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First published online 26 September 2007
doi: 10.1242/dev.008250
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Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA.
* Author for correspondence (e-mail: rserra{at}uab.edu)
Accepted 20 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wnt5a, Mammary gland, TGF-ß, Ddr1, Non-canonical Wnt, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ewan et al., 2002;
Serra and Crowley, 2003;
Serra and Crowley, 2005;
Silberstein and Daniel, 1987;
Wakefield et al., 2001).
Our laboratory previously showed that dominant-negative interference of
TGF-ß signaling (DNIIR) in the mammary gland results in accelerated
ductal extension and increased lateral branching of the ductal tree compared
with wild-type glands (Crowley et al.,
2005; Joseph et al.,
1999). Stemming from these studies, cDNA-based microarray and
suppression-subtractive hybridization (SSH) assays were performed on glands
from wild-type and DNIIR mice to identify genes with expression profiles that
were altered when TGF-ß signaling was interrupted. The hypothesis was
that some of the genes that were regulated by TGF-ß would mediate a
subset of TGF-ß inhibitory actions within the mammary gland. From this
screen a gene involved in non-canonical Wnt signaling, Wnt5a, was
identified. Wnt5a was of special interest because, like TGF-ß, it has
been suggested to act as a tumor suppressor in many tumor types, including
those of the breast (Dejmek et al.,
2005; Jonsson et al.,
2002; Leris et al.,
2005). Nevertheless, the role of Wnt5a in normal mammary
development has gone largely unexplored.
The Wnt family of proteins consists of at least 19 members with functions
that contribute to the regulation of a wide range of cellular processes,
including proliferation and differentiation
(Kikuchi et al., 2006;
Veeman et al., 2003). Wnts
have also been implicated in tumor formation
(Brennan and Brown, 2004;
Katoh, 2005). Wnts activate
many signaling cascades, which can be divided broadly into two general
categories: (1) the canonical, ß-catenin-dependent pathway; and (2) the
non-canonical ß-catenin-independent pathway
(Kikuchi et al., 2006;
Kuhl et al., 2000;
Veeman et al., 2003;
Widelitz, 2005). In addition,
it has been shown that some non-canonical Wnts can directly antagonize
canonical signaling (Mikels and Nusse,
2006; Topol et al.,
2003; Westfall et al.,
2003). Wnt5a has been shown to signal through non-canonical
signaling pathways in many cell types.
Previously, it was shown using northern blot analysis that Wnt5a
is expressed at all stages of mammary development except lactation
(Gavin and McMahon, 1992;
Weber-Hall et al., 1994).
During embryonic development, Wnt5a mRNA was localized by whole-mount
in situ hybridization to a broad strip of mesenchyme underlying the area where
the mammary placodes form (Chu et al.,
2004). In adult virgin mice, expression was detected by northern
blot in RNA isolated from cleared fat pads, suggesting that Wnt5a is
expressed in the mammary stroma
(Weber-Hall et al., 1994).
Recently, it was shown using microarray screens that Wnt5a is
preferentially expressed in the TEB relative to the mature ducts
(Kouros-Mehr and Werb,
2006).
Many of the observations that address the potential role of Wnt5a in the
mammary gland come from in vitro studies using mammary cell lines. Early
studies show that WNT5A expression decreases when cells of an immortalized
human mammary epithelial cell line, HB2, are transferred from a
two-dimensional monolayer culture to an embedded three-dimensional culture,
and even more when three-dimensional cultures undergo branching morphogenesis
in response to hepatocyte growth factor (HGF) treatment
(Huguet et al., 1995).
Overexpression studies show that WNT5A can abolish HGF-induced branching of
HB2 cells in three-dimensional culture and enhance cell-to-collagen adhesion
(Jonsson and Andersson, 2001).
By contrast, siRNA-mediated repression of WNT5A enhances cell scattering,
impairs cell-collagen interaction and increases motility of HB2 cells
(Jonsson and Andersson, 2001).
A correlation between discoidin domain receptor 1 (DDR1) phosphorylation and
WNT5A expression has also been observed, and data exist that suggest a
requirement of WNT5A for collagen-induced DDR1 activation/phosphorylation and
proper mammary cell adhesion (Dejmek et
al., 2003). Together, the results imply an inverse correlation
between Wnt5a activity and branching.
