolar budding can occur resulted in a corresponding decrease in the number of

100

 

 

 

Figure 3.2. Morphological response of mammary glands to hormone treatment.
Mammary gland whole mounts were prepared from 18 wk old OVX Stat5a+/+, Stat5a+/-
and Stat5a-l- mice treated for 5d with E+P. Arrows indicate enlarged tips where alveoli
will form. Scale bar = 1 mm. ‘

10]

enlarged tips in the Stat5a-/- glands. Again, the Stat5a+/- mice displayed a phenotype
intermediate between the knockout and wild-type animals. This finding suggests that the
blunted response to E+P treatment is at least partially due to the pre-existing defect in

side-branching observed in the virgin Stat5-/- mice.

Proliferative responses to hormone treatment

To determine if the proliferative response to E+P differed between the genotypes,
mice were treated with a pulse of BrdU 2 h before they were euthanized. The analysis of
BrdU incorporation in response to the 5 day E+P treatment is shown in Fig. 3.2. In the
wild-type glands, 15% of all epithelial cells were BrdU positive (+). Approximately half
as many BrdIF cells (7%) were present in glands from Stat5a-/- mice. The percentage of
BrdU+ cells in heterozygous mice (12%) was higher than the knockouts and lower than
the wild-types, but was not significantly different from either.

Although BrdU+ cells were present throughout the entire mammary gland, the
areas of highest proliferation were seen at the enlarged tips of ducts and side-branches,
regardless of StatSa genotype (Fig. 3.3). When duct and tip structures were analyzed
independently, the difference in the percentage of BrdU+ cells between the Stat5a
genotypes was the same (Fig. 3.3B). Thus, fewer proliferating cells were present in all

Stat5a-/- mammary gland structures compared to wild-type glands.

102

 

 

 

Figure 3.3. Proliferative response of mammary glands to hormone treatment.
Irnmunoperoxidase detection of incorporated BrdU was performed on mammary gland
sections from 18 wk old OVX mice treated for 5 d with E+P. A. Representative images
from BrdU staining is Shown for Stat5a+/+ and Stat5a-/- ducts and alveolar buds. Scale
bar = 30 pm. B. Quantitation of the percent BrdU-positive luminal epithelial cells was
carried. out. Proliferation was significantly higher in the Stat5a+/+ glands compared to
Stat5a-/- glands. Results are Shown for quantitation carried out on duct and alveolar bud
structures independently, as well as for all structures types combined. The values
represent the mean :1: SEM from five mice per group with a minimum of 1000 cells
analyzed per mouse. Images in this dissertation are presented in color.

103

neat.
gland
rages
Scale
5 was
ed to
r bud
'zrlues

cells

Duct

Tips

% BrdU positive cells

 

 

 

 

 

 

 

 

  

-l- +/- +/+ -/- +/- +/+ -/- +l- +/+
Tips Ducts All Stmdures

Downstream targets of Stat5a in the mammary gland

Data from chapter 2 demonstrated that Stat5a+ cells rarely proliferate in response
to E+P treatment, and are ~5 times less likely to proliferate than adjacent Stat5a' cells
(11). Thus, we hypothesized that Stat5a elicits its proliferative effect through a paracrine
mechanism. PRL treatment has been shown to induce RANKL expression in the
mammary gland in vivo (12), and studies in heterologous cell culture models suggest this
is through the J ak2/Stat5a pathway (13). Preliminary studies in chapter 2 showed
evidence that RANKL is downstream of Stat5a Signaling in vivo, since the two proteins
were highly colocalized in mammary epithelial cells after E+P treatment (11). To
determine if RANKL is regulated by Stat5a, mammary gland sections from the 5 day
E+P treated mice were examined by immunofluorescent staining for RANKL (Fig. 3.4A).
Overall, the intensity of RANKL staining was much weaker in Stat5a-/- sections than
Stat5a+/+ sections, although there was some variability between individual mice. In
addition, the amount of secreted RANKL in duct lumens was much lower in the Stat-l-
mice. To confirm and quantitate these findings, Western blot analysis for RANKL in
whole mammary gland homogenates fiom individual mice was carried out (Fig. 3.4B).
In agreement with the immunohistochemical analysis, RANKL levels were variable
between animals of the same genotype, particularly in the knockouts. However, 4 out of
5 Stat5a-/- mice had less expression of RANKL than Stat5a+/+ mice, confirming a
defect. A strong correlation was observed between the immunofluorescent staining and

