1 Department of Anatomy, University of California, 513 Parnassus Ave., San
Francisco, CA 94143-0452, USA
2 Department of Biochemistry and Biophysics and UNC Lineberger Comprehensive
Cancer Center, University of North Carolina School of Medicine, Chapel Hill,
NC 27599-7260, USA
* Author for correspondence (e-mail: sternli{at}itsa.ucsf.edu)
Accepted 1 July 2005
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SUMMARY |
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Key words: Mammary gland, Branching morphogenesis, Metalloproteinase, ADAMs, TNF converting enzyme, ERBB, Stromal-epithelial interactions, Epidermal growth factor receptor, Mouse
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Introduction |
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Epidermal growth factor receptor (EGFR/ERBB1) is a transmembrane tyrosine
kinase that is activated upon binding EGF, transforming growth factor
(TGF
), amphiregulin (AREG), heparin-binding EGF-like growth factor
(HB-EGF), betacellulin (BTC), epiregulin (EPIR) or epigen (EPGN), each of
which is expressed as a transmembrane precursor that is proteolytically shed
from the cell surface (Harris et al.,
2003
). Once occupied, EGFR dimerizes with another EGFR monomer or
one of three related receptors, ERBB2, ERBB3 or ERBB4. Notably, mammary
development is impaired in waved 2 mutant mice that harbor a kinase-impaired
EGFR (Fowler et al., 1995
;
Sebastian et al., 1998
) and in
transgenic mice that express a mammary-targeted, dominant-negative EGFR
(Xie et al., 1997
).
Egfr mRNA and protein are abundant in mammary stroma
(Luetteke et al., 1999
;
Schroeder and Lee, 1998
).
Indeed, EGF induces EGFR phosphorylation in gland-free fat pads
(Sebastian et al., 1998
) and
significantly more 125I-EGF binds to stromal cells surrounding TEBs
than to any other area of the developing gland
(Coleman et al., 1988
).
Notably, Egfr/ glands show impaired ductal
outgrowth when grown under the renal capsules of host mice, and when wild-type
or Egfr/ ducts are surgically recombined
with fat pads of the same or opposite genotype, the ducts grow regardless of
genotype if the stroma contains EGFR, but not if it lacks EGFR. This indicates
that stromal rather than epithelial EGFR is essential for ductal development
(Wiesen et al., 1999
).
Nevertheless, Egfr/ transplants do undergo
alveolar differentiation in response to prolactin from nearby pituitary
isografts, suggesting that EGFR is essential for ductal, but not alveolar,
development.
The importance of EGFR also means that one or more of its ligands must
influence mammary development. At least six EGFR agonists are expressed during
mammary development, but only AREG is strongly upregulated at puberty and
dramatically downregulated during and after pregnancy
(D'Cruz et al., 2002;
Schroeder and Lee, 1998
), a
pattern consistent with the importance of EGFR in post-pubertal mammary
development. Indeed, ductal outgrowth is severely impaired in triple-null mice
lacking AREG, EGF and TGF
, which are lactation incompetent, and
variably impaired in mice lacking only AREG
(Luetteke et al., 1999
). As
neither ductal outgrowth nor lactation is affected by elimination of EGF,
TGF
, HB-EGF or BTC alone or in various combinations
(Jackson et al., 2003
;
Luetteke et al., 1999
), AREG
must be uniquely required for this process.
Like all EGFR ligands, AREG is expressed as a transmembrane precursor that
is generally cleaved and released to activate its receptor. Extensive data
indicate that various members of the ADAM (a disintegrin and
metalloproteinase) family of cell surface enzymes, including ADAM17
(TNF-converting enzyme or TACE), are responsible for the release of
EGFR ligands, including AREG, in vitro
(Hinkle et al., 2004
;
Sahin et al., 2004
;
Sunnarborg et al., 2002
) and
even cell contact-dependent juxtacrine activation of EGFR may require
ADAM17-mediated processing of EGFR agonists
(Borrell-Pages et al., 2003
).