In the current study, we show that TGF-ß regulates the expression of Wnt5a, which in turn, mediates a subset of TGF-ß inhibitory actions on ductal elongation and lateral branching during puberty and in non-pregnant adults. We demonstrate that Wnt5a exhibits inhibitory effects on mammary gland development similar to those of TGF-ß. We go on to show the necessity of Wnt5a for normal ductal extension and branching, and subsequently provide evidence for a functional interaction between TGF-ß and Wnt5a, through Ddr1 activation. We also show that Wnt5a is required for TGF-ß-mediated inhibition of branching. The current study demonstrates a functional link between TGF-ß and Wnt5a, which provides insight into the coordination of signaling events that contribute to mammary development and how alterations in these events might promote tumor progression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). DNIIR
and wild-type mice were administered 25 mM zinc sulfate in the drinking water
for 2 weeks to induce DNIIR transgene expression. This time frame allowed for
DNIIR expression before any phenotypic changes in branching occurred. Balb/c
mice were obtained from Jackson Laboratories (Bar Harbor, Maine).
Wnt5a disrupted mice (Wnt5atm1/Amc) were acquired
from Jackson Laboratories and kept on a 129S7/C57B6 background to obtain
Wnt5a-/- embryos. ICR/SCID mice were purchased from
Taconic Farms.
Primary mammary cell isolation and culture
Adult virgin Balb/c mice (8-10 weeks of age) were the source of primary
mammary epithelial cells and fibroblasts. Cell fractions were isolated by
enzymatic dissociation and Percoll gradient density centrifugation as
previously described (Xie and Haslam,
1997). Fibroblasts were plated in DMEM/F12 supplemented with 5%
FBS, 100 µg/ml penicillin and 50 µg/ml streptomycin, were allowed to
attach for 2 hours, were rinsed with 1xPBS, and were refreshed with
growth medium. The epithelial cells were plated 1x105
cells/cm2 in DMEM/F12 supplemented with 5% FBS, 20 ng/ml EGF, 0.5
µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin, 100
µg/ml penicillin, 5 µg/ml streptomycin. Both epithelial cells and
fibroblasts were grown to 80% confluence and serum-starved overnight before 5
ng/ml TGF-ß1 treatment. For cycloheximide experiments, cells were
pretreated overnight in basal conditions with 5 µg/ml cycloheximide.
Serum-free medium was used for fibroblast cultures and basal medium (DMEM/F12
supplemented with 0.1 mM non-essential amino acids, 0.1 µg/ml insulin, 1
mg/ml fatty acid-free BSA (fraction V) and 100 µg/ml penicillin, 50
µg/ml streptomycin) was used for epithelial cultures. For IP studies,
primary epithelial cells were plated on rat-tail collagen type I (BD
Biosciences).
For three-dimensional cultures, primary mammary epithelial cells were suspended and plated in 24-well culture dishes in neutralized rat-tail collagen type I (2 mg/ml, 200 µl per well). Neutralized collagen was prepared according to the manufacturer's instructions (BD Biosciences). Cultures were allowed to gel for 30 minutes at 37°C, at which point 1 ml of overlay basal medium was added to each well. Treatments included 30 ng/ml HGF (R&D) alone or in combination with 40 ng/ml Wnt5a (R&D). For wild-type and Wnt5a-/- organoid experiments, cultures were treated with 30 ng/ml HGF alone or in combination with 5 ng/ml TGF-ß1 (R&D). Analysis of branching was performed after 3 days.
RNA isolation and semi-quantitative RT-PCR
RNA isolation was performed using Trizol reagent. Relative levels of
Wnt5a mRNA were determined using semi-quantitative RT-PCR. cDNA was
synthesized from 2 µg total RNA using random primers. cDNA was then
amplified using primers specific for Wnt5a and 18S. Primer
sequences are as follows: Wnt5afwd
5'-GAATCCCATTTGCAACCCCTCACC-3', Wnt5arev
5'-GCTCCTCGTGTACATTTTCTGCCC-3', 18Sfwd
5'-ACGGAAGGGCACCACCAGG-3', 18Srev
5'-CACCAACTAAGAACGGCCATGC-3'. Amplification of cDNA was performed
over varying cycles to arrive at product formation in the linear range.