immunoblot data from individual mice. For example among the five Stat5a-/- mice, the

105

l

 

 

Figure 3.4. RANKL expression in response to hormone treatment.
Immunofluorescent staining for RANKL was performed using mammary gland samples
from 18 wk old OVX mice treated for 5 d with E+P. A. Representative images of
RANKL staining (green) is shown for Stat5a+/+ and Stat5a -/- ducts. Nuclei were
stained with DAPI (blue). Images were captured with identical exposure times. Scale
bar = 15 um. B. Western analysis of RANKL levels in whole mammary gland
homogenants from Stat5a+/+ and Stat5a-/- mice. Each lane represents a single mammary
gland from independent mice (6 Stat5a+/+ mice and 5 Stat5a-/- mice). Images in this
dissertation are presented in color.

106

RANKL RANKL + DAPI

+/+ -/—

12 3 4 5 612 a 4
RANKLi-a-J- whiz-'- ‘ -3

Actin firm

+/+

 

-/- #4

-/- #5

 

 

107

lowest levels of RANKL were detected by both methods in mouse #4, while the highest
levels of RANKL were detected by both methods in mouse #5.

The data shown here and in chapter 2 support the model that E+P induces
expression of Stat5a, and activated Stat5a leads to RANKL expression. P treatment can
induce the expression of RANKL in the mouse mammary gland (12), and knockout
studies have shown that PR is necessary for the increased expression of RANKL in the
mouse mammary gland after E+P treatment (14). To examine whether the defect in
mammary cell proliferation or RANKL expression in Stat5a-/- mice was due to altered
PR levels, staining for PRA was carried out on the 5 day E+P treated Stat5a-/- and
Stat5a+/+ mammary gland sections. The two genotypes exhibited similar PRA staining
intensities (Fig. 3.5A), and contained a similar percentage of PRA+ cells (~ 47%) (Fig.
3.5B). Thus, the altered response to E+P is not due to a lack of PRA expression, and
these findings support the model that Stat5a can directly induce RANKL expression.
Analysis of the PRB isoform was not carried out Since this protein is not readily
detectable in the mammary gland until ~10 days of E+P treatment (15).

We next examined a potential downstream target of the Stat5a —> RANKL
pathway, Id2. The Id proteins are inhibitors of basic helix-loop-helix (bHLH)
transcription factors, since they contain a helix-loop-helix motif, but lack a DNA-binding
domain (16). AS a consequence, they act as dominant-negative regulators of bHLH
transcription factors via heterodimerization with these proteins. Id2-/- mice exhibit
defective alveolar development and lactation similar to RANKL-/- and Stat5a-/- mice
(17, 18). Recently, it was reported that Id2 nuclear localization is induced by RANKL,

and is critical for RANKL-mediated proliferation in primary mouse mammary epithelial

108

 

 

PRA + DAPI

-/- +/+

Figure 3.5. Progesterone receptor expression in response to hormone treatment. A.
Immunofluorescent detection of PRA (red) in mammary gland samples from 18 wk old
OVX mice treated for 5 d with E+P was performed. Nuclei were stained with DAPI
(blue). Representative images from sections of Stat5a+/+ and Stat5a -/- mammary glands
are shown. Scale bar = 30 mm. B) Quantitation of the percentage of PRA-positive
epithelial cells was conducted. The values represent the mean i SEM from five mice per
group, with a minimum of 1000 cells analyzed per mouse. Images in this dissertation are

presented in color.