However, no genetic evidence supporting a role for ADAM17 as a physiological
AREG sheddase has yet been provided. By contrast,
Adam17/ mice
(Peschon et al., 1998a
;
Shi et al., 2003
) display the
altered eyelid, hair and whisker development of TGF
-deficient mice
(Luetteke et al., 1993
;
Mann et al., 1993
), the
aberrant heart valve development of HB-EGF-null and uncleavable HB-EGF
knock-in mice (Iwamoto et al.,
2003
; Jackson et al.,
2003
; Yamazaki et al.,
2003
), and the broad epithelial defects and perinatal lethality of
Egfr/ mice
(Miettinen et al., 1995
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
Moreover, studies using single-, triple- and quadruple-gene knockout mice
lacking ADAM9, ADAM12, ADAM15 and/or ADAM17 show that only ADAM17 is
responsible for the eyelid and heart phenotypes and is thus required for
efficient processing of TGF
and HB-EGF in these tissues
(Sahin et al., 2004
).
Although the above data suggest that ADAM17 processes EGFR ligands in certain embryonic situations, its role in mediating paracrine crosstalk between differing cell-types postnatally remains unexplored. In this study, we use tissue recombination methods to show that EGFR is indeed required in the stroma of the developing mammary gland, whereas the requirement for AREG to form a competent ductal tree rather than an inadequate bush resides in the epithelium, and that ADAM17, which can process AREG, is also required in the epithelium in a paracrine pathway that is essential for normal branching morphogenesis.
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Materials and methods |
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Recombined mammary transplants were prepared as previously described
(Wiesen et al., 1999). The
rudimentary ductal tree was microdissected from the abdominal 4 fat pad,
trimmed of excess stroma and placed onto a gland-free embryonic or neonatal
fat pad. The recombined epithelium and stroma were allowed to adhere to one
another by overnight culture on solidified agar plates containing 0.5% Bacto
agar (Difco), 10% fetal bovine serum, 100 U/ml penicillin G and 100 µg/ml
streptomycin in DME-H16 medium enriched with 6 mM L-glutamine. Recombined and
non-recombined glands were placed under the renal capsules of nude mice with
intact ovaries and allowed to grow for 2-6 weeks with or without subcutaneous
1.7 mg 60-day slow-release 17ß-estradiol pellets or adjacent 10 µg
21-day AREG micropellets (Innovative Research of America). Embryonic mammary
glands were also transplanted to surgically cleared host fat pads as described
elsewhere (Wiesen et al.,
1999
). Morphometry was performed on digital images of
carmine-stained mammary whole mounts using FoveaPro3. All experiments were
performed in accordance with protocols approved by the UCSF and UNC Committees
on Animal Research.
Mammary organoid culture
Embryonic and neonatal mammary organoids were prepared in a similar manner
to that described for adult organoids
(Simian et al., 2001).
Rudimentary ductal trees were microdissected from the surrounding stroma,
pooled and swirled at 100 rpm for 30 minutes at 37°C in DMEM/F12 medium
containing 0.2% collagenase A (Sigma), 0.2% trypsin (Life Technologies), 5%
fetal bovine serum, 5 µg/ml insulin and 50 µg/ml gentamicin. The
resulting suspension was spun at 300 g for 5 minutes and the
pellets gently agitated at room temperature for 2 minutes in DMEM/F12 with 40
U/ml DNase I (Sigma). Cell clusters and dissociated single cells were pelleted
at 300 g, washed with DMEM/F12, re-centrifuged and gently
resuspended in ice-cold growth factor-reduced Matrigel (Becton Dickinson). The
suspended organoids were transferred to 48-well plates over a thin cell-free
layer of Matrigel and allowed to gel at 37°C. Following gel formation, 300
µl of basal medium (5 mg/ml insulin, 5 mg/ml transferrin, 5 µg/ml
selenium, 100 U/ml penicillin G and 100 µg/ml streptomycin in DMEM/F12) was
added with or without growth factors, and the organoids cultured in a
humidified 5% CO2 incubator at 37°C. Growth was observed over 7
days in the presence or absence of 5 nM murine TNF
or human AREG,
HB-EGF or NRG1ß1 (R&D Systems), or 5 nM rat TGF
or human EGF,
FGF1, FGF2, FGF7 or FGF10 (Sigma).
Expression profiling
TEB, duct and distal stroma regions of mammary glands 2-5 were
independently microdissected from anesthetized 5-week-old ß-actin-GFP
reporter mice (Jackson Laboratory) using a Leica MZFLIII fluorescence
microscope. RNA was extracted with Trizol Reagent (Tel-Test), reverse
transcribed in the presence of amino-allyl-dUTP, coupled to CyScribe dyes
(Amersham), and the unamplified Cy5-labeled TEB or duct cDNAs and Cy3-labeled
stromal cDNAs were hybridized to 70-mer oligonucleotide microarrays with
19,500 features (Operon, mouse version 2.0), as described elsewhere
(Barczak et al., 2003).