Immunoprecipitation and western blot analyses
Protein from cells and tissue was extracted using RIPA buffer supplemented
with a protease inhibitor cocktail (Roche, Complete Mini) and sodium
orthovanadate (Sigma). For Wnt5a analyses, 50 µg protein lysate was
fractionated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane
and probed with 0.2 µg/ml polyclonal Wnt5a antibody (R&D Systems,
AF645) and rabbit anti-goat polyclonal secondary IgG-HRP (1:1000; Santa Cruz,
sc-2922). To ensure equal protein loading between lanes, membranes were
stripped and re-probed with anti-ß-tubulin polyclonal antibody (1:1000;
Santa Cruz H-325, sc-9104) and secondary goat anti-rabbit IgG-HRP (1:2000;
Santa Cruz, sc-2054). Blots of the tissue lysate were also probed with
-HGF (Santa Cruz, sc-7949) to demonstrate the increase in HGF
expression in the DNIIR phenotype. Also, blots of the cell culture experiments
were probed with anti-Vimentin (Santa Cruz, sc-5565) to confirm
fibroblast-rich populations. For immunoprecipitation studies, 500 µg
protein lysate was pre-cleared with agarose-bound protein A (Vector, sp-0050)
and 2 µg normal rabbit IgG (Santa Cruz, sc-2027), and immunoprecipitated
with 1 µg polyclonal anti-Ddr1 antibody (Santa Cruz C-20, sc-532). The
immunoprecipitated proteins were resolved on a 10% SDS-PAGE gel, transferred
to a nitrocellulose membrane and probed with a rabbit polyclonal
anti-phosphotyrosine antibody (Upstate, 06-427) to detect changes in the
phosphorylation status of Ddr1. Membranes were stripped and re-probed with
anti-Ddr1 as an internal control for loading.
Elvax slow-release pellet preparation and surgical implantation
The use of Elvax in slow-release delivery of proteins has been previously
described (Silberstein and Daniel,
1987). Wnt5a pellets were prepared using 6 µg of Wnt5a protein
and 20 µg BSA as a carrier. The final stock pellet was cut into 
1 mg
pellets containing 170 ng Wnt5a protein. BSA control pellets were prepared
with 20 µg BSA and no additional protein. Mice were anesthetized and
incisions were made in the lower abdomen and along the hind legs to expose the
#4 inguinal mammary glands. Pellets were implanted into the glands by making a
pocket within the fat pad with fine surgical forceps and carefully placing the
pellet within the pocket. BSA pellets were inserted into one gland, while
Wnt5a pellets were implanted into the contralateral gland of the same mouse.
Mammary glands were removed and evaluated 1 week after implantation of the
pellets.
TGF-ß1 pellets were prepared with 1 µg of protein. To test TGF-ß1 pellet efficacy and demonstrate ductal inhibition by TGF-ß1 in wild-type glands, TGF-ß1 and BSA pellets were implanted into contralateral #4 glands of Balb/c mice at the onset of puberty just ahead of the leading edge of the ductal tree. Glands were evaluated after one week by whole-mount staining. For Wnt5a-/- experiments, surgical implantation of the pellets was performed by clearing contralateral fat pads of endogenous epithelium followed by implantation of TGF-ß1 or BSA pellets and transplanted Wnt5a-/- epithelium. The mammary glands were allowed to develop for 2 weeks and then removed and evaluated by whole-mount preparation.
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Wnt5a-/- and wild-type anlagen isolation and transplantation
Wnt5a-/- and wild-type embryos [embryonic day 16.5
(E16.5)] were the source of the mammary tissue for renal capsule grafting and
fat pad transplantation experiments. Wnt5a-/- embryos were
phenotypically distinguishable from wild-type embryos. Briefly, mammary
epithelial primordia were microdissected from female embryos as previously
described
(http://mammary.nih.gov).
Mammary buds were then grafted onto mesenchyme and cultured overnight to allow
the two tissue types to form one transplantable unit. Grafts were cultured on
Whatman Nucleopore Track-Etch Membranes in DMEM/F12 medium supplemented with
10% FBS, 100 µg/ml penicillin and 50 µg/ml streptomycin. After overnight
culture, grafts were either implanted under the kidney capsule of ICR/SCID
mice (or syngeneic hosts) or within the cleared fat pad of ICR/SCID mice
(DeOme et al., 1959).