>

+/+

 

./-

 

 

w
5‘:

 

 

% PRA+ Cells

 

 

109

cells (17). It is believed that Id2 mediates proliferation in mammary cells through its
ability to inhibit transcription of p21, a cell cycle inhibitor (17). To examine the levels
and cellular localization of Id2 in the mammary glands from 5 day E+P treated Stat5a-/-
and Stat5a+/+ mice, immunohistochemical analysis was carried out. Id2 staining was
predominantly nuclear in all wild-type animals (Fig. 3.6). In contrast, Id2 was largely
cytoplasmic in sections from the Stat5a-/- glands that had low RANKL expression,
especially mice 2-4 (see Fig. 3.5B) (staining from mouse 3 shown). Interestingly, Id2
localization appeared nuclear in mice 1 and 5 (staining from mouse 5 shown), which had
levels of RANKL similar to wild-type animals. Thus, Id2 nuclear localization correlated

with high RANKL levels in both Stat5a+/+ and Stat5a-/- mice.
DISCUSSION

Numerous studies have defined a functional role for Stat5a in the pregnant
mammary gland. Data from chapter 2 demonstrated that Stat5a is expressed and active in
the virgin gland, but very little is known about its function outside of pregnancy.
Evidence from previous knockout studies was contradictory, with some reporting no
phenotype in the virgin gland, and others reporting a defect in ductal branching. To
resolve this issue and to examine potential fimctions of Stat5a in the virgin gland,
mammary whole mounts from Stat5a-l- and Stat5a+/+ mice were compared at the
pubertal (6 wk) and mature (18 wk) stages of development. The results demonstrated
that Stat5a was dispensable for ductal elongation, but was necessary for normal primary

branching and side-branching (Fig. 3.1). This finding presents an intriguing new role for

110

 

 

Id2 Id2 + TOPRO

    

+/+

 

-/- #4

-/- #5

 

Figure 3.6. Id2 localization in response to hormone treatment. Representative
confocal images are Shown for immunofluorescent detection of Id2 (green) and nuclei
stained with TOPRO (blue). The mammary glands shown correspond to Stat5a+/+
mouse #4 and Stat5a-/- mice #4 and #5 (see Fig. 3.4B) from 18 wk old OVX mice treated
for 5 d with E+P. Optical Slice = 0.5 pm. Scale bar = 5 pm. Images in this dissertation
are presented in color.

111

Stat5a in mammary development. The mechanisms by which branching is regulated are
not yet fully understood, but many interesting ductal morphogenesis phenotypes have
been described that are associated with overexpression or deletion of specific genes. The
regulators thus identified to date include hormones, grth factors and growth factor
receptors, cell cycle regulators, transcription factors, cytokines' and cell adhesion
molecules (19).

It is interesting to note that while primary branching is accomplished mainly
through the bifurcation of TEBS, Stat5a is not expressed in TEB structures (Fig. 2.1 of
chapter 2) (11), suggesting a possible role for stromal Stat5a in this process. Stat5a
signaling in leukocytes may explain its function in mammary branching. Macrophages
and eosinophils have been shown to be located specifically to the stromal area
surrounding TEBS in pubertal mammary glands, and are necessary for normal outgrowth
and branching (20). These hematopoietic cells are recruited to TEBS by colony
stimulating factor-1 (CSF-l) (20). Immunohistochemical and in situ hybridization
studies have shown that mammary epithelial cells synthesize CSF-l (17), while the CSF-
1 receptor (CSF-lR) is exclusively expressed in macrophages in the mammary gland
tissue of virgin mice (20). CSF-l activation of the CSF -1R is capable of inducing Stat5a
phosphorylation (21) and DNA binding (22). Furthermore, virgin CSF -1 knockout mice
display a mammary phenotype similar to virgin Stat5a knockout mice, including limited
primary branching and a disorganized ductal network (20). However, a role for epithelial
Stat5a in primary branching should not be precluded since branching defects were

reported in Stat5a-/- epithelial outgrowths in wild-type stroma (10). It is therefore

112

possible that multiple branching mechanisms are regulated by Stat5a in the mammary
gland, and finther investigation is needed to elucidate the specific pathways involved.

