THE EFFECT OF PROGESTIN ON MAMMARY GLAND BRANCHING
MORPHOGENESIS IN VITRO
By
Gabriele M. Meyer

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Genetics
2011

ABSTRACT
THE EFFECT OF PROGESTIN ON MAMMARY GLAND BRANCHING
MORPHOGENESIS IN VITRO
By
Gabriele M. Meyer
Mouse mammary organoids that express progesterone receptor A (PRA) produce tubules
in response to hepatocyte growth factor (HGF) when cultured in collagen gels. These structures
resemble ducts in the mouse mammary gland. When treated with the combination of HGF and
the synthetic progestin, promogestone (R5020), tubulogenesis is stunted to form shorter tubules
that resemble sidebranches in early pregnancy. It was hypothesized that R5020 reduces early
HGF/cMet signaling to produce sidebranch-like tubules. Therefore, the HGF-induced pathway
controlling early extension formation was determined and how R5020 altered HGF/cMet
signaling was analyzed. Using molecular inhibitors and shRNA, it was found that HGF activates
Rac1 to form extensions in the first step of tubulogenesis and that Rac1 activity is Src and FAK
dependent. In addition, it was discovered that R5020 increases extracellular laminin to reduce the
Src, FAK and Rac1 pathway leading to reduced extensions. This is likely mediated through PRA,
as PRA is the predominant PR isoform expressed in organoids. This may in part explain blunted
tubulogenesis observed with combined HGF and R5020 treatment and further supports a role for
PRA in sidebranching during pregnancy.

DEDICATION
To my father Bill, mother Carolyn, and my sisters and brothers Karen, Bill, Stephanie, David and
Nicholas. I could not have done all this without you.

iii

ACKNOWLEDGEMENTS

I would like to thank my mentor, Dr. Sandra Haslam, for support and guidance on my
project and graduate school. Thanks to members of my committee, Dr. Susan Conrad, Dr.
Michele Fluck, Dr. Richard Miksicek, Dr. Chengfeng Yang for additional guidance on my
project. In addition, thanks to Dr. Miksicek on help and feedback with the design of PRAKO and
PRBKO mice, and Dr. Yang for his advice on the mouse mammary organoid project. Thanks to
Dr. Bruce Uhal for his guidance on my committee during my comprehensive and oral exams.
Thanks to Jeff Leipprandt, Jianwei Xie and other members of the Haslam Lab (past and present)
who helped me with my project. Thanks to Dr. Rich Schwartz, Dr. John Fyfe, Dr. Karen
Frederici, Dr. Karl Olsen and members of the Breast Cancer Signaling Network consortium for
advice and technical assistance. Thanks to Dr. Thomas Saunders, Elizabeth Hughes and the
University of Michigan Transgenic Animal Model Core Facility for assistance with the PRAKO
and PRBKO mice. Thanks to staff of the University Laboratory Animal Resources for assistance
with mouse breeding. Thanks to Dr. Andrea Almalfitano, Sarah Roosa and members of the
Amalfitano Lab for assistance on generating adenovirus. Finally thanks to the support of all my
friends and family.

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TABLE OF CONTENTS

LIST OF TABLES ……………………………………………………………….….….……..…vi
LIST OF FIGURES ……………………………………………………………….….……...….vii
LIST OF ABBREVIATIONS …………………………………………………………………....ix
CHAPTER 1
LITERATURE REVIEW .…………………………………………………………...………….. 1
CHAPTER 2
MATERIALS AND METHODS ………………………………………………………………... 9
CHAPTER 3
RESULTS ……………………………………………………………………………………… 16
CHAPTER 4
DISCUSSION ………………………………………………………………………………….. 23
APPENDICES
APPENDIX A, Progesterone Receptor Isoform Knockouts …...……………………..... 30
APPENDIX B, Figures …...……………………………………………………………. 35
REFERENCES ………………………………………………………………………………… 67

v

LIST OF TABLES

TABLE 1
Quantitative RT-PCR analysis of fold change of Laminin-γ2 mRNA after knockdown
with Lmγ2 shRNA ……………………………………………………………………………... 66

vi

LIST OF FIGURES

FIGURE 1
Tubulogenesis of mammary organoids ....……………………………………………… 35
FIGURE 2
Time course of morphological changes induced by HGF……………………………… 36
FIGURE 3
The effect of Rho and Rac1 inhibition on HGF-induced tubulogenesis ……………….. 37
FIGURE 4
Rac1 inhibition reduces HGF-induced tubulogenesis ………...………………………... 38
FIGURE 5
Rac1 shRNA blocks HGF-induced tubulogenesis …………..……….………………….40
FIGURE 6
Src inhibition blocks HGF-induced tubulogenesis ………..…….……………………... 42
FIGURE 7
FAK inhibition blocks HGF-induced tubulogenesis ………………………………….... 44
FIGURE 8
The effect of MEK inhibition on HGF induced tubulogenesis ……………………….... 46
FIGURE 9
The effect of PI3K inhibition on HGF-induced tubulogenesis ………………………… 48
FIGURE 10
R5020 reduces HGF-induced tubulogenesis ………..………………………………….. 50
FIGURE 11
The effects of collagen I vs. laminin on HGF-induced tubulogenesis …………………. 52
FIGURE 12
R5020 induces Laminin-γ2 expression in mammary organoids ……………...………... 53
FIGURE 13
Laminin-γ2 shRNA reverses the effect of HGF+R5020 response in organoids …...…... 55
FIGURE 14
The effect of a neutralizing antibody against α6-integrin on the HGF+R5020 response in
organoids ……………………………………………………………………………….. 57
vii

FIGURE 15
Proposed mechanism for R5020-blunted tubulogenesis ……………………………….. 59
FIGURE 16
Strategy for developing PRA and PRB knockout Balb/c mice ……………………….... 61
FIGURE 17
Identification of PRAKO and PRABKO alleles in ES cells …………………………… 63
FIGURE 18
Identification of germline PRBKO allele …………………………………………….... 64
FIGURE 19
Timeline for generating PRAKO and PRBKO mice …………………………………... 65

viii

LIST OF ABBREVIATIONS

Amphiregulin (AREG)
Basal media (BM)
Collagen (Col)
Estrogen (E)
Epidermal growth factor (EGF)
Estrogen receptor (ER)
Extracellular matrix (ECM)
Fibroblast growth factor (FGF)
Focal adhesion kinase (FAK)
FAK inhibitor-14 (FAK-i14)
Growth factors (GF)
Hepatocyte growth factor (HGF)
Laminin (Lm)
Luminal epithelial cell (LEC)
Madine Darby canine kidney cells (MDCKs)
Map kinase inhibitor (U0126)
Myoepithelial cell (MEC)
Phosphoinositide-3 kinase (PI3K)
PI3K inhibitor (LY294002)
Progesterone/progestin (P)
Progesterone receptor (PR)

ix

Progesterone receptor A (PRA)
Progesterone receptor B (PRB)
PRA knockout (PRAKO)
PRB knockout (PRBKO)
Promogestone (R5020)
Rac1 inhibitor (NSC23766)
Rho kinase inhibitor (Y-27632)
Terminal end bud (TEB)
Transforming growth factor (TGF)
Src kinase inhibitor (PP2)