Differential expression values in the text were obtained by converting the
lowest normalized, log2-transformed gene expression ratios
M=log2(Cy5/Cy3) and average overall signal intensities
A=0.5[log2(Cy5)+log2(Cy3)] to linear values for each of
six TEB versus distal stroma and six duct versus distal stroma arrays.
Protein analysis
Tissues were extracted on ice in four volumes (w/v) of 20 mM HEPES, pH 7.4,
150 mM NaCl, 1% Triton X-100 buffer containing protease and phosphatase
inhibitors (2 mM EDTA, 2 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM PMSF, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 50
µM sodium molybdate, 2.5 mM sodium pyrophosphate and 1 mM
ß-glycerophosphate) by Polytron homogenization and centrifugation.
Supernatant proteins were resolved by SDS-PAGE and transferred to PVDF
membranes for western blots or immunoprecipitated with rabbit anti-mouse EGFR
(Upstate, 1 µg/125 µg of protein) and protein A agarose in Tris-buffered
saline containing 0.5% NP-40 and protease and phosphatase inhibitors.
Immunoblotting was performed using mouse anti-phosphotyrosine (4G10, Upstate,
1:1000), rabbit anti-phosphoEGFR (Y1068, Cell Signaling, 1:1000), rabbit
anti-mouse EGFR (Upstate, 1:1000), rabbit anti-mouse keratin 14 (Covance,
1:20,000), goat anti-actin (Santa Cruz, 1:2000), and HRP-conjugated donkey
anti-rabbit, mouse and goat IgG secondary antibodies (Amersham, 1:2000)
followed by enhanced chemiluminescence autoradiography.
Statistical analysis
Mean values are provided with standard deviations and were compared by
unpaired, two-tailed t-tests. Array-based statistics were adjusted
for multiple comparisons using the Benjamini-Hochberg method of controlling
for the false discovery rate (Benjamini and
Hochberg, 1995).
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Results |
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|
Stromal EGFR is required for mammary development
Our data show that during mammary development the crucial EGFR ligand AREG
comes from the epithelium. However, prior studies suggest that EGFR is
enriched in the periepithelial mammary stroma
(Coleman et al., 1988;
Schroeder and Lee, 1998
) and
that stromal rather than epithelial EGFR is required for mammary epithelial
development in vivo (Wiesen et al.,
1999
). Moreover, EGFR ligands are epithelial (and stromal)
mitogens, yet epithelial EGFR is not needed for mammary epithelial development
in vivo. Thus, we revisited the tissue recombination studies that led to this
somewhat paradoxical finding, this time in the presence of estradiol pellets
in order to also assess the role of EGFR in estrogen-induced alveolar
differentiation, and again found that wild-type and
Egfr/ epithelium grew in fat pads that
contained EGFR, but not in Egfr/ fat pads
(Fig. 2C,D). After 3 weeks,
non-recombined Egfr/ glands occupied only
4% of the area occupied by paired wild-type glands (0.5±0.2 versus
11.2±2.9 mm2; P<0.0001), and wild-type
epithelium grown in Egfr/ fat pads occupied
areas that were
11% of those occupied by
Egfr/ epithelium in wild-type fat pads
(1.3±0.7 versus 11.6±5.7 mm2; P=0.0002).
Thus, in contrast to AREG, stromal EGFR indeed is required for mammary
epithelial development, whereas epithelial EGFR is dispensable. However, like
AREG, EGFR was not required for estrogen-induced lobuloalveolar development
(Fig. 2E).