Wild-type and Wnt5a-/- mammary explants were surgically
implanted within contralateral kidney capsules or fat pads of the same host to
ensure the same hormonal environment for the transplanted tissue to develop.
For the renal grafts, sometimes more than one graft was put into a single
kidney. In some cases epithelium transplanted into the fat pad was allowed to
fill the fat pad so that Wnt5a-/- cells could be isolated
and used in cell culture experiments as described below.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wnt5a inhibits ductal extension during puberty
As TGF-ß regulated Wnt5a expression, we sought to investigate the role
of Wnt5a in the mammary gland in vivo and determine whether Wnt5a had similar
effects on branching as those reported for TGF-ß. Elvax slow-release
pellets containing BSA as a control or Wnt5a were implanted into contralateral
#4 inguinal mammary glands of pubertal Balb/c mice at 3-4 weeks of age, in
front of the terminal end buds of the developing ductal tree. After 1 week #4
inguinal glands receiving BSA pellets and their contralateral #4 glands
receiving Wnt5a pellets were removed from mice to evaluate the effect of Wnt5a
on ductal extension. In those glands that received Wnt5a pellets
(Fig. 2B), ductal extension was
inhibited compared with the contralateral gland that received BSA
(Fig. 2A). Ductal extension was
quantified by measuring the distance of the leading edge of the terminal end
buds relative to the lymph node over the total length from the lymph node to
the end of the fat pad. This ratio was expressed as a percentage of the
terminal end bud distance traveled from node to the end of the gland.
Quantification and statistical significance of the inhibition of ductal
extension by Wnt5a is illustrated in Fig.
2C (n=11 mice; paired Student's t-test,
P<0.05).
Terminal end buds are reduced in size in glands receiving Wnt5a
In addition to the inhibition of ductal extension during puberty, mammary
glands that received Wnt5a pellets (Fig.
2E,G) exhibited a regression in terminal end bud size relative to
contralateral glands that received BSA pellets
(Fig. 2D,F). H&E stained
sections showed that the TEBs were smaller in size and had lost normal
structure (Fig. 2F,G). The
effects elicited by Wnt5a on the end buds could be a result of decreasing
proliferation, increasing apoptosis or a combination of both, so we carried
out BrdU and TUNEL analysis on glands that received slow-release pellets and
specifically analyzed proliferation and apoptosis in the terminal end buds
between Wnt5a and BSA groups. BrdU incorporation in the terminal end buds
demonstrated a fourfold decrease in proliferation in glands receiving Wnt5a
pellets versus BSA pellets (Fig.
2H; n=31 end buds from BSA group, n=48 end buds
from Wnt5a group, two-tailed Student's t-test, P<0.05).
TUNEL staining did not indicate any significant changes in the percentage of
cells undergoing apoptosis in the terminal end buds between Wnt5a and BSA
groups (Fig. 2I; n=31
end buds from each group). The regression in size of the terminal end buds was
therefore, at least in part, a result of a decrease in proliferation and not
because of an increase in apoptosis.
Wnt5a inhibits branching morphogenesis in vivo and HGF-induced branching in vitro
The inhibitory effect of Wnt5a on ductal extension during puberty led us to
speculate that Wnt5a might also inhibit lateral branching. To test this
hypothesis, Wnt5a pellets were implanted within the ductal tree at the end of
puberty, around 6 weeks of age. After 1 week, #4 inguinal glands that received
Wnt5a pellets displayed fewer secondary and higher order branches in
comparison to the contralateral glands that received BSA pellets
(Fig. 3A-C). The number of
lateral branches along the primary ducts was measured in three fields from
each of the glands and the average number of secondary branches per 0.5 cm
length of the primary branch was calculated. A paired Student's
t-test indicated that the difference in branching was significant
(Fig. 3C; n=4,
P=0.002). To better quantify branching, we used a three-dimensional
in vitro model consisting of primary mammary epithelial organoids embedded in
a type I collagen matrix. Under basal conditions, organoids formed spherical
`cysts' without branches (Fig.
3D). Upon stimulation with HGF, sphere-shaped organoids formed
branching processes after a few days (Fig.
3D). When treated with HGF in conjunction with Wnt5a, organoid
branching was reduced (Fig.
