1 Department of Structural and Cellular Biology, Tulane Cancer Center, Tulane
University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana
70112-2699, USA
2 Eppley Institute for Research in Cancer and Allied Diseases, University of
Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA
3 Center for Comparative Medicine, School of Veterinary Medicine, University of
California, Davis, California 95616, USA
4 Faculté de Médecine Necker, Paris 75730, France
5 Laboratory of Genetics and Physiology, National Institute of Diabetes,
Digestive, and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland 20892, USA
* Author for correspondence (e-mail: fjones{at}tulane.edu)
Accepted 9 July 2003
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SUMMARY |
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Key words: Differentiation, ERBB, Lactation, Mammary gland development, STAT5, Tissue specific gene deletion, Mouse
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Introduction |
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The ERBB family consists of four receptors, EGFR, ERBB2/HER2/NEU (referred
to here as ERBB2), ERBB3 and ERBB4, which are activated through binding of
EGF-like ligands and subsequent receptor dimerization
(Yarden and Sliwkowski, 2001).
Although expression of ERBB receptors and EGF-like ligands can be detected at
multiple stages of mammary gland development
(Schroeder and Lee, 1998
),
recent biochemical experiments and genetic models suggest that one or more of
the heregulin (HRG) receptors, which include ERBB2, ERBB3 and ERBB4, regulate
mammopoiesis during crucial stages of pregnancy and lactation. For example,
enhanced receptor activation profiles within mammary tissue during pregnancy
and lactation indicate active ERBB receptor signal transduction during these
developmental stages (Schroeder and Lee,
1998
). Furthermore, we addressed the roles of ERBB2 and ERBB4
during mammary development through the independent overexpression of
dominant-negative mutant forms of these receptors within the mammary glands of
transgenic mice (Jones and Stern,
1999
; Jones et al.,
1999
). These experiments suggested that ERBB2 and ERBB4 contribute
to the function of milk-producing alveolar structures during pregnancy and
lactation, respectively. Interestingly, mice with mutations affecting
expression or processing of the ERBB4 ligands, heregulin
(HRG
;
NRG1 - Mouse Genome Informatics) (Li et
al., 2002
) or heparin-binding epidermal growth factor (Hb-EGF)
(Yu et al., 2002
) also exhibit
defects in alveolar functional development. These genetic results, coupled
with the fact that ERBB2 is an orphan receptor and as such must be activated
through heterodimer formation with a ligand-bound ERBB family member, raise
the possibility that ligand-activated ERBB4 plays an important regulatory role
in the mammary gland.
STAT5 belongs to the family of signal transducers and activators of
transcription (STAT), and is an ERBB-activated signaling protein required for
mammary gland development. In the mammary gland, STAT5 is believed to be
activated following a cascade of events involving binding of the lactogenic
hormone prolactin (PRL) to the prolactin receptor (PRLR) (reviewed by
Hennighausen et al., 1997).
Patterns of STAT5 expression and activation are tightly coupled to epithelial
proliferation and differentiation during pregnancy
(Liu et al., 1996
). Indeed,
the essential role of STAT5 activity in mammary development and lactogenesis
was confirmed in mice containing genetic disruptions of the STAT5 isoforms,
STAT5A or STAT5B or both (Liu et al.,
1997
; Miyoshi et al.,
2001
; Teglund et al.,
1998
). Recently, members of the ERBB family have also been shown
to activate STAT5 (Jones et al.,
1999
; Kloth et al.,
2002
; Olayioye et al.,
2001
). Similar to the PRLR, ERBB4 phosphorylates STAT5A at the
regulatory amino acid Y694 in a STAT5A SRC-homology 2 (SH2) domain-dependent
manner (Jones et al., 1999
).
Furthermore, ERBB4 phosphorylates STAT5A at a tyrosine(s) in addition to at
Y694 (Jones et al., 1999
),
raising the intriguing possibility that ERBB4 regulates novel STAT5 activities
through multiple phosphorylation events.