x

CHAPTER 1

LITERATURE REVIEW
Overview
In the mouse mammary gland, estrogen (E) and progesterone (P) regulate the epithelium
and stroma to influence ductal elongation, sidebranching and alveologenesis. From puberty to
sexual maturity, E stimulates growth factor (GF) production from stromal cells and epithelial
cells (1-4). GFs, in turn, act on the epithelium leading to ductal elongation and branching until
the ducts have reached the limits of the fat pad (5-7). In pregnancy, increases in E and P are
concurrent with sidebranching and alveologenesis. Although E is present, P is responsible for the
formation of sidebranches and alveoli (8). It is likely that crosstalk between E-dependant GFs
and P promotes sidebranching and alveologenesis. However, this interaction is not well
understood. Complicating this issue is that there are conflicting reports about the function of
progesterone receptor isoforms PRA and PRB in sidebranching.
In order to understand specific P and GF interactions, cell culture systems have been
useful. However, care must be taken when interpreting information from monolayer culture as it
does not represent the three-dimensional (3D) architecture of the gland. Cell signaling is likely
altered in such cases. To address this problem, cells cultured in 3D extracellular matrices are
used. In collagen gel culture, the mesenchymal factor hepatocyte growth factor (HGF) causes
tubule formation in primary mammary epithelial organoids (3, 9, 10). These structures resemble
mammary ducts observed in vivo. The synthetic progestin promogestone (R5020) stunts HGFinduced tubulogenesis to cause shorter tubes that resemble sidebranches (10). This response is
mediated through PRA in organoids from virgin adult mice (11) and is inhibited by the PR

1

antagonist RU486 (10). The mechanism of R5020-mediated blunting of tubulogenesis might
mimic a potential mechanism of sidebranching during early pregnancy. Therefore the aim of this
research is to understand the mechanism of action of P/PRA on tubule blunting and to determine
how P/PRA signaling influences HGF signaling to cause sidebranches in vitro.
Mouse mammary gland composition
The architecture of the mammary gland and the distribution of hormone receptors
highlight the complexity of crosstalk controlling development. In the epithelial component, an
inner layer of luminal epithelial cells (LECs) line the lumen of ducts while basal myoepithelial
cells (MECs) surround the LECs (12). Only a subset of the LECs are estrogen receptor (ER) and
progesterone receptor (PR) positive (2, 13, 14). Most proliferation occurs in hormone receptor
negative cells, indicating that paracrine signaling is responsible for hormone-induced
proliferation and differentiation of the gland (13-16).
A specialized extracellular matrix (ECM) rich in collagen, laminin and fibronectin
separates the epithelium from the stromal compartment of the gland. Both epithelial cells and
stromal cells deposit this basement membrane (17) and the composition is developmentally
regulated (18, 19). The role of the ECM is to regulate cellular organization and polarity
supporting integrity of the gland, and to regulate epithelial responsiveness to hormones (20, 21).
Regulation of the epithelium by the ECM may be through autocrine and paracrine mechanisms
and is mediated by integrins expressed on the surface of cells. In the mammary gland, the
composition of the ECM may be structure dependent. It has been noted that collagen I is
concentrated near the large main ducts while laminin expression appears near shorter branches
and alveoli (18). This implicates specific ECMs in directing the formation of distinct mammary
structures.

2

Finally, in the stroma, ER-positive cells produce essential growth factors necessary for
epithelium development (1, 3, 22). Any disruption between the epithelium, stroma and basement
membrane results in impaired mammary gland organogenesis, highlighting the importance of the
microenvironment in development of the gland.
Hormonal and Growth Factor influence on mouse mammary gland development in vivo
Ductal elongation and branching: the role of E/ER and HGF/cMet
At birth a rudimentary ductal system exists that grows minimally until the animal reaches
puberty. Despite the presence of hormone receptors the gland is non-responsive to steroid
hormones prior to puberty (23). At about 3-4 wks of age, with the onset of estrus cycles, E
stimulates ductal development. Terminal end buds (TEBs) develop and proliferate to elongate
the ducts. Ductal elongation and branching continues with each estrous cycle until the mouse
reaches sexual maturity. At this point, the ducts have reached the limits of the mammary fat pad.
It is known that E signaling through ERα is primarily responsible for this process (2).
Studies have indicated that E action on the epithelium is both direct and indirect (1, 2). E
stimulates both the epithelium and neighboring stromal cells to produce a number of GFs that act
on the epithelium to promote ductal elongation and branching. Epidermal growth factor (EGF),
amphiregulin (AREG), fibroblast growth factor (FGF), and transforming growth factor TGFβ
have all been identified as factors affecting proliferation and morphogenesis during ductal
development (6, 24-27).
Yet another E-dependent stroma-derived growth factor implicated in ductal branching is
HGF (5, 28-31). HGF action in the epithelium is mediated through the tyrosine kinase receptor,
cMet. When HGF binds, cMet activates a number of downstream pathways that affect
proliferation, cell survival, and motility (32). Expression of HGF and cMet during development

3

is coordinated. From puberty to sexual maturity, levels of HGF increase in the stroma while
cMet levels increase in the epithelium (33). This is concurrent with ductal elongation and
branching of the ducts. Recent evidence from mammary specific ablation of cMet suggests that
HGF/cMet signaling is important for secondary branching off of main ducts during development
(5). The role of cMet in the mammary gland during pregnancy was not reported for these mice.
However, during pregnancy, expression of HGF and cMet decline and expression is absent
during late pregnancy and lactation (33). This suggests that the HGF/cMet role in alveologenesis
and lactation is less important. Yet, a role in sidebranch formation during early pregnancy cannot
be dismissed.
Sidebranching: the role of P/PR
In the fully mature virgin gland and during pregnancy, E and P promote the second phase
of development, sidebranching and alveologenesis (34). Short branches form laterally off ducts,
and expand to form alveoli. During this time the level of estrogen receptor (ER) declines while
the level of progesterone receptor (PR) increases (35, 36) indicating that P/PR signaling is a
major contributor to this stage.
PR exists as two isoforms, PRA and PRB. The receptors are encoded within a single gene
under the control of two different promoters (37, 38). PRA and PRB are nearly identical with the
exception that PRB has an additional 164 amino acids at the N-terminus (37-39). The receptor
may follow a classical nuclear receptor signaling pathway to activate gene transcription (40).
Information from cell lines suggest that PRB is a strong activator of transcription while PRA has
a repressing function (40-43). Additionally, PR may activate non-genomic pathways through
interactions with Src in the cytoplasm (44-46).