In contrast to primary branches, side-branches are produced via proliferation of
epithelial cells in pre-established ducts. It was previously reported that Stat5 is necessary
for normal mammary epithelial cell proliferation since the Stat5-/- knockout epithelium
exhibited less BrdU incorporation after E+P treatment (6, 7). To investigate if this
requirement was specific to Stat5a, 5 day E+P treated OVX Stat5a+/+, Stat5a+/- and
Stat5a-/- mice were examined. The use of hormone treatments in OVX mice allowed us
to avoid any possible complications due to altered endogenous ovarian hormone levels,
and the 5 day treatments allowed us to examine the morphological response to hormone
treatment. Although enlarged distal tips were present in glands from all genotypes, there
was a notable lack of enlarged side-branch tips in the Stat5a-/- glands. This is likely due
to the fact that there are fewer side-branches in Stat5a-/- glands to begin with, and
therefore, fewer structures on which alveolar budding can take place. Thus, the
defective alveolar development previously reported in pregnant Stat5a-/- mice might be
at least partially the result of the side-branching defect seen in virgin mammary
development. However, the defective E+P response in StatSa-/- glands was not simply
due to the scarcity of pre-existing side-branches. Structure specific analysis of
proliferation demonstrated that the percentage of BrdU-labeled cells was lower in Stat5a-
/- glands compared to Stat5a+/+ glands by ~40% in ducts and ~42% in tips,
demonstrating that Stat5a regulates mammary epithelial cell proliferation throughout the

entire gland. Although pregnancy levels of hormones were used in this experiment, it is

113

possible that the lack of side-branches seen in virgin Stat5a-/- mice could be due to a
decreased proliferative response to E+P during the normal estrous cycle.

It was demonstrated in chapter 2 that Stat5a+ cells rarely proliferate in response to
E+P treatment, suggesting that Stat5a induces proliferation via a paracrine mechanism.
The data presented here indicate that Stat5a is required for the maximal induction of
RANKL expression in response to E+P treatment. One important target of RANKL’S
receptor, RANK, in mammary epithelial cells is the inhibitor of bHLH transcription
factors, Id2 (17). RANKL treatment induces the nuclear localization of Id2, which
inhibits transcription of p21, and ultimately leads to cell proliferation ( 17). The nuclear
localization of Id2 was decreased in the mammary epithelial cells in Stat5a-/- mice
compared to Stat5a+/+ mice following E+P treatment. Along with data from chapter 2,
this evidence supports a pathway where E+P —> Stat5a —) RANKL ——> RANK —> Id2 —>
cell proliferation (see Figure 3.7). This model is consistent with the fact that RANKL-/-,
Id2-/- and Stat5a-/- mammary glands display similar phenotypes during pregnancy,
although the defective alveolar development seen in Stat5a-/- mice is less severe than the
other knockout models (4, 5, 12, 23).

There was considerable heterogeneity observed in mammary gland morphology
between individual animals of all StatSa genotypes (+/+, +/-,-/-). This variability is likely
to be at least partially due to the mixed background of the Stat5a mice. Likewise, the
induction of RANKL was variable from gland to gland, especially in the StatSa-/- mice.
Along with the fact that the mammary defect is less severe in Stat5-/- mice than in
RANKL-l- mice, this finding suggests that other pathways leading to RANKL expression

may exist. Stat5b might be capable of inducing RANKL similar to Stat5a. It is also

114

Stat5a+
ER+
PR+

Stat5a-
ER-
PR-

 