3D). The extent of branching was quantified based on primary
branch number and primary branch length. Primary branches were those extending
directly from the sphere-shaped organoid. There was a statistically
significant decrease in the number of primary branches
(Fig. 3E; n=63
organoids per group, pooled from three independent experiments,
P<0.05) and in the length of the branch
(Fig. 3F; n=63
organoids per group, pooled from three independent experiments,
P<0.05) when cultures were treated with Wnt5a. In addition,
mammary organoids subjected to Wnt5a in combination with HGF displayed a
decrease in proliferation as measured by BrdU incorporation compared with
those organoids treated with HGF alone
(Fig. 3G, n=32
organoids per group, pooled from two independent experiments,
P<0.05). Together, these data illustrate the inhibitory effect of
Wnt5a on branching. TGF-ß has similar effects on primary and lateral
branching, suggesting a model in which at least some of the effects of
TGF-ß on branching could be mediated by Wnt5a.
Wnt5a-/- mammary explants exhibit accelerated development
The experiments described until now establish the capacity of Wnt5a to
inhibit ductal extension and lateral branching based on exogenous
administration of Wnt5a, yet do not address a functional requirement for Wnt5a
in development of the mammary gland. Phenotypic examination of the postnatal
Wnt5a-/- mammary gland is not feasible as a result of
severe developmental defects rendering the Wnt5a-/- mouse
perinatal lethal. To address this issue, mammary buds and mesenchyme were
harvested from E16.5 female embryos, cultured together overnight, and
subsequently grafted under the kidney capsules of 3-week-old ICR/SCID mice, or
transplanted into cleared fat pads of 3-week-old ICR/SCID mice. It was
previously shown that embryonic development of Wnt5a-/-
glands is normal (Chu et al.,
2004), and we confirmed that result here
(Fig. 4A). Subrenal grafting of
the mammary tissue was an attractive strategy to rescue
Wnt5a-/- embryonic tissue, given that the kidney is a
highly vascular organ and the take rate of the graft reaches nearly 100%.
Also, evaluation of the absence of Wnt5a in both epithelial and
stromal compartments is achieved using this strategy. By contrast,
transplantation of tissue into cleared mammary fat pads addresses the absence
of Wnt5a in the epithelium with intact expression in the stroma
(DeOme et al., 1959). After 1
week under the kidney capsule, Wnt5a-/- explants exhibited
accelerated development compared with wild-type explants that were inserted
under the contralateral kidney of the same host
(Fig. 4B; n=8).
Wnt5a-/- mammary development was highlighted by the
presence of larger terminal end buds, increased ductal extension and increased
branching relative to wild-type explants. In addition, Brdu analysis of
Wnt5a-/- and wild-type explants demonstrated a 2.2-fold
increase in proliferation of the Wnt5a-/- explants
compared with wild-type control explants
(Fig. 4C; n=5
wild-type explants and n=6 Wnt5a-/- explants,
Student's t-test, P=0.0005). Transplantation of wild-type
and Wnt5a-/- mammary explants into cleared fat pads
revealed that the absence of Wnt5a in the epithelium is enough to
promote accelerated mammary development
(Fig. 4D,E). Two weeks after
surgical implantation of the explants into the fat pad, whole-mount evaluation
of Wnt5a-/- transplants revealed a greater percentage of
the fat pad filled, accompanied by an increase in terminal end bud size and an
increase in lateral branching relative to the contralateral gland containing
wild-type epithelium (Fig. 4D,E
n=2 mice each containing wild-type and Wnt5a-/-
epithelium in contralateral #4 glands), consistent with the results obtained
from the subrenal grafting experiments. Together, these data suggest that
Wnt5a from the epithelium facilitates proper growth and patterning of
the mammary ductal tree by acting as a negative regulator of branching.
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|
;
Vogel et al., 1997). Ddr1 is
expressed at all stages of mammary development
(Barker et al., 1995).
Previously, it was shown that repression of WNT5A in HB2 cells results in
enhanced cell motility and impaired binding to collagen
(Jonsson and Andersson, 2001).