4

The two isoforms function differently in mouse mammary gland development. Knockout
studies have shown that PRB is important for sidebranching and alveologenesis but that PRA is
non-essential(8, 47). However, it should be noted that the PR-isoform knockouts were in a mixed
genetic background of C57/black x 129SV, and that the genetic background of a mouse dictates
hormonal response. It was reported that adult C57/black mice have a reduced response to P
compared to the Balb/c strain of mice (48) and developmental studies in Balb/c mice suggest that
the role of PRA in mammary gland development might have been overlooked. Only PRA is
expressed in the gland in the virgin mouse during ductal development (14). In addition PRA is
the only isoform detected in early pregnancy, as sidebranches begin to appear (14). PRB is
detected later in pregnancy with the onset of alveologenesis (14). Furthermore, sidebranch
formation can be induced in ovariectomized virgin adult mice treated with P alone (49). Taken
together, this suggests that P signaling through PRA can promote sidebranching while PRB is
responsible for alveologenesis.
In vitro model of mammary gland branching
The mammary gland can be modeled in vitro by placing primary mammary epithelial
organoids in a 3 dimensional matrix. This can then become a platform for analyzing the
interaction between hormone and GF signaling. When primary organoids containing LECs and
MECs are placed in collagen gels and treated with HGF, the cells are able to form tubes (3, 10).
These tubules resemble ductal structures in the mouse prior to pregnancy. Interestingly, the
process of HGF-induced tubulogenesis can be modified with the inclusion of a synthetic
progestin, promogestone (R5020). This results in shorter tubules resembling sidebranches in the
pregnant mouse (10, 11). The combination of both HGF and R5020 is needed for this effect, as
R5020 alone promotes only lumen formation (10). Furthermore, R5020 blunting of HGF-

5

induced tubulogenesis was blocked by RU486, a PR-specific inhibitor (10) indicating that this is
a PR-specific signaling event. Analysis of PR isoform expression revealed that PRA is the
predominant isoform expressed in organoids (11). Therefore R5020 blunting of tubulogenesis is
likely mediated through PRA. The HGF signaling pathway(s) regulating tubule formation and
morphology in organoids is not known. Nor is the effect of R5020 on the morphology pathway
known. However, information from other tubulogenesis models can help identify potential
signaling mechanisms.
Tubulogenesis has been observed for other epithelial cells including those from kidney,
lung and salivary gland (50-52). Key information about tubulogenesis has been obtained from
Madine Darby canine kidney cells (MDCK; (53, 54). After HGF stimulation, epithelial cells send
out cytoplasmic extensions into the surrounding matrix. A single layer chain of cells invades
further, lengthening the growing tube. Following this period of tubule development, the
epithelial cells undergo a re-differentiation stage where they form bi-layered cords of cells and
finally mature tubules with complete lumens (54). This same sequence of events has also been
observed for HGF-treated mammary organoids (11). However, mainly LECs produce extensions
to lead tubulogenesis, while MECs follow (11).
Tubule length is determined by the distance epithelial cells migrate from the body of the
organoid, and this depends on changes in actin polymerization. Members of the Rho family
GTPases, Rac1 and Rho, regulate actin dynamics. Rac1 contributes to actin protrusions at the
leading edge of migrating cells and is implicated in extension and chain formation during
tubulogenesis (55-59). Rho controls the formation of stress fibers and focal adhesions at the rear
of migrating cells (55, 56). Rho contributes to later stages of tubule development leading to
mature tubules (57, 60). A number of critical pathways acting upstream of Rac1 and Rho for

6

tubulogenesis and cell migration have been identified including MAP kinase (MAPK; (54)),
phosphoinositide-3 kinase (PI3K; (60, 61), Src kinase (62, 63), and focal adhesion kinase (FAK;
(58). Though the signaling pathways driving mammary organoid tubulogenesis are not known,
those established in MDCK cells may also be relevant. Because R5020/PR signaling was shown
to blunt HGF-induced tubulogenesis in PRA-expressing mammary organoids (10, 11) it was
hypothesized that R5020 signaling through PRA reduced Rac1-GTP activity to inhibit extension
formation and blunt HGF-induced tubulogenesis. Therefore, the goal of this project was (1) to
determine the HGF-induced signaling pathway responsible for early morphological events during
tubulogenesis, (2) to determine the effects of R5020 on that pathway, (3) to determine the
mechanism of how R5020 alters the morphological pathway and (4) to generate PRA and PRB
knockout mice in Balb/c background to confirm the role of PRA in sidebranching in vitro and in
vivo.

7

CHAPTER 2

MATERIALS AND METHODS
Animals
Balb/c virgin adult females (16-24 week old) from our colony were used as the source of
mammary gland tissue for primary cell culture. Animal use was 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.
Cell Culture
Collagen gel 3-D Culture
For 3D collagen cultures, primary mouse mammary epithelial organoids were isolated as
previously published (11). Prior to plating cells, 96-well or 24-well culture plates were first
coated with an underlay of 40 µl/well or 250 µl/well, of neutralized rat tail collagen I (2 mg/ml,
BD Biosciences, Bedford, MA). For 96-well culture plates, the isolated organoids were
5

suspended in 2 mg/ml neutralized collagen I and plated at a density of 1 x 10 cells/well and a
total volume of 75 µL/well. For 24-well culture plates, organoids were suspended in the same
6

concentration of collagen but plated at a density of 1.4 x 10 cells/well and a total volume of 600
µL/well. The cell/collagen suspensions were allowed to set for 30 minutes at 37°C before
addition of media. Treatments were done in triplicates for 96-well culture plates, and in
quadruplicates for 24-well culture plates. Cultures were maintained in serum-free medium with
or without HGF or progestin (basal medium: serum- and phenol red-free DMEM/F12 (Sigma, St.
Louis, MO), supplemented with 100 ng/ml human recombinant insulin, 1 mg/ml fatty acid-free

8

BSA (fraction V), 100 µg/ml penicillin, and 50 µg/ml streptomycin. Treatments included 50
ng/ml HGF, (Sigma, St. Louis, MO), and 20 nM of the synthetic progestin, R5020, (Perkin
Elmer, Boston, MA) that were added at the time of plating. Organoid cultures were maintained in
5% CO2 at 37°C for up to 3 days and culture media was replaced every 48h.
For all inhibitor studies, 100 µM Rac1 inhibitor NSC23766 (Tocris Bioscience, Ellisville,
MO), 50 µM Rho kinase inhibitor Y27632 (Tocris Bioscience), 10 µM FAK inhibitor-14 (Tocris
Bioscience), 10 µM PI3K inhibitor LY294002 (Cell Signaling Technology, Danvers, MA), 10
µM MEK inhibitor U0126 (Cell Signaling Technology), or 20 µM Src inhibitor PP2 (Cell
Signaling Technology) were added to the cell/collagen suspensions prior to plating as well as
included in media to maintain a constant concentration. Cell viability was assessed after 24h and
48h of treatment. Organoids were labeled with Alexa Fluor 555 conjugated annexin V
(Invitrogen, Carlsbad, CA) as per manufacturer’s recommendation and cell viability was
assessed using a Nikon inverted epifluorescence scope (Mager Scientific, Dexter, MI).
Matrigel 3-D culture
Freshly isolated organoids were resuspended in growth factor reduced Matrigel (BD
5

Biosciences, Bedford, MA) and plated at a density of 1 x 10 cells/well in a 96-well plate. The
cell/Matrigel suspensions were allowed to set for 30 minutes at 37°C before addition of media.
Treatments were done in triplicates for 96-well culture plates. Treatments included BM, HGF,
R5020 or HGF+R5020 at the concentration indicated above that were added at the time of
plating. Organoid cultures were maintained in 5% CO2 at 37°C for up to 7 days and culture
media was replaced every 48h.
Monolayer Cell Culture