Figure 3.7. Proposed mechanism of Stat5a mediated proliferation in response to
E+P. E+P treatment directly induces the expression of Stat5a, and indirectly activates
Stat5a via PRL in ER+, PR+ mammary luminal epithelial cells. Activated Stat5a induces
the expression of RANKL, which is secreted from the cell. RANKL acts through its
receptor RANK on neighboring Stat5a-, ER-, PR- epithelial cells to induce the nuclear
localization of Id2. Nuclear Id2 inhibits the expression of p21, leading to a proliferative
response. In addition to Stat5a, other pathways may lead to the induction of RANKL in
response to E+P treatment, such as Stat5b.

115

possible that PR might directly regulate RANKL, since PR-/— mice are defective in
inducing RANKL following E+P treatment (14). The levels or activation of these and
other molecules could potentially vary between animals due to their mixed background,
causing a variable response to E+P treatment independent of Stat5a. However, this
heterogeneity allowed us to draw some conclusions by examining individual mammary
glands. Specifically, the strong correlation between RANKL expression levels and Id2
nuclear localization within individual animals supports the model of Stat5a-induced

proliferation proposed here.

116

 

10.

REFERENCES

Darnell JE, Jr. 1997 STATS and gene regulation. Science 277:1630-5

Ihle JN 1996 STATS: signal transducers and activators of transcription. Cell
84:331-4

Lin X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning
and expression of Stat5 and an additional homologue (Stat5b) involved in

prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci U S
A 92:8831-5

Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D,
Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b proteins have
essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841-

50

Lin X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A,
Hennighausen L 1997 Stat5a is mandatory for adult mammary gland
development and lactogenesis. Genes Dev 112179-86

Miyoshi K, Shillingford JM, Smith GH, Grimm SL, Wagner KU, Oka T,
Rosen JM, Robinson GW, Hennighausen L 2001 Signal transducer and
activator of transcription (Stat) 5 controls the proliferation and differentiation of
mammary alveolar epithelium. J Cell Biol 155:531-42

Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW,
Hennighausen L 2004 Inactivation of Stat5 in mouse mammary epithelium

during pregnancy reveals distinct functions in cell proliferation, survival, and
differentiation. Mol Cell Biol 24:8037-47

Iavnilovitch E, Cardiff RD, Groner B, Barash I 2004 Deregulation of Stat5
expression and activation causes mammary tumors in transgenic mice. Int J
Cancer 112:607-19

Hogan B, Beddington R, Costantini F, Lacy. E 1994 Manipulating the Mouse
Embryo: A Laboratory Manual, second edition ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York

Liu X, Gallego MI, Smith GH, Robinson GW, Hennighausen L 1998

Functional rescue of Stat5a-null mammary tissue through the activation of
compensating Signals including Stat5b. Cell Growth Differ 9:795-803

117

 

ll.

12.

13.

14.

15.

16.

17.

l8.

19.

20.

21.

22.

Santos SJ, Haslam SZ, Conrad SE 2007 Estrogen and Progesterone are Critical
Regulators of StatSa Expression in the Mouse Mammary Gland. Endocrinology

Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, Elliott R,
Scully S, Voura EB, Lacey DL, Boyle WJ, Khokha R, Penninger JM 2000
The osteoclast differentiation factor osteoprotegerin-ligand is essential for
mammary gland development. Cell 103241-50

Srivastava S, Matsuda M, Hon Z, Bailey JP, Kitazawa R, Herbst MP,
Horseman ND 2003 Receptor activator of NF-kappaB ligand induction via J ak2
and Stat5a in mammary epithelial cells. J Biol Chem 278:46171-8

Mnlac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM 2003 Defective
mammary gland morphogenesis in mice lacking the progesterone receptor B
isoform. Proc Natl Acad Sci U S A 100:9744-9

Aupperlee MD, Haslam SZ 2007 Differential hormonal regulation and function
of PR isoforms in normal adult mouse mammary gland. Endocrinology