Alterations in binding and migration after depletion of WNT5A were correlated
with impaired phosphorylation of DDR1. Activation and subsequent
phosphorylation of DDR1 was shown to require both collagen and WNT5A. Given
that TGF-ß regulates Wnt5a, we sought to investigate whether TGF-ß
could alter the activation status of Ddr1, and additionally, determine whether
interruption of TGF-ß signaling in the mammary gland affects Ddr1
phosphorylation in vivo. First, we determined if interruption of TGF-ß
signaling in the mammary gland impaired Ddr1 phosphorylation in vivo. Ddr1 was
immunoprecipitated from protein lysates derived from DNIIR and wild-type
mammary glands followed by western blot for phosphotyrosine or total Ddr1. We
found that Ddr1 tyrosine phosphorylation was diminished in DNIIR mammary
glands relative to wild-type glands, whereas total Ddr1 levels remained fairly
constant (Fig. 5A). These
results suggest that TGF-ß signaling is required for proper Ddr1
activation. We next hypothesized that Wnt5a was required for
TGF-ß-mediated effects on Ddr1 activation. First, to show Ddr1 receptor
activation in response to Wnt5a, primary epithelial cells cultured on type I
collagen were treated with Wnt5a over a period of 6 hours. Immunoprecipitation
of Ddr1 and western blotting for phosphotyrosine showed an increase in Ddr1
phosphorylation within 30 minutes of Wnt5a treatment
(Fig. 5B). Primary epithelial
cells (plated on type I collagen) treated with TGF-ß1 also demonstrated
an increase in Ddr1 phosphorylation, but after 30 hours. This activation
corresponded to a time after the induction of Wnt5a protein levels seen at 24
hours (Fig. 5C). To test
whether the activation of Ddr1 by TGF-ß1 was directly mediated through
the upregulation of Wnt5a, Wnt5a-/- epithelium was
transplanted into cleared fat pads of wild-type mice and allowed to fill the
fat pad. The Wnt5a-/- cells were then isolated and
cultured on type I collagen followed by treatment with TGF-ß1 for up to
48 hours. Compared with wild-type primary epithelium, which exhibited an
increase in Ddr1 phosphorylation after TGF-ß1 treatment,
Wnt5a-/- cells failed to undergo Ddr1 phosphorylation in
response to TGF-ß1 (Fig.
5C). These findings illustrate that TGF-ß can affect the
phosphorylation/activation status of Ddr1 through the regulation of Wnt5a.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Silberstein and Daniel, 1987).
Moreover, loss-of-function experiments involving subrenal grafting and fat pad
transplantation of Wnt5a-/- mammary tissue revealed a
requirement for Wnt5a in normal mammary development. In the absence of Wnt5a,
development was accelerated, marked by the presence of larger terminal end
buds, more extensive ductal invasion, increased lateral branching and elevated
proliferation within the epithelium relative to wild-type glands.
Additionally, we were able to identify a functional connection between
TGF-ß and Wnt5a. TGF-ß facilitated the phosphorylation of Ddr1 via
the regulation of Wnt5a expression and interruption of TGF-ß signaling in
vivo reduced both Wnt5a expression and the phosphorylation status of Ddr1.
Lastly, we show that TGF-ß1 is unable to inhibit ductal extension and
growth in Wnt5a-/- mammary transplants or branching in
Wnt5a-/- organoids, suggesting that Wnt5a is required for
TGF-ß-mediated inhibition of mammary development. A key finding made here is that Wnt5a is required for many of the effects of TGF-ß on branching. TGF-ß pellets placed in front of the end bud normally inhibit ductal extension through the fat pad accompanied by a decrease in end bud size; however, Wnt5a-/- epithelium was resistant to the inhibitory effects of TGF-ß. Additionally, HGF-induced branching in Wnt5a-/- organoids was not inhibited by TGF-ß. Wild-type organoids, by contrast, exhibit a dramatic reduction in HGF-induced branching when treated with TGF-ß. This is the first evidence that TGF-ß requires a secondary effector, Wnt5a, for some of its inhibitory actions in the mammary gland.
It is possible, based on our data and those of other studies
(Dejmek et al., 2003;
Jonsson and Andersson, 2001),
that the inhibitory effects of TGF-ß and Wnt5a are based on the ability
of these proteins to modify cell adhesive properties, thus dictating the
proliferative or migratory capacity of the epithelium in response to other
signals. Ddr1, which we demonstrate can be activated by TGF-ß via
upregulation of Wnt5a, has been shown to inhibit migration of breast cancer
cells in vitro (Hansen et al.,
2006) and tubulogenesis/migration of MDCK cell in
three-dimensional culture (Wang et al.,
2005; Wang et al.,
2006). Moreover, data suggest that Ddr1 tyrosine kinase activity
can mediate the expression of MMPs (Hou et
al., 2002), which are involved in the orchestration of mammary
development. It is known that one of the main functions of TGF-ß is to
mediate cell adhesion and shape (Massague
et al., 1992; Sporn and
Roberts, 1988). It has also been suggested that WNT5A is a key
regulator of mammary cell adhesion through its ability to act as a co-factor
for the activation and phosphorylation of DDR1
(Dejmek et al., 2003;
Jonsson and Andersson, 2001).