9

6-well plates were precoated with 50 µg/mL of collagen I or laminin-1 (BD Biosciences,
Bedford, MA) prior to plating of cells. Isolated primary mammary epithelial organoids were
6

plated as monolayer with 4 x 10 cells/well and cultured in BM for 48h to allow attachment of
the cells to the different ECMs. After 48h, media was replaced with fresh BM or BM containing
HGF. Cultures were then harvested after 4h of treatment using the Rac1 lysis buffer. The
supernatants were used for Rac1-GTP assay as outlined below.
Adenovirus Vector and Virus production
shRNA sequences targeting mouse Rac1, Laminin-5 (γ-2 subunit) and scramble
sequences were designed using shRNA Explorer™ program (Gene Link, Hawthorne, NY).
Design included 5’ Bgl II and 3’ Cla I overhangs that allowed subcloning into pSuper® vector
(Oligoengine, Seattle, WA; gift from Dr. Amalfitano) and placed the shRNA sequences under
the control of a pol II H1-promoter. The resulting H1-shRNAs were subsequently removed from
pSuper® with Sal I and BamH I restriction enzymes and placed within pShuttle-IRES-hrGFP®
vector (Agilent Technologies, Santa Clara, CA; gift from Dr. Miranti) creating an H1-shRNACMV-GFP vector. This was linearized using Pme I and recombined with pAdEasy® (Agilent
Technologies - Gift from Dr. Amalfitano) in BJ-5183 bacterial cells. Adenovirus was produced
and purified as described in Luo et al., (64). Briefly, the adenovirus vectors were used to
transfect HEK293 cells. After several serial passages in HEK293 cells, amplified virus was
purified by sequential CsCl2 gradient centrifugations. The purified virus was dialyzed against
10mM Tris (pH 8.0) and stored at -80°C in 1% sucrose, 1x PBS until use. The tissue culture
9

11

infectious doses (TCID50) were in a range of 10 -10

10

infectious particles/ml.

Knockdown of proteins by adenovirally delivered shRNA
Freshly isolated organoids were infected in suspension in BM with adenovirus (MOI 50)
carrying GFP-vector, shRNA vectors, or scramble vector with gentle agitation for 3.5h at 37°C.
The infected organoids were centrifuged at 200 x g for 5 min. and the viral supernatant was
removed. Organoids were then washed with 1xHBSS, and resuspended in basal media (BM).
Total cell numbers were determined by Crystal Violet staining as previously described (11) and
viability was analyzed by Trypan Blue exclusion. Organoids were plated in collagen gel as
described before and maintained in BM prior to treatment. To determine infectivity, GFP
expression was analyzed using a Nikon inverted epifluorescence scope (Mager Scientific, Dexter,
MI) 24h post infection. 80-90% infection was obtained. Cell viability was assessed 24h and 48h
after infection by annexin V labeling as was described above. For Rac1 knockdown, cultures
were cultured in BM at for 48h to achieve knockdown of proteins, as analyzed by
immunoblotting. For Laminin-γ2 (Lmγ2) knockdown, organoids were maintained in BM for 24h
and knockdown was analyzed by quantitative RT-PCR. Following knockdown, cultures were
treated with BM, HGF or the combination of HGF and R5020. Cultures were maintained for 3
days changing the media after 48h as before.
Morphometrics
Images of primary mammary organoids were captured with a 20x lens using a Nikon
TMS-F inverted scope (Nikon, Melville, NY) equipped with a Q imaging Micropublishing 5.0
RTV camera and Qcapture pro software (QImaging Corporation, Surrey, B.C., Canada). Images
were analyzed for extension and tubule number per organoid with ImageJ software (National
Institutes of Health, Bethesda, MD). The width and length of projections from the organoid body
were used to classify structures into extensions, chains and tubes. In the α6-integrin blocking

11

antibody experiments, structures longer than the average tubule length (>140 pixels) were
considered long tubules. For shRNA cultures, images of organoids expressing 80-90% GFP
were captured using a Nikon inverted epifluorescence scope with 10x lens (Mager Scientific,
Dexter, MI) and analyzed with MetaMorph software. Quantitation of extension and tubule
number per organoid was determined from 45 or more organoids per treatment from three or
more separate experiments. For Lmγ2shRNA experiments, structures longer than the average
tubule length (>50 pixels) were considered long tubules. The results are reported as the mean +
SEM, and differences are significant at P< 0.05 with ANVOA.
Rac1-GTP assay
Rac1-GTP assays were performed following a modified method from Yamazaki et al.,
2009. Collagen gels containing organoids were homogenized in Rac1 lysis buffer (50mM Tris
pH 7.5, 150mM NaCl, 5mM MgCl2, 1% NonidetP-40, 0.5% deoxycholate, 1mM Na3VO4, 0.1%
SDS, protease inhibitor cocktail; Sigma, St. Louis, MO). Lysates were cleared by centrifugation
and the protein supernatant was reserved. Small aliquots of the supernatants were set aside for
total Rac1 and Erk 1/2 immunoblots. The remaining supernatants were used for Rac1 pull down
assays using PAK-PBD protein agarose beads (Cytoskeleton, Denver, CO) as per manufacturer’s
recommendation. Beads were washed in 1 x PBS and resuspended in NuPAGE Sample Reducing
Agent and Loading dye (Invitrogen, Carlsbad, CA) then heated for 10 min at 70°C. Denatured
beads and proteins were separated on a 4-12% Bis-Tris NuPAGE gel (Invitrogen) then
transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was
blocked in Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NB) diluted in 1 x TTBS
(1:1), and incubated overnight in anti-Rac1, clone 23A8 antibody (Millipore, Billerica, MA,
1:1000 dilution), and anti-Erk 1/2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000
12

dilution) for a loading control. Membranes were washed with 1 x TTBS and incubated for 1h
with 1:5000 dilutions of IRDye® 680 or IRDye® 800 secondary antibodies against mouse or
rabbit (Licor Biosciences). Membranes were scanned using an Odessey scanner (Licor
Biosciences) and densitometric measurements of protein bands were assessed using ImageJ
(National Institutes of Health, Bethesda, MD) to determine the ratio of Rac1-GTP to total Rac1.
Since actin levels changed with treatments and Erk1/2 did not change, Erk1/2 level was used as a
loading control.
Immunoblotting
Protein supernatants were prepared as outlined above and were directly used for western
blotting of total and phosphorylated proteins using the following antibodies: pAKT 1/2/3 (Ser
473, 1:200 dilution), PI3 Kinase p85α (B9, 1:500 dilution), pErk (E-4, 1:200 dilution), Erk 1 (C16, 1:1000 dilution), Lamininγ-2 (Lmγ2; c-20, 1:200 dilution) and FAK (A-17, 1:1000 dilution)
from Santa Cruz Biotechnology, Santa Cruz, CA. The pFAK (pY397, 1:1000 dilution) antibody
was from Invitrogen (Carlsbad, CA). The pSrc (Y416, 1:200 dilution) and Src (1:1000 dilution)
antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA). For phospho-FAK,
p-Tyr proteins were immunoprecipitated using pTyr antibody (PY99, Santa Cruz Biotechnology,
Santa Cruz, CA) and an immunoblot was performed to detect pFAK. Erk1/2 was used as a
loading control for all immunoblots. IRDye® 680 or IRDye® 800 secondary antibodies against
mouse, rabbit or goat were used at 1:5000 dilutions. Erk1/2 level was used as a loading control
and protein levels were normalized to Erk 1/2.
Antibody labeling
The procedure for antibody labeling of organoids in collagen gels was previously
described (Haslam et al., 2008). Briefly, gels containing organoids were washed with PBS+