To date, experiments designed to examine the function of ERBB receptors
during mammary gland development have generated compelling functional data
(reviewed by Stern, 2003;
Troyer and Lee, 2001
);
however, mechanistic information is required to firmly establish the
contribution of ERBB signaling to mammopoiesis. Although we have previously
reported a mammary gland phenotype in mice expressing a dominant-negative
ERBB4 protein (Jones et al.,
1999
), pervasive ERBB-heterodimer formation means that a
dominant-negative mutant receptor could, theoretically, inhibit signaling by
all co-expressed ERBB family members. Analysis of ERBB4 function in the
developing breast is further hampered because genetic deletion of
Erbb4 alleles results in an embryonic lethal phenotype
(Gassmann et al., 1995
). To
overcome the limitations of dominant-negative mutant receptors and the early
lethality of Erbb4-null embryos, we defined the function of ERBB4 in
the mammary gland by deleting both epithelial Erbb4 alleles using a
CRE-LOX recombination strategy (Gu et al.,
1994
). Our results indicate that ERBB4 functions in the pregnant
mammary gland by regulating STAT5 induced epithelial differentiation, and we
demonstrate that ERBB4 is essential for the successful engagement of
lactation. We propose a new mechanism for STAT5 regulation in the developing
breast and provide crucial evidence implicating the ERBB family as essential
local mediators of mammary gland development.
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Materials and methods |
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Embryonic stem cells and the generation of homologous recombinant
clones
Following an established protocol (Nagy
et al., 1993), R1 embryonic stem (ES) cells were transfected with
the pFLRT-ERBB4-targeting construct linearized at a unique restriction site in
the vector sequence. Cells were plated on dishes containing mitomycin
C-treated embryonic fibroblasts (EMFI) and grown in LIF-supplemented ES media.
After positive/negative selection of FLRT-ERBB4 with G418 and FIAU, clones
that had successfully undergone homologous recombination with the
ERBB4-targeting construct (floxed) were identified by PCR, and confirmed by
Southern digests of genomic DNA hybridized with a 32P-labeled neo
probe and an external probe (A probe) generated by PCR of genomic sequence
contained upstream of and outside the targeting construct. ES cell clones that
were confirmed to have undergone homologous recombination were frozen in
liquid nitrogen and used for blastocyst injection.
Germline transmission of the floxed ERBB4 allele
Following an established protocol
(Klein et al., 1993), floxed
ES cell clones were resuspended in phosphate buffered saline at 4°C in
preparation for injection into blastocysts harvested from C57Bl/6 females.
Under microscopic control,
20-30 R1 cells were microinjected into each of
12-16 blastocysts. After injection,
6-10 blastocysts were transferred
surgically into the uterus of timed pseudopregnant CD1 females. Ten to 14 days
after birth (approximately 30 days after injection), chimeras were detected by
their dominant agouti coat color. At 8 weeks of age, chimeric males were bred
with C57Bl/6 females to establish germline transmission of the targeted
allele. Tail genomic DNA was extracted and tested by PCR for presence of the
homologous recombinant floxed allele. The male agouti-colored offspring that
were heterozygous for the floxed allele
(Erbb4Flox/+) were saved for breeding to female
mice that were heterozygous for the floxed allele
(Erbb4Flox/+). Twenty-five percent of offspring
from this mating have two floxed alleles (Erbb4Flox/Flox)
and were used in later matings to generate the tissue-specific deletion of the
Erbb4 gene. Functional transcription of the floxed Erbb4
allele was tested by RT-PCR of mRNA extracted from tissues obtained from
Erbb4Flox/Flox mice. One
Erbb4Flox/Flox line was used for the analysis described in
this report.
Crosses and genotype analysis
All mouse strains were crossed and maintained in a FVB background. Genomic
DNA was isolated from mouse tail biopsies and genotyped by PCR, exactly as
described elsewhere (Jones and Stern,
1999). A 210 bp fragment in the Wap-Cre allele was
amplified using the forward primer (5'-TAGAGCTGTGCCAGCCTCTTC-3')
and the reverse primer (5'-CATCACTCGTTGCATCGACC-3'). Control
(Erbb4+/+Wap-Cre) and experimental
(Erbb4Flox/FloxWap-Cre) mice were generated by crossing
mice with the identical genotype Erbb4+/FloxWap-Cre/+. The
extent of CRE-mediated excision of Erbb4 exon 2 was determined by
Southern blot analysis of 6 µg of mammary gland DNA, digested with
BamHI and probed with a 32P-labeled Erbb4 gene
fragment harboring exon 2. The Erbb4 exon 10 32P-labeled
probe 5'-GCCTCTGAAGGAAATCAGTGCGGG-3' representing nucleotides (nt)
87377-87400 from the mouse Erbb4 gene was used as an internal
control.
Whole-mount staining of mouse mammary glands
The entire number four inguinal mammary gland was excised. The tissue was
spread onto a glass microscope slide, fixed in acidic ethanol and stained in
carmine solution exactly as described previously
(Jones et al., 1996).