|
Adam17/ pups, which exhibit perinatal lethality, had 65-69% fewer mammary branches and 64-68% shorter ductal trees than their wild-type littermates at E18.5 and birth (P<0.0001; Fig. 3). Likewise, Egfr/ neonates had 77% fewer branches (P<0.005) and 74% shorter ductal trees (P<0.001) than their own wild-type littermates, indicating that they too had impaired fetal mammary development (Fig. 3B,C). Two weeks after renal transplantation, Adam17/ glands lacked normal TEBs and had 90% less overall ductal length than contralateral wild-type glands when exogenous estradiol was absent (P<0.0001; Fig. 4A,I). Adam17/ transplants to cleared mammary fat pads also underwent little or no growth in the absence of exogenous estradiol, and even after 5 weeks, were still not significantly larger than the rudiments of newborn wild-type mice (Fig. 4E,I). When estradiol was added, the wild-type renal transplants often filled the fat pads by 3 weeks, whereas the Adam17/ epithelium occupied only 20-30% of the area of wild-type transplants at all time points up to six weeks (P<0.0001; Fig. 4B,C,J,K). Indeed, the slope of the regression line for growth of the Adam17/ glands in the presence of added estradiol was not significantly different from that of a flat line (P=0.75), again indicating that they were not catching up and that other ADAMs are unable to compensate for the absence of ADAM17. Adam17/ epithelium also consistently failed to grow in wild-type stroma in tissue recombination or cleared fat pad experiments, whereas wild-type epithelium grew readily in Adam17/ stroma (P<0.002; Fig. 4F,J) and cleared contralateral fat pads (P=0.0002; Fig. 4D,E,I). Thus, like AREG, ADAM17 is only required in the epithelium. Moreover, like AREG and EGFR, its absence had no apparent effect on estrogen-induced lobuloalveolar development (data not shown).
|
EGFR ligands induce branching in cultured Adam17/ mammary organoids
If EGFR regulates mammary development downstream of ADAM17 and AREG, then
its other ligands should also foster the growth and branching of
Adam17/ and
Areg/ mammary epithelium in culture. Indeed,
when embryonic and neonatal mammary organoids were grown in three-dimensional
basement membrane gels, the wild-type,
Adam17/ and
Areg/ organoids underwent robust growth and
branching in the presence of EGF (91±8% of organoids), TGF
(93±4%), HB-EGF (55±26%) and AREG (72±15%). By contrast,
no growth was seen when insulin was the sole growth or survival factor
provided or when Egfr/ organoids were
cultured in the presence of EGFR agonists
(Fig. 5). When heparin-acrylic
beads saturated with AREG were embedded in Matrigel to mimic the AREG pellets
in vivo, 67% of organoids within 400 µm of the pellets displayed definitive
growth, whereas those that were more than 1 mm away did not grow (Fisher's
exact test P<0.001). This distinction may again reflect
sequestration of AREG.
Because ADAM17 can cleave multiple substrates, it may also influence
mammary development via other targets. TNF, the substrate for which
ADAM17 was originally named, can stimulate growth and branching of cultured
mammary epithelial cells in an EGFR-independent, but
metalloproteinase-dependent manner (Lee et
al., 2000
; Varela et al.,
1997
). In our study, however, TNF
failed to support
organoid growth in six independent experiments
(Fig. 5) and its mRNA was
undetectable in developing glands (Fig.
1). Moreover, mice that lack TNF
or either of its receptors
(which are also shed by ADAM17) (Peschon
et al., 1998a
) have no overt phenotype
(Marino et al., 1997
;
Pasparakis et al., 1996
;
Peschon et al., 1998b
) and are
lactationally competent (J. Peschon, L. Old and G. Kollias, personal
communications). Thus, TNF
is not required for mammary morphogenesis or
function.
|
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Discussion |
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|
Clearly, many inputs influence the expression and activity of ADAM17, AREG
and EGFR during mammary development. For example, AREG is strongly induced by
estrogens (Vendrell et al.,
2004) and is the only EGFR ligand that is adequately expressed and
enriched in developing mammary epithelium. However, several ADAMs are
expressed during mammary development, at least two of which (ADAMs 15 and 17)
can process AREG (Schafer et al.,
2004
). However, only ADAM17 appears to be required, as other ADAMs
are unable to compensate for its absence and triple-null mice lacking ADAM9,
ADAM12 and ADAM15 are fully able to nurse their pups (C. Blobel, personal
communication). Thus, either ADAM17 is the only physiologic AREG sheddase or
it must be regulated independently of the other available ADAMs, or both.
Several potential avenues are available for the differential regulation of
ADAM17. ADAM17 is active at the cell surface, as the removal of its propeptide
domain by furin-like proprotein convertases occurs in the trans-Golgi network
(Srour et al., 2003). Notably,
our results indicate that the only known natural inhibitor of ADAM17, TIMP3
(Lee et al., 2004
), is
specifically downregulated in and around invading TEBs. Thus, even though
ADAM17 is ubiquitously expressed, local downregulation of its inhibitor would
tend to increase its net activity in and around TEBs, thereby augmenting the
local release of its only readily available substrate, AREG, and enhancing
EGFR activation on nearby cells. However, the upregulation of TIMP1 in TEBs
may offset the absence of TIMP3 as far as other metalloproteinases are
concerned, while having no direct effect on ADAM17-mediated signaling.