13

(PBS, 1 mM CaCl2, 0.5 mM MgCl2; ph 7.2). Gels were fixed in 4% paraformaldehyde in PBS+
and pearmeablized with P buffer (PBS+, 0.025% saponin). Gels were then quenched with Q
buffer (PBS+, 75 mM NH4Cl, 20 mM glycine) and blocked 10 min in B buffer (PBS+, 0.025%
saponin, 0.3% gelatin). Gels were incubated overnight at 4°C with the goat polyclonal Lmγ2
antibody and a mouse monoclonal antibody against cytokeratin-18 (K18; ab668-100-Abcam,
Cambridge, MA) diluted 1:200 in B buffer. Gels were washed with P buffer and incubated for 1h
with rabbit-anti-goat biotin (Dako, Denmark) diluted 1:400 in B buffer. Gels were rinsed with
PBS+ and incubated overnight at 4°C with goat-anti-mouse Alexa 488 and streptavidin-Alexa
546 (Molecular Probes, Eugene, OR) diluted 1:200 in B buffer. Samples were washed P buffer
then post-fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and
mounted to slides with fluorescence mounting media. A Pascal laser scanning confocal
microscope (Carl Zeiss Inc., Thornwood, NY) was used to capture images. A 3-D Z plane series
with a step size of 4 µM was used to generate each image.
RT-PCR
For RT-PCR of Lmγ2, organoids were infected and maintained in BM for 24h, then
treated with HGF or HGF+R5020. 24h later, gels were removed and RNA was extracted using
the Trizol method, described previously (65). cDNA was prepared from isolated RNA using
2

random hexamer primers from RT First Strand Kit (Qiagen, Valencia, CA) per manufacturer’s
instructions. A 7500 FAST RT-PCR System (Applied Biosystems, Carlsbad, CA) was used for
2

RT-PCR. Gene expression assays were performed in triplicate with RT SYBR Green ROX
qPCR Mastermix (Qiagen) and prevalidated primers for Lmγ2 (LAMC2) and GAPDH. The

14

expression values were normalized to GAPDH and the Comparative Ct Method was used to
determine the change in gene expression.
Blocking antibody cultures
Isolated primary organoids were pretreated in BM suspension with 10 µg/mL of GoH3 or
IgG2a Isotype control (Becton-Dickinson, Franklin Lakes, NJ) for 30 min. Organoids were
plated in collagen I containing 10 µg/mL GoH3 or IgG2a, and allowed to gel for 20 min prior to
addition of HGF or HGF+R5020. Organoids were maintained as described above.
Statistical Analysis
All experiments were repeated up to 8 times. Values are presented as mean ± S.E.M.
Statistical significance was determined by Student’s t-Test or ANOVA using SYSTAT
(SYSTAT Software Inc., Chicago, IL) as appropriate and results considered significant at p <
0.05.

15

CHAPTER 3

RESULTS
HGF induces extensions to initiate tubulogenesis in mammary organoids by Rac1
activation
It was previously reported that organoids treated with HGF form duct-like tubules by 72h
and that the addition of R5020 blunts tubule formation (10). As was described, LECs produce
extensions that lead to tubulogenesis (11). The extensions progress to form chains of cells, cords
and mature tubules (FIGURE 1). The blunting of tubulogenesis is hypothesized to be relevant to
the mechanism through which progesterone promotes sidebranch development. It is not known
when the blunting effect of R5020 occurs in the development of tubules. Therefore, as a first step,
a time course of HGF-induced tubulogenesis was analyzed in organoids from 0-24h (FIGURE 2
A-B). When first plated, organoids have a rounded morphology. By 4h, HGF increased the
number of cytoplasmic extensions. By 24h, chains and tubules were observed in HGF-treated
organoids.
The pathway responsible for initiating tubulogenesis in organoids is not known and
identifying this pathway can define where R5020 might act to blunt the HGF response. Rho
GTPases Rac1 and Rho have been reported to regulate cell morphology in the MDCK model of
tubulogenesis (57). To determine the roles of Rac1 and Rho in mammary organoid tubulogenesis,
organoids were treated for 24h with HGF in the presence of the Rho kinase (ROCK) inhibitor,
Y-27632, or the Rac1 inhibitor, NSC23766 (FIGURE 3 A). By 24h, ROCK inhibition did not
prevent the formation of extensions or chains in HGF-treated organoids. After 48h, mature
tubules were unable to form and migrating cells pinched off the organoid body (data not shown).

16

Similar results have been published for MDCK cells treated with Y-27632 (60).This suggests
that Rho signaling regulates tubule maturation, the second stage of tubulogenesis. In contrast,
Rac1 inhibition significantly reduced HGF-induced formation of chains and tubules by 24h
(FIGURE 3 A, FIGURE 4 B). This suggests that Rac1 activity promotes extension and chain
formation in the early steps of tubulogenesis. Consistant with this observation, HGF significantly
increased levels of Rac1-GTP at 4h (FIGURE 3 B) when extensions were first observed. Though
24h treatment with the Rac1 inhibitor dramatically reduced chains and tubules, 4h treatment did
not reduce extension number or Rac1-GTP (FIGURE 4 A-C). This indicates that a longer
exposure time to the inhibitor was necessary to block tubulogenesis.
The observation that HGF increased Rac1-GTP during extension formation indicated that
Rac1 could contribute to extension formation. As an alternative method to the Rac1-GTP
inhibitor, Rac1 was silenced in organoids with shRNA to determine the role of Rac1 in extension
formation. Two separate adenoviruses were created that contained a CMV-GFP cassette and
shRNA against Rac1. The presence of GFP (GFP+) allowed infected and shRNA-producing
cells to be differentiated from non-infected cells (GFP-). 48h after infection, organoids were
treated with BM or HGF for 4h. Total Rac1 levels were assessed after 4h treatment and GFP+
extensions, chains and tubules were measured over 24h (FIGURE 5 A-C). Organoids infected
with vector alone or scramble shRNA were able to make GFP+ and GFP- extensions, chains and
tubules. The only extensions, chains and tubules that were able to form in shRNA treated
organoids were GFP-. The absence of GFP indicated that cells in these structures were
uninfected and thus not likely to contain Rac1 shRNA. There were virtually no GFP+ extensions
in the shRNA treated organoids at 4h. By 24h, the number of GFP+ extension, chains and
tubules were significantly reduced by 24h. Western blot confirmed that total Rac1 was reduced