Norton JD, Deed RW, Craggs G, Sablitzky F 1998 Id helix-loop-helix proteins
in cell grth and differentiation. Trends Cell Biol 8:58-65

Kim NS, Kim HJ, Koo BK, Kwon MC, Kim YW, Cho Y, Yokota Y,
Penninger JM, Kong YY 2006 Receptor activator of NF-kappaB ligand
regulates the proliferation of mammary epithelial cells via Id2. Mol Cell Biol
26:1002-13

Mori S, Nishikawa SI, Yokota Y 2000 Lactation defect in mice lacking the
helix-loop-helix inhibitor Id2. Embo J 19:5772-81

Howlin J, McBryan J, Martin F 2006 Pubertal mammary gland development:
insights fi'om mouse models. J Mammary Gland Biol Neoplasia 11:283-97

Gouon-Evans V, Rothenberg ME, Pollard JW 2000 Postnatal mammary gland
development requires macrophages and eosinophils. Development 127:2269-82

Yenng YG, Wang Y, Einstein DB, Lee PS, Stanley ER 1998 Colony-
stimulating factor-1 stimulates the formation of multimeric cytosolic complexes

of signaling proteins and cytoskeletal components in macrophages. J Biol Chem
273:17128-37

Novak U, Mui A, Miyajima A, Paradiso L 1996 Formation of STATS-

containing DNA binding complexes in response to colony-stimulating factor-1
and platelet-derived grth factor. J Biol Chem 271:18350-4

118

 

23.

Miyoshi K, Meyer B, Gruss P, Cni Y, Renou JP, Morgan FV, Smith GH,
Reichenstein M, Shani M, Hennighausen L, Robinson GW 2002 Mammary
epithelial cells are not able to undergo pregrrancy-dependent differentiation in the
absence of the helix-loop-helix inhibitor Id2. Mol Endocrinol 16:2892-901

119

 

CONCLUDING REMARKS

Stat5a is a signaling molecule shown to be critical for normal mammary gland
development, and has been implicated in breast cancer. In this dissertation, I set out to
explore two aspects of Stat5a biology in the mouse mammary gland. First, the regulation
of Stat5a expression in mammary epithelial cells was examined. Although the pathways
leading to Stat5a activation in the mammary gland had been elucidated, the mechanisms
that regulate its expression in this tissue were relatively unknown. The results presented
in chapter 2 demonstrated that the ovarian steroid hormones E and P are critical
regulators of Stat5a expression in the pubertal and adult virgin mammary gland. Based
on the fact that the majority of Stat5a+ cells also express ERa and PR, I argue that this
regulation likely occurs at the cellular level through epithelial ERoc and PR. E and P are
sufficient to induce StatSa expression, but activation and nuclear localization of the
protein is dependent on PRL Signaling, leading to the regulation of StatSa responsive
genes. This pathway offers an important new mechanism by which StatSa signaling is
regulated in mammary epithelial cells.

In chapter 2 of this dissertation, I demonstrated that Stat5a iS present and activated
in the pubertal and adult virgin mouse mammary gland. Although the function of Stat5a
in the pregnant and lactating gland was well understood, its roles in the virgin gland were
relatively unexplored. The data presented in chapter 3 revealed that Stat5a is required for
both primary branching and side-branching in the virgin, developing mammary gland.
Additionally, Stat5a—/- mice exhibit a defect in the proliferative response to E+P, and this

may partially explain the lack of side-branches in the virgin gland. The results presented

120

in this dissertation also shed light on the mechanism by which Stat5a induces
proliferation in response to E+P treatment. The finding that Stat5a+ cells do not
themselves proliferate, suggest that they produce a paracrine mediator which can
stimulate adjacent StatSa- cells to proliferate. In chapter 3, evidence is presented that
RANKL is one such paracrine mediator, and that it stimulates proliferation in responsive
cells, at least in part, by inducing nuclear localization of Id2. In conclusion, the findings
presented here represent a significant advance to the existing knowledge about Stat5a
Si gnaling, including insight into the regulation of its expression as well as the mechanism

by which it induces proliferation during mammary gland development.