At 3 months of age, Ddr1-null mammary glands exhibit an increase in
lateral branching and proliferation when compared with wild-type glands with
some similarities to the effects of dominant-negative interference of
TGF-ß signaling or loss of Wnt5a, suggesting that TGF-ß, Wnt5a and
Ddr1 may interact to regulate proliferation and branching in the adult mammary
gland (Crowley et al., 2005;
Joseph et al., 1999;
Vogel et al., 2001). We
previously showed that dominant-negative interference of TGF-ß signaling
results in elevated expression of HGF
(Joseph et al., 1999).
Overexpression of DDR1 (Wang et al.,
2005) or WNT5A (Jonsson and
Andersson, 2001) inhibits HGF-induced branching in vitro. It seems
likely that elevated HGF expression from the stroma in conjunction with a
decrease in Wnt5a expression and subsequent loss in Ddr1 activation might
facilitate branch formation and result in increased branching seen when
TGF-ß signaling is interrupted. These observations create a potential
mechanism for the regulation of branching by TGF-ß through its regulation
of both negative and positive mediators of branching, Wnt5a and HGF.
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; Kuhl et al.,
2000; Veeman et al.,
2003; Widelitz,
2005). Wnt5a has been shown to act primarily through non-canonical
signaling pathways. Among the non-canonical pathways, Wnt5a has been shown to
activate both the Wnt/Ca+2 and planar cell polarity (PCP) pathways.
Wnt5a can also antagonize canonical signaling but the mechanism is not clear
and varies between cell types. The non-canonical signaling pathways are not
very well characterized in general and not at all understood in the context of
normal mammary development. However, the role of canonical Wnt signaling has
been studied in mouse mammary gland development
(Brennan and Brown, 2004).
Expression of a Wnt reporter, TOPGAL, and characterization of mice with
mutations or deletions of components of the canonical pathway suggest that
canonical signaling is important in early embryonic development of the mammary
gland and then again during pregnancy (Chu
et al., 2004; Hsu et al.,
2001; Brisken et al.,
2000; Tepera et al.,
2003). Although several canonical signaling Wnts are expressed
during puberty and in the adult mammary gland, there is no evidence that
canonical signaling is required for normal development at these stages;
nevertheless, inappropriate canonical signaling in adult mice results in
hyperplasia or metaplasia in the ducts and is correlated with tumorigenic
transformation (Benhaj et al.,
2006; Gavin and McMahon,
1992; Imbert et al.,
2001; Kouros-Mehr and Werb,
2006; Miyoshi et al.,
2002). Wnt5a has been shown to antagonize canonical signaling in
several cell types, and it is possible that its role in normal development is
to suppress inappropriate canonical signaling. Furthermore, even though
several non-canonical signaling Wnts are expressed in the mammary gland
(Benhaj et al., 2006;
Kouros-Mehr and Werb, 2006),
the fact that loss of Wnt5a alone is sufficient to alter normal branching and
growth suggests that Wnt5a has unique roles in mammary development. It should
also be noted that experiments in which Wnt5a-/-
epithelium is transplanted into the cleared fat pads of wild-type mice
indicate that Wnt5a synthesized in the epithelium is required for normal
branching and growth. Although Wnt5a expression has been detected in the
stroma, stromal Wnt5a alone is not sufficient to mediate normal growth and
branching. This study is the first to show a functional requirement for Wnt5a in the mammary gland and the first to show that Wnt5a acts to mediate many of the effects of TGF-ß on branching. We demonstrate that Wnt5a is required for TGF-ß to regulate mammary branching and phosphorylation of Ddr1. The task at hand lies in identifying downstream signaling targets of Wnt5a and Ddr1 that could provide insight into how these factors regulate development of the mammary gland. Future work will be directed at understanding the role of Wnt5a in tumor formation and progression.
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ACKNOWLEDGMENTS |
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