17

by shRNAs (FIGURE 5 C).Taken together, these data confirmed a role for Rac1 in extension,
and chain formation during HGF-induced tubulogenesis.
HGF-induced Rac1-GTP in mammary organoids is Src and FAK dependant
Src, FAK, PI3K, and MapK have all been reported to regulate Rac1, tubulogenesis and
cell migration in other models (54, 58, 60-63, 66). Therefore, molecular inhibitors targeting Src,
FAK, PI3K or MEK signaling were used to determine the regulator(s) of HGF-induced Rac1 in
organoids. When organoids were treated with HGF in the presence of the Src inhibitor (PP2),
extensions were significantly reduced compared to HGF-controls (FIGURE 6 A-B). By 24h, the
Src inhibitor significantly reduced the number of chains and tubules. Furthermore, Src inhibition
significantly reduced the level of Rac1-GTP at 4h compared to the HGF-control (FIGURE 6 C).
This indicates that Src signaling downstream of HGF/cMet regulates Rac1 activity and extension
formation in organoids. Similar results were observed with the FAK inhibitor (FAK inhibitor-14).
After 4h, FAK inhibition significantly reduced extension formation (FIGURE 7 A-B). Chains
and tubules were also significantly inhibited after 24h. In addition, FAK inhibition significantly
reduced the level of Rac1-GTP compared to the HGF control (FIGURE 7 C). However,
phosphorylated FAK levels were too low to be detected by immunoblot to confirm inhibition. In
contrast to Src and FAK inhibition, when organoids were treated with HGF in the presence of the
MEK inhibitor (U0126; FIGURE 8 A-C) or the PI3K inhibitor (LY294002; FIGURE 9 A-C)
there was no significant inhibition of extensions and chains by 4 and 24h. In addition, the MEK
and PI3K inhibitors did not significantly reduce the level of Rac1-GTP. The MEK inhibitor did
reduce tubule formation by 60% (FIGURE 8 A-B) whereas the PI3K inhibitor did not (FIGURE
9 A-B). This is not surprising since sustained Erk activity was necessary for tubulogenesis in
MDCK cells (54, 59). Although MEK signaling is important for tubulogenesis, results presented

18

here suggest that MEK signaling may not be essential for 4h activation of Rac1 or extension
formation in organoids. These data indicate that Src and FAK mediate 4h Rac1 activity and
extension formation in organoids.
Promogestone (R5020) reduces HGF-induced extensions and Rac1-GTP in mammary
organoid tubulogenesis
To determine when R5020/PR signaling reduces tubulogenesis, the timing of the R5020
effect on tubulogenesis was analyzed (FIGURE 10 A-B). At 4h, R5020 significantly decreased
the number of HGF-induced extensions. By 24h, HGF- and HGF+R5020-treated organoids had
similar numbers of chains. However, the inclusion of R5020 significantly decreased the number
of HGF-induced tubules. These results reveal that the R5020 effect on tubulogenesis in
mammary organoids occurs by 4h.
The reduced extensions observed in HGF+R5020-treated organoids suggested Rac1-GTP
might also be reduced. Indeed, HGF+R5020-treated organoids had lower Rac1-GTP than HGFtreated organoids (FIGURE 10 C). Because Src and FAK were necessary for Rac1-GTP and
extension formation in tubulogenesis, the levels of phospho-Src and phospho-FAK were also
analyzed. There was a trend for reduced phospho-Src and phospho-FAK in HGF+R5020-treated
organoids. (FIGURE 10 D). However, the levels of phospho-Erk1/2 and phospho-AKT did not
appear to be affected by R5020. These data suggest that R5020 decreases HGF-induced
extensions through reduced activation of Src, FAK and Rac1
R5020-regulated Laminin-5 reduces HGF-induced Rac1-GTP and tubulogenesis
Next, we investigated how R5020 reduced Src, FAK and Rac1 activity after HGF
treatment. An important contributor to tubulogenesis is the ECM which can influence a cell’s
response to hormones and GFs. It was reported that MDCK cells embedded in collagen I (Col I)

19

form tubules in response to HGF, but do not when embedded in laminin (Lm)-1 rich Matrigel
(67, 68). In previously published microarray analysis of mouse mammary organoids treated with
R5020, Lmγ2 (subunit of Lm-5) mRNA was upregulated (65). Also MCF7 human breast cancer
cells cultured on Lm-1 have reduced Rac1-GTP compared those cultured on Col I (69). This led
to the hypothesis that laminin acts as an R5020-induced paracrine factor to reduce Rac1-GTP
level and extension formation in organoids undergoing tubulogenesis.
First, to determine if laminin could block tubulogenesis of mammary organoids,
organoids were cultured in Matrigel and treated with BM, HGF, R5020 or HGF+R5020
(FIGURE 11 A). Compared to organoids in Col I, organoids in Matrigel were unable to make
tubules. This was observed even after 7 days of treatment with HGF (data not shown). Thus, a
laminin-rich environment has an inhibitory effect on HGF-induced tubulogenesis. To determine
if laminin could alter HGF-induced Rac1-GTP, primary mammary epithelial cells were cultured
on Col I or Lm-1 coated plates and treated with HGF for 4h (FIGURE 11 B). HGF failed to
increase Rac1-GTP in cells cultured on Lm-1 compared to cells on Col I. These results suggested
that altered composition of the ECM can modulate the HGF-response.
Elevated Lmγ2 protein level in response to R5020 treatment was then verified by
immunoblot of organoid extracts and in-gel antibody labeling of intact organoids after 4h and
24h treatment (FIGURE 12 A-C). Lower levels of Lmγ2 were observed for HGF-treated
organoids. The combination of HGF and R5020 resulted in an intermediate level of Lmγ2
(FIGURE 12 B-C). These results are consistent with our previous report that R5020 induced
Lmγ2 mRNA expression and indicates that R5020 signaling might alter the ECM composition.
To determine if R5020-increased Lmγ2 is a paracrine factor responsible for stunted
tubulogenesis, organoids were infected with adenovirus to deliver a GFP+ Lmγ2-shRNA vector

20

or GFP+ Scramble control. Following knockdown, organoids were treated with HGF or
HGF+R5020 and the effect on morphology and Rac1 activity was assessed (FIGURE 13 A-D).
Compared to HGF+R5020 controls, organoids that had been infected with Lmγ2-shRNA were
able to produce significantly more tubules at 24h suggesting that extensions were also increased.
However, an increase of extensions at 4h was not observed. It is likely that infection with
adenovirus alters the tubulogenesis time-course of mammary organoids. The percentage of
organoids with long tubules was higher in Lmγ2-shRNA treated organoids compared to the
HGF+R5020-treated controls. In addition, there was a trend for increased Rac1-GTP and
phospho-Src in Lmγ2-shRNA treated organoids compared to HGF+R5020 controls. Quantitative
RT-PCR was performed to verify knockdown of Lmγ2 mRNA, and showed a 3-fold reduction of
Lmγ2 mRNA compared to scramble controls (Table 1). Taken together, these results suggest that
Lm-5 is an R5020-mediated paracrine factor that inhibits HGF-induced tubulogenesis through
inhibition of Src and Rac1.
Lm-5 signals through α6β1 and α6β4 integrins. As further support that Lm-5 negatively
regulates tubulogenesis in mammary organoids, α6-integrin was blocked using a neutralizing
antibody (FIGURE 14 A-D). The organoids were then treated with HGF or HGF+R5020 and the
effect on tubulogenesis was analyzed. There was a trend for increased number of extensions,
chains and tubules after blocking α6 integrins in HGF+R5020 treated organoids. In addition, it
appeared that a higher percentage of HGF+R5020-treated organoids were able to produce long
tubules in the presence of the α6-integrin blocking antibody compared to HGF+R5020 controls
at 24h. This suggested that blocking α6-integrin might also prevent the R5020-blunting effect of
tubulogenesis. Furthermore, there was a trend for increased Rac1-GTP and phospho-Src after
blocking α6-integrin in HGF+R5020-treated organoids. These results are consistent with the

21

interpretation that R5020 upregulates Lm-5/α6-integrin signaling which appears to have an
inhibitory effect on Src and Rac1 resulting in blunted extension formation and tubulogenesis.