121

 

 

APPENDIX

REGULATION OF STATSA EXPRESSION BY R5020 IN PRIMARY
MAMMARY EPITHELIAL CELLS

122

 

INTRODUCTION

Several lines of evidence suggest that progesterone (P) can regulate the
expression of Stat5a in the mammary gland. Stat5a protein and RNA levels have been
shown to increase during pregnancy, a period of increased systemic P levels. In addition,
it has been demonstrated that P can directly induce the expression of Stat5a mRN A in the
human breast cancer cell line T47D (1). Interestingly, the increased Stat5a expression in
response to P treatment was much greater in cells expressing only PRB than cells
expressing only PRA. While PRA is detectable in mammary epithelial cells (MEC)S in
the virgin mammary gland, PRB is only detectable during pregnancy (2). Based on these
facts, we hypothesized that P can induce expression of Stat5a in normal mouse mammary
gland cells from mature adult mice to a greater degree than in cells from pubertal mice.
This was tested by measuring the mRNA levels of Stat5a from MECs grown in a serum-
free, three-dimensional in vitro culture system, treated with R5020, by quantitative RT-

PCR.

MATERIALS AND METHODS

Reagents: Collagenase HI and pronase were obtained from Worthington
Biochemical Corp. (Freehold, NJ) and Calbiochem (La Jolla, CA), respectively.
DMEM/Ham’s nutrient mixture F-12 (1 :1; DMEM/F12), Hanks’ Balanced Salt Solution,
and trypsin were purchased from Sigma (St. Louis, MO). L-Glutamine, penicillin, and
streptomycin were purchased from Life Technologies, Inc. (Gaithersburg, MD). Rat

collagen 1 (>90% pure) was purchased from BD Biosciences. The synthetic progestin,

123

R5020 (promegestone), was purchased from NEN Life Science Products (Boston, MA).
Superscript HI first strand cDNA synthesis kit and Trizol reagent was obtained from
Invitrogen (Carlsbad, CA). DNA-free reagent was from Ambion (Austin, TX). Taqman
probes and universal PCR master mix were obtained from Applied Biosystems (A/B)

(Foster City, CA).

Mammary epithelial cells were isolated from pubertal (6 wk old) and adult virgin
(18 wk-old) BALB/c female mice from our own colony using enzymatic dissociation
methods as previously described (3). Cell viability was approximately 95% as
determined by trypan blue exclusion. Freshly isolated epithelial cells (8 x 105/well) were
suspended in neutralized collagen I (2 mg/ml, 600 pl/well), plated on top of a layer of
collagen I (250 til/well), and allowed to gel for 30 min at 37 C. Preparation of collagen
gel was according to the manufacturer’s instructions. All serum-free cultures were carried
out in basal medium (BM): serum- and phenol red-free DMEM/F-12 supplemented with
0.1 mM nonessential amino acids (product 11140-050, Life Technologies, Inc., Grand
Island, NY), 2 mM L-glutamine, 0.] pg/ml insulin, 1 mg/ml fatty acid-free BSA (fraction
V), 100 ug/ml penicillin, and 50 pg/ml streptomycin. Cultures were kept in 5% C02 at 37
C for 48 h. Cells were treated with 10 nM of the synthetic progestin, promegestone

(R5020). R5020 was used instead of progesterone because it is metabolized less rapidly.