G-protein-coupled receptors can induce ADAM17-mediated release of AREG and
transactivation of EGFR in culture
(Gschwind et al., 2003
;
Lemjabbar et al., 2003
);
however, it remains unclear how they do so, whether they regulate ADAM17 in
mammary epithelium or which receptor agonists may be physiologically relevant.
Phosphorylation of the cytoplasmic domain of ADAM17 appears to regulate
processing of some substrates
(Diaz-Rodriguez et al., 2002
;
Fan et al., 2003
), whereas the
cytoplasmic domain is dispensable for the processing of others
(Reddy et al., 2000
). Integrin
5ß1 may also influence ADAM17 activity
(Bax et al., 2004
), and
Eve-1/Sh3d19, which binds to the cytoplasmic domain of various ADAMs, appears
to promote the processing of EGFR ligands, including AREG
(Tanaka et al., 2004
).
Interestingly, our microarray data indicate that Sh3d19 expression mirrors
that of ADAM17 in developing mammary gland.
Does EGFR act alone or in concert with other ERBB receptors?
It is unclear whether EGFR forms homodimers or heterodimers with other ERBB
receptors during mammary development. One argument favoring the formation of
homodimers is that EGFR is enriched in the mammary stroma, whereas ERBB2 is
mainly expressed in the epithelium, ERBB3 is not detected until mammary glands
mature, and ERBB4 is only expressed during pregnancy and lactation
(Schroeder and Lee, 1998). Our
data indicate that stromal EGFR regulates mammary development, yet ductal
development is also impaired in transgenic mice that express dominant-negative
EGFR in the epithelium alone (Xie et al.,
1997
). Although this could reflect downregulation of ERBB2
signaling, transgenic expression of dominant-negative ERBB2 causes alveolar
defects that only become apparent at parturition
(Jones and Stern, 1999
).
Nevertheless, Erbb2/ mammary glands do
exhibit delayed ductal penetration and TEB defects when transplanted to
cleared fat pads, but eventually catch up and undergo lactational
differentiation (Jackson-Fisher et al.,
2004
). In this case, only epithelial ERBB2 is required, as the
host fat pads contain ERBB2 (and EGFR). Indeed, selective ablation of ERBB2 in
mammary epithelial cells yields a similar phenotype
(Andrechek et al., 2005
).
Because ERBB2 has no known ligand, it requires a co-receptor; yet ERBB3 and
ERBB4 are in short supply during ductal development and our data suggest that
epithelial EGFR is expendable. Thus, epithelial EGFR-ERBB2 interactions,
though not absolutely essential, may still influence the rate of ductal
development, a parameter not specifically addressed in our study. Although our
organotypic culture experiments show that the mutant epithelium is competent
to grow and branch, the ability of the organoids to respond to EGFR ligands in
culture may also reflect the possibility that epithelial EGFR signals
contribute to normal ductal development in a non-essential way. Nevertheless,
prior studies (Simian et al.,
2001
) and our examination of the current organoid cultures show
that at least 12% of the cells in these organotypic cultures are stromal in
origin and may, therefore, also contribute to organoid growth and
branching.
EGFR may also interact with ERBB3 or ERBB4, although the latter only
appears to affect lobuloalveolar development. Genetically rescued
Erbb4/ mice develop alveolar defects during
pregnancy and lactation, yet their ductal development often surpasses that of
their wild-type siblings (Tidcombe et al.,
2003). Likewise, mice that express a mammary-targeted,
dominant-negative ERBB4 or lack the ERBB4 ligand NRG1
display impaired
alveolar differentiation but normal ductal development
(Jones et al., 1999a
;
Li et al., 2002
). Although
these alveolar effects could also involve EGFR, our data suggest that EGFR is
not necessary for alveolar development in response to estradiol or prolactin
(Wiesen et al., 1999
).
However, EGFR ligand-deficient dams do display more compact alveoli than
wild-type mothers during true pregnancy and lactation, when other important
stimuli, such as placental lactogens, also participate, although this could
also reflect crowding as a result of impaired ductal outgrowth
(Luetteke et al., 1999
). Thus,
some EGFR ligands may affect lactational differentiation. Indeed, EGF is
strongly upregulated during late pregnancy and lactation
(D'Cruz et al., 2002
;
Schroeder and Lee, 1998
),
although any effects it might have could still be independent of EGFR, as
ERBB4 has high affinity for EGF in the presence of ERBB2
(Jones et al., 1999b
).