Prolactin injections
PRL (National Hormone and Pituitary Program, Torrance, CA), at a
concentration of 5 µg/g body weight, was injected intraperitnoeally (ip)
into biparous Erbb4Flox/FloxWap-Cre mice at P18. After 15
minutes the mice were sacrificed and mammary tissue was processed for
immunohistochemistry.
Progesterone implants
Progesterone with biodegradable carrier (Innovative Research of America)
was administered to pregnant Prlr-/- and
Erbb4Flox/FloxWap-Cre mice as described previously
(Binart et al., 2000).
Tissue preparation for histological analysis
For Hematoxylin and Eosin staining and immunohistochemistry, a portion of
the number four inguinal mammary gland was spread onto a glass microscope
slide and fixed with freshly prepared 4% paraformaldehyde in PBS at 4°C
overnight. Fixed tissue was embedded in paraffin wax and 6 µm sections were
dried onto Snowcoat X-tra slides (Surgipath) using standard procedures.
Immunohistochemistry
Immunohistochemical detection of ERBB4, STAT5 and STAT5 phosphorylated at
Y694 (phospho-STAT5) was performed as described elsewhere
(Jones et al., 1999) with the
following modifications. To detect phospho-STAT5, the primary antibody, goat
anti-P-STAT5 (Santa-Cruz Biotechnology), was diluted between 1-2 µg/ml and
the biotinylated rabbit anti-goat (Vector Labs) secondary antibody was diluted
to 15 µg/ml. Detection of expression of NKCC1 and NPT2B by
immunofluorescence has been described elsewhere
(Miyoshi et al., 2001
).
Injection of mice with Bromodeoxyuridine (BrdU) Cell Labeling Reagent
(Amersham), BrdU immunohistochemistry, and statistical analyses were performed
exactly as described elsewhere (Li et al.,
2002
). Significant differences between data sets was determined by
calculating the means and standard deviations of at least 250 epithelial
nuclei from at least four independent animals at each time point. The
Student's t-test was performed at each developmental time point. In
all experiments, the DAB substrate was prepared fresh before use by adding
hydrogen peroxide to 0.03% in 50 mM Tris (pH 7.6) containing 1.7 mM
3'-diaminobenzidine tetrahydrochloride (Sigma).
Sections were lightly counterstained in Hematoxylin (Polysciences) according to the manufacturer's instructions, then dehydrated in ethanol, cleared in xylene and coverslipped with Permount (Fisher).
RNA isolation and northern blot analysis
Total mammary gland RNA was isolated by TRIzol (Invitrogen) extraction,
according to the manufacturer's instructions, using 200 mg of tissue from the
number four inguinal mammary gland that was previously snap-frozen in liquid
nitrogen and stored at -80°C. Expression of ß-casein, WAP and
-lactalbumin was detected in 10 µg of total mammary gland RNA by
northern blot exactly, as described previously
(Li et al., 2002
).
Immunoprecipitation and western blot analysis
Tissue protein lysates were prepared from mammary glands and
immunoprecipitated proteins were analyzed by western blot essentially as
described elsewhere (Schroeder and Lee,
1998), with the following modifications. The Triton X-100 lysis
buffer contained 1 mM phenylmethylsulfonyl fluoride and Complete (Roche
Diagnostics) as protease inhibitors, and the phosphatase inhibitors 10 mM NaF,
1 mM sodium orthovanadate and Phosphatase Inhibitor Cocktail II (Sigma).
Immunoprecipitation and western blot analysis was performed as described
elsewhere (Jones et al.,
1999
), using the same primary antibodies described for
immunohistochemistry. Proteins containing phosphotyrosine residues were
detected using the primary antibody p-Tyr (Santa-Cruz Biotechnology).
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Results |
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ERBB4 contributes to lobuloalveolar development
To determine the impact of ERBB4 ablation on mammary gland development, we
examined the number four inguinal mammary glands from biparous
Erbb4Flox/FloxWap-Cre and
Erbb4+/+Wap-Cre mice at multiple stages of
pregnancy and lactation. Development of the normal breast during pregnancy and
through parturition is characterized by a dramatic increase in epithelial
proliferation, which results in the accumulation of lobuloalveolar structures.
Fully developed lobuloalveoli with differentiated epithelium become the
milk-producing structures during lactation. Mammary tissue from
Erbb4+/+Wap-Cre mice accumulated lobuloalveoli at
P13.5 of the second pregnancy (Fig.