22

CHAPTER 4

DISCUSSION
It is known that P is important for sidebranching and alveologenesis in development of
the mammary gland. Yet, the mechanism of how P mediates sidebranching is not well
understood. Using a 3D cell culture model of mammary gland ductal development and
sidebranching, it was determined that HGF/c-Met activates Src, FAK and Rac1 pathway to
promote extension formation, the first step in in vitro ductal elongation. It was found that
activation of this pathway is reduced by R5020 to produce fewer extensions and stunted tubule
formation, representing shortened ducts of sidebranches. In addition, it was determined that this
is mediated by R5020-induced changes in laminin expression.
HGF activates a Src/FAK-Rac1 pathway to affect early tubule morphology
As a first step to identifying how R5020 blunts the tubulogenic response of organoids to
HGF, the key pathway controlling early steps in tubule formation was determined. It was
observed that HGF induced extension formation, the first step of tubulogenesis, occurred by 4h,
and that Rac1-GTP was elevated at this time. Through the use of shRNA and an inhibitor against
Rac1, the GTPase was identified as a regulator of extension and chain formation. For MDCK
cells, extension formation and tubulogenesis were mediated through Rac1 signaling (57). It
appears that mammary organoids, composed of two cell types, behave similarly to MDCKs in
tubulogenesis. We have previously reported that LECs rather than MECs form extensions (11).
Therefore it is likely that Rac1-induced changes during extension formation is occurring mainly
in the LECs.

23

Using specific inhibitors, Src and FAK were identified as key regulators of Rac1 activity
and extension formation in tubulogenesis. Although HGF-induced Src had been implicated in
motility and proliferation of mouse mammary carcinoma cells (62), a role for Src in
tubulogenesis of normal mouse mammary cells had not been established. Inhibition of Src
reduced Rac1-GTP levels and severely impaired extension formation, suggesting that Src is
upstream of Rac1 in tubulogenesis. Inhibition of FAK resulted in a similar reduction of
extensions and Rac1-GTP. FAK and Src have been documented to cooperate at focal adhesions
and to mediate both integrin and RTK signlaling (70). Therefore, it is likely that both kinases
interact upstream of Rac1 to mediate extension formation of organoids. Interestingly, a role for
Src in tubulogenesis in MDCK cells has not been reported. In contrast, MEK and PI3K mediate
cell migration and tubulogenesis of MDCK cells (54, 60, 61, 71). However, MEK and PI3K
inhibition failed to significantly reduce Rac1-GTP levels and extension formation in the present
study of mammary organoids. It is probable that these kinases contribute to later stages of
tubulogenesis of mammary organoids but are nonessential for Rac1-induced extension
formation.
R5020 reduces the Src-Rac1 pathway and extension formation
R5020 reduced the number of extensions, the early stage of tubulogenesis, and decreased
the level of Rac1-GTP in organoids treated with HGF+R5020. Furthermore, R5020 reduced the
level of phosphorylated Src and FAK. Therefore it is likely that R5020 blunts HGF-induced
tubulogenesis through inhibiting activation of the Src/FAK-Rac1 pathway. This early effect of
R5020 contributes to the ultimate blunted morphology reported previously (10). To the best of
our knowledge, this is first evidence of progestin regulating Rac1 to control tubulogenesis of
primary mammary organoids.

24

R5020 increases Lm-5 expression to decrease HGF-induced tubulogenesis
The role of ECM composition was investigated based on information that (1) MDCK
cells in laminin-rich Matrigel do not form tubules in response to HGF (67, 68), (2) that R5020
signaling induces expression of laminin mRNA in organoids in collagen (65), and (3) that breast
cancer cells have reduced Rac1-GTP when cultured on Lm-1 (69). In the present study, it was
found that Matrigel inhibited HGF-induced tubulogenesis and that Lm-1 reduced Rac1-GTP
level in organoids. In addition, it was observed that R5020 increased expression of Lmγ2
(subunit of Lm-5) in mammary organoids. When Lmγ2 was silenced, Rac1-GTP, tubule length
and number were restored, indicating that Lm-5 is an R5020-induced paracrine factor that
negatively regulates tubulogenesis. Blocking α6-integrin also restored Rac1-GTP and
tubulogenesis in HGF+R5020-treated organoids, further confirming the role of Lm-5 signaling.
Taken together, the present findings suggest that Lm-5/α6-integrin signaling is a negative
regulator of tubulogenesis and Rac1-GTP, and that Lm-5/α6-integrin signaling contributes to
shorter tubules observed in HGF+R5020 treated organoids. Since the adult mammary gland that
was the source of our organoids express only PRA (11) we postulate that the effects of R5020
are mediated through PRA. These findings lead us to propose the following model of mammary
organoid tubulogenesis and sidebranching (FIGURE 14). Under HGF treatment and increased
Rac1-GTP, LECs in collagen I become motile and begin migration outwards to form tubules.
However, in the presence of R5020, PRA upregulates Lm-5 and blunts the tubulogenic response
to HGF through an Lm-5/α6-integrin and Rac1 mediated pathway. We interpret blunting of
tubulogenesis to be a surrogate for the side branching response induced by P in vivo.
Non-genomic signaling of progestin in tubulogenesis

25

The effects of R5020 mediated by PRA are likely occurring through genomic signaling
since RU486 blocks the R5020 inhibitory effect on tubulogenesis (10). However, it is important
to note that other mechanisms might be at play in organoid sidebranching. One possibility is that
R5020 might initiate a non-genomic PRA pathway in addition to a classical genomic pathway.
There is evidence from human breast cancer cell lines that non-genomic signaling of PR
influences activity of Src. PRB has been shown to interact with the SH3 domain of Src leading to
activated MAPK signaling and higher proliferation of T47D cells (45, 72). In contrast to what
was observed in breast cancer cells, R5020/PRA reduced the level of phosphorylated Src in
mammary organoids. This difference might be a reflection of the lack of significant PRB in the
adult virgin mouse mammary gland compared to the co-expression of PRA and PRB in human
normal and cancerous breast cells. Additional differences might be due to culture of mammary
organoids in collagen vs. human cancer cell lines on plastic.
Conclusion
In summary, we identified an ECM/integrin and HGF/cMet activated Src/FAK-Rac1
pathway that mediates extension formation as an early step of tubulogenesis in mammary
organoids in vitro. This pathway was inhibited by R5020 and resulted in blunted tubulogenesis
believed to be analogous to the sidebranching response to P in vivo. A potential mechanism was
highlighted through which R5020 acting through PRA increases expression of extracellular
laminin to blunt tubulogenesis. PRA has been considered by some to be non-essential in
mammary gland development. However, in vivo studies of its expression during development
(14, 49) and the findings presented here suggest that it may play an important role in
sidebranching.