Clearly, the unique growth pattern we observed in culture in response to
NRG1ß1 did not require EGFR, as it occurred in
Egfr/ organoids. However, the implication
that ERBB3 or ERBB4 can affect mammary cell growth independently of EGFR does
not necessarily mean that EGFR signaling occurs independently of ERBB3 or
ERBB4. Thus, it remains unclear whether EGFR and ERBB4 interact during
pregnancy and lactation, whereas it is unlikely that they do so during ductal
development, as ERBB4 is neither necessary nor expressed at that time.
How do AREG-activated stromal cells regulate mammary epithelial development?
Because mammary epithelial development requires stromal EGFR, reciprocal
stromal-to-epithelial responses must also contribute. Other metalloproteinases
undoubtedly affect branching downstream of ADAM17, as TIMP1 inhibits branching
in culture and in vivo (Fata et al.,
1999), even though it does not inhibit ADAM17. Moreover,
broad-spectrum metalloproteinase inhibitors block organoid growth in response
to EGF and KGF (Simian et al.,
2001
; Wiseman et al.,
2003
), yet the absence of ADAM17 alone does not. Notably, AREG
administration induces expression of the matrix metalloproteinase (MMP)
inducer EMMPRIN, MMP2 (gelatinase A) and MMP9 (gelatinase B) in cultured
breast epithelial cells (Menashi et al.,
2003
). Moreover, the activator of latent MMP2, MMP14 (MT1-MMP), is
strongly induced by EGFR activation in neonatal lung and cultured embryonic
fibroblasts (Kheradmand et al.,
2002
), and is present at high levels in the stromal cells adjacent
to invading mammary TEBs (Wiseman et al.,
2003
). Indeed, our data show that MMP2 and MMP3 (stromelysin 1)
regulate mammary ductal morphogenesis in vivo
(Wiseman et al., 2003
) and
that MMP14 promotes ductal development by activating MMP2 and degrading type I
collagen (M. Egeblad, B. S. Wiseman, M.D.S. and Z.W., unpublished).
Nevertheless, the collagen accumulation that characterizes
Mmp14/ mammary glands is absent in
Adam17/, Areg/ and
Egfr/ glands (M.D.S. and Z.W., unpublished),
either because EGFR does not regulate MMP14 during mammary development or
because collagen deposition and remodeling are not elicited in the absence of
ductal development itself. Moreover, because MMP14 is membrane bound, it can
influence epithelial behavior only indirectly, unless it is also shed.
Stromal FGF2 and FGF7/KGF may also act downstream of EGFR and may do so
directly, as they support the growth and branching of
Egfr/ mammary organoids. Indeed, their
receptor, FGFR2B, is expressed on mammary epithelial cells and is required for
the initial formation of embryonic mammary placodes, as is FGF10
(Veltmaat et al., 2003). Thus,
a full understanding of their role in subsequent processes, such as branching,
will require the analysis of conditional deletion models. However, no mammary
phenotype has been described in FGF7-deficient mice, possibly owing to
compensatory mechanisms. Nevertheless, stromal FGFs and their epithelial
receptors have been shown to play critical roles in branching of the tracheal
system in Drosophila and in mammalian lung, salivary gland and kidney
branching, suggesting that similar signaling mechanisms may influence mammary
branching as well (Affolter et al.,
2003
).
The pathway that we have elucidated is undoubtedly part of a larger cascade
of signals that pass back and forth between neighboring cells of the
developing mammary gland. In addition, similar pathways undoubtedly contribute
to other biological processes and may be hijacked or corrupted during the
onset and evolution of disease. Indeed, ADAM17, AREG, TGF and EGFR are
often upregulated in human breast cancer, with co-expression of the latter two
indicating a worse prognosis (Desruisseau
et al., 2004
; Lendeckel et
al., 2005
; Umekita et al.,
2000
). Moreover, experimental data show that these molecules can
actively contribute to the development and progression of cancer
(Borrell-Pages et al., 2003
;
Brandt et al., 2000
;
Gschwind et al., 2003
). Thus,
a clearer understanding of the mechanisms that regulate ADAM17-AREG-EGFR
signaling under normal circumstances will be crucial to overcoming them when
they go awry.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/17/3293/DC1
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