3A, arrow), culminating in extensive lateral and terminal
lobuloalveolar outgrowths by P18.5 (Fig.
3C, arrow). At parturition, engorged lobuloalveoli
(Fig. 2E, arrow) masked the
underlying ductal system (Fig.
2E, arrowhead). By contrast, a reduction in lobuloalveolar
outgrowth was observed in biparous Erbb4Flox/FloxWap-Cre
mice at P13.5 (Fig. 3B, arrow),
the earliest pregnancy time-point examined. By P18.5 large regions of mammary
tissue exhibited sparse lobuloalveolar expansion with ducts bearing few
lateral and terminal alveoli (Fig.
3D, arrow). At parturition, fully distended ducts were observed
(Fig. 3F, arrowhead); however,
they bore few engorged lobuloalveoli (Fig.
3F, arrow). Mammary glands from
Erbb4Flox/FloxWap-Cre dams that did not support litters
underwent extensive involution by L3 and were completely regressed by L10
(data not shown).
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We have previously observed NKCC1 expression at high levels on the
basolateral membrane of mammary ductal epithelium from nulliparous mice. The
specification of ductal epithelium to a secretory phenotype is accompanied by
a diminution of NKCC1 levels during pregnancy and at parturition
(Miyoshi et al., 2001).
Likewise, relatively high levels of NKCC1 expression were observed in biparous
Erbb4+/+Wap-Cre and
Erbb4Flox/FloxWap-Cre mammary epithelium at P13
(Fig. 6A,B; arrowheads), and
lower levels were observed at P18 (Fig.
6C,D). NKCC1 expression was dramatically reduced at L1
(Fig. 6E,F). These results
indicate that ductal epithelium from biparous
Erbb4Flox/FloxWap-Cre mice successfully undergoes cellular
specification, forming alveolar secretory epithelium. Smooth muscle actin
(SMA) was expressed within myoepithelial cells of all mammary glands examined
(Fig. 6A-F)
|
ERBB4 regulates STAT5 activation
The defects in lobuloalveolar accumulation, absence of epithelial NPT2B
expression and lactational failure observed in biparous
Erbb4Flox/FloxWap-Cre mice are similar to the mammary
gland phenotypes described for Stat5a-null mice
(Liu et al., 1997;
Miyoshi et al., 2001
;
Teglund et al., 1998
). To
determine whether ERBB4 is required for the functional activation of STAT5 in
mammary epithelium, we examined STAT5A phosphorylation at the regulatory amino
acid Y694 (phospho-STAT5) by immunohistochemistry and western blot.
Prominent nuclear staining of phospho-STAT5 was observed within alveolar epithelium of biparous Erbb4+/+Wap-Cre mammary glands at P13.5 (Fig. 7A, arrowhead), P17.5 (Fig. 7C) and L1 (Fig. 7E). Immunohistochemistry using an antibody that detects both phosphorylated and unphosphorylated STAT5 populations (STAT5) indicated that, as expected, the majority of STAT5 protein was localized within epithelial nuclei of Erbb4+/+Wap-Cre mammary glands at L1 (Fig. 7G, arrowhead). Although phospho-STAT5 was detected within alveolar epithelium of biparous Erbb4Flox/FloxWap-Cre mammary tissue at P13.5, the majority of the activated STAT5 protein remained cytoplasmic (Fig. 7B, inset arrow), with less than 50% of nuclei showing phospho-STAT5 staining (Fig. 7B, inset arrowhead). Strikingly, phospho-STAT5 immunohistochemistry failed to reveal activated STAT5 within ERBB4-deficient mammary epithelium at either P17.5 (Fig. 7D) or L1 (Fig. 7F). Furthermore, STAT5 protein was expressed but was excluded from nuclei of Erbb4Flox/FloxWap-Cre mammary epithelium at L1 (Fig. 7H, arrow).
Inactivation of STAT5 in Erbb4Flox/FloxWap-Cre mammary tissue was confirmed by the analysis of ERBB4 and STAT5 proteins immunoprecipitated from biparous Erbb4+/+Wap-Cre and Erbb4Flox/FloxWap-Cre mammary tissue at L1. Anti-ERBB4 immune complexes, analyzed by western blot with an ERBB4 antibody, confirmed the absence of ERBB4 in Erbb4Flox/FloxWap-Cre mammary tissue