Primary cultures were flash frozen in liquid nitrogen and stored at -80 C. Frozen
gels were homogenized with a Brinkman Polytron in Trizol reagent for ~3 min. RNA
was extracted following the standard Trizol protocol. Quantitation of RNA was carried

out using a Nanodrop Spectrophotometer. Genomic DNA was eliminated by treating the

124

 

 

RNA with DNA-free reagent. Reverse transcription of the RNA was carried out using
Superscript III enzyme, and the samples were treated with RNase H. Quantitative PCR
was performed using commercially available Taqman assay probes and primer sets for
Stat5a and 18s rRNA. Fluorescence was detected by an A/B 7900HT therrnocycler, and
data was analyzed on SDS software (MB). 188 rRNA levels were used to normalize
cDNA input amounts. Data represent the average of a minimum of 3 independent cell

cultures, with cDNA from each culture analyzed in triplicate.

RESULTS

Results from these culture experiments showed a ~2-2.5 fold increase in the
Stat5a mRNA level in response to a 48 h R5020 treatment. This finding was supported
by a microarray analysis of primary cultures carried out independently of the experiments
Shown here. The microarray data demonstrated a similar ~2 fold increase in StatSa
expression when cells were treated with R5020 for 24 h (data not shown). Contrary to
our hypothesis, cells from the pubertal and adult mice exhibited an equal increase in
Stat5a mRNA expression in response to R5020. This finding is consistent with the in
vivo data from chapter 2, which showed that Stat5a expression is induced by E+P
treatment in cells containing either PRA or PRB.

The results from the culture experiments are not completely consistent with those
from the in vivo studies. Expression of Stat5a in vivo required both E and P, while R5020
was able to induce Stat5a expression in the absence of E in the primary cell culture

system. A possible explanation for this contradiction could be a decrease in the amount

125

 

of PR in the mammary glands following ovariectomy. To remove the confounding
effects of systemic E and P, the mice were ovariectomized prior to hormone treatment.
However, it has been demonstrated that ovariectomy results in decreased PR levels in the
mammary epithelial cells (4). In contrast, the primary cells used in the culture studies
were isolated fiom intact mice. P might be able to induce Stat5a expression in vivo, but
could require a minimum amount of PR above the level present one wk after
ovariectomy. PRA levels are increased in response to E treatment ovariectomized
animals (4). Therefore, E treatment may have been required in the animal experiments

from chapter 2 to increase the amount of PR above a threshold level.

126

 

 

Stat5a mRNA Expression

 

 

 

Relative mRNA Level

 

 

Basal Media R5020 Basal Media R5020

 

 

Treatment

7 wk old 18 wk old

 

 

 

Figure 4.1. Stat5a mRNA expression in response to R5020. Mammary epithelial cells
were isolated from pubertal (7 wk old) and mature (18 wk old) virgin mice and were
grown as organoids suspended in collagen gel with Basal Media alone or with R5020 for
48 h. Total RNA was extracted from the organoids, RNA was DNaseI treated, and
reversed transcribed. The cDNA was amplified using a Taqman probe and primer set for
StatSa. 18S rRNA was used to normalize cDNA input amounts. The data is expressed as
the average fold induction over basal media from 3-5 independent cell culture
experiments. PCR amplification was carried out in triplicate from a single cDNA
preparation for each culture date.

127

REFERENCES

Richer JK, Jacobsen BM, Manning NG, Abel MG, Wolf DM, Horwitz KB
2002 Differential gene regulation by the two progesterone receptor isoforms in
human breast cancer cells. J Biol Chem 277:5209-18

Aupperlee MD, Smith KT, Kariagina A, Haslam SZ 2005 Progesterone
receptor isoforms A and B: temporal and spatial differences in expression during
murine mammary gland development. Endocrinology 146:3577-88

Haslam SZ, Levely ML 1985 Estrogen responsiveness of normal mouse
mammary cells in primary cell culture: association of mammary fibroblasts with

estrogenic regulation of progesterone receptors. Endocrinology 1 16: 1835-44

Aupperlee MD, Haslam SZ 2007 Differential hormonal regulation and function
of PR isoforms in normal adult mouse mammary gland. Endocrinology

128

 

   

lljlllgjijjjjjlljjjjl