26

APPENDICES

27

APPENDIX A

PROGESTERONE RECEPTOR ISOFORM KNOCKOUTS

28

Introduction
The question of PRA function in mammary gland development has not been fully
resolved. Isoform specific PR knockout mice revealed that PRB contributes to alveologenesis
while PRA is important for normal uterine and ovary function (8, 47). However, the isoform
knockouts were generated in the mixed genetic background of C57BL/6 x 129SV. C57BL/6
strain is known to have a delayed mammary gland development and reduced progesterone
response compared to the Balb/c strain that is often used in studies of the gland (48, 73).
Developmental studies from Balb/c suggest there might be a role for PRA in sidebranch
development (11, 14, 49). Therefore, a role for PRA in mammary gland development may have
been missed in the PRA knockout in C57BL/6 context. To address this, Balb/c DNA constructs
for PRA-specific and PRB-specific knockouts were designed. C-Terminal epitope tags were
included in the design to facilitate easy detection of PRA or PRB in developmental studies.
Materials and Methods
PRA- and PRB-knockout design
Targeting vectors were generated to create PR isoform specific mini genes with epitope
tags. Vectors contained 5.5 Kb genomic Balb/C DNA with the PR promoters and exons 1-2. Site
directed mutagenesis was used to mutate the separate PR initiation sites from ATG (Met) to
GCG (Ala) for the PRAKO or CTG (Leu) for the PRBKO. A loxP-flanked PGK-Neo selection
cassette (gift from Dr. Pam Swiatek) was inserted within intron 1. An HA tag or Flag tag was
included at the C-terminal end of PR cDNA encoding exons 3-8. The HA-tagged cDNA was
then added to exon 2 of the PRAKO vector while the Flag-tagged cDNA was added to exon 2 of
the PRBKO vector. An SV40 polyadenylation sequence was included after the tagged cDNA and
a 3’ homologous recombination arm was added that consisted of 5 Kb of intron 2.

29

Recombination of the constructs with wild type PR DNA would replace the PR promoters and
exons 1-2 with an isoform specific tagged mini PR gene, interrupting the wild type gene
(FIGURE 15).
The PRAKO and PRBKO constructs were submitted to the University of Michigan
Transgenic Animal Model Core (TAMC) for electroporation into Oz-Balb/C embryonic stem
(ES) cells. G418 resistant clones were isolated by the TAMC. ES cell DNA received from the
TAMC was analyzed by Southern blotting and PCR to identify PRAKO and PRBKO
recombinants.
Southern blot and PCR analysis of recombinant ES cells
Two Bcl I restriction sites flank the endogenous PR gene resulting in a 15 Kb fragment
when the DNA is digested with the restriction enzyme. Recombination with the PR mini genes
introduces a two additional Bcl I restriction site within the Neo cassette and the cDNA portion of
the constructs, resulting in 7.4 Kb, 3.6 Kb and 7.8 Kb fragments. A 510 bp 5’ DNA probe was
generated upstream of the 5’ recombination site, and a 448 bp 3’ DNA probe was created
downstream of the 3’ recombination site. The 5’ and 3’ probes recognize the 7.4 Kb and 7.8 Kb
fragments, respectively.
ES DNA on 96-well plates was digested overnight with Bcl I (20 U; Roche Diagnostics,
Indianapolis, IN) and RNase I (50 µg/mL) in a humidified chamber. DNA was separated by
electrophoresis overnight on a 0.8% agarose gel. DNA within the gel was depurinated in 0.25 N
HCl for 15 minutes, rinsed in ddH2O, and denatured in 0.5 N NaOH for 30 minutes. DNA was
transferred in 10x SSC (1.5 M sodium chloride, 1.5 M sodium citrate, pH 7.0) by vacuum
blotting to Hybond-N nylon membrane (GE-Amersham, Piscataway, New Jersey) using 785
Vacuum Blotter (BioRad, Hercules, CA) at 5 inches Hg. The membrane was washed with 2x

30

SSC and baked 1 hour at 65°C and wetted with 2x SSC and pre-hybridized with QuckHyb
hybridization solution (Stratagene, La Jolla, CA) 30 minutes at 65°C prior to hybridization.
32

DNA probes were labeled with (α- P)dCTP using Random Primer Labeling Kit as per
manufacturers recommendation (Invitrogen, Carlsbad, CA). Labeled probes were purified using
Chroma Spin columns (Clonetech, Mountain View, CA). Probes were boiled with 10 mg/mL
sonicated herring sperm DNA, and added to QuickHyb hybridization solution. Probes were
hybridized to the membrane overnight at 55°C. The membrane was washed the following day
twice for 15 minutes at room temperature with 2x SSC and 0.1% SDS and washed once for 30
minutes at 55°C with 2x SSC and 0.1% SDS. Membranes were exposed to a PhosphorImaging
screen (GE Healthcare Biosciences), and scanned using a Storm Molecular Imager (GE
Healthcare Biosciences). Because of difficulties in analyzing the 3’ recombination event in
PRAKO clones by Southern blots, PCR analysis was used to confirm the presence of the HA
epitope tag.
Generation of chimeric mice
Recombinant ES cells were expanded by the TAMC and chromosomes were counted to
verify euploid chromosome numbers. Clones were injected into C57BL/6NCrl x (C57BL/6J X
DBA/2J) blastocysts by the TAMC resulting in PRAKO- and PRBKO- chimeric mice. Chimeric
mice were put into breeding with Balb/c mice to generate Balb/c PRAKO +/- and PRBKO +/mice.

31

PCR Genotyping
For screening the 5’ end of the PRAKO and PRBKO allele, primers were generated
flanking the start sites. PCR generates a 1289 bp product for both wild type (WT) and mutant
alleles. Following PCR amplification, the products are digested with Bsp HI to distinguish WT
allele from mutant alleles. For screening the 3’ end of the PRAKO and PRBKO allele, primers
were designed to amplify a region of intron 2. The primers flank the insertion site of the SV40
polyadenylation signal. The size of the PCR product was used to distinguish the WT allele from
mutant alleles.
Results
PRA and PRB are encoded within a single gene containing two promoter sites. In order to
create PRA knockout and PRB knockout Balb/c mice, targeting vectors were generated that
introduce a PRA-specific or PRB-specific mini-gene into the WT PR locus (FIGURE 15). The 5’
recombination arm consisted of the Balb/c PR promoter through exon 1. The ATG sites for PRA
or PRB were mutated so that only one isoform would be expressed. A loxP-flanked Neomycin
(Neo) selection cassette was included within intron 1 for selection of recombinant ES cells.
Epitope tagged cDNA encoding exons 3-8 was fused to exon 2 to complete the PR mini-genes.
Presence of an HA- or Flag-epitope tag will make it easy to identify PRA or PRB protein in
future PRAKO or PRBKO mice. Finally, the 3’ recombination arm consisted of DNA from
within intron 2 and included an SV40 polyadenylation signal. These targeting vectors should
replace the WT promoter and exons 1-2 and interrupt the WT PR gene after homologous
recombination. The endogenous PR exons 3-8 remain.
After the targeting vectors were introduced into OZ-Balb/c ES cells, Neo-containing ES
cells were screened by Southern blotting and PCR analysis to identify correct recombinants. 2

32

PRA knockout ES clones and 3 PRB knockout clones were identified by Southern blot analysis
(FIGURE 16). These were validated by PCR analysis using primers specific for the epitope tags.
These clones were injected into C57BL/6NCrl x (C57BL/6J X DBA/2J) blastocysts resulting in
14 PRBKO chimeric mice and 11 PRAKO chimeric mice. The chimeras were put into breeding
with wt Balb/c and germline PRB-/+ Balb/c have been identified by PCR genotyping (FIGURE
17). These animals have been put into breeding to amplify the colony. Heterozygoteheterozygote breeding will be done to generate homozygous PRB -/- mice. To date, no PRA -/+
heterozygotes have been identified from breeding of the chimeric PRA -/+ animals.

33

APPENDIX B

FIGURES

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

REFERENCES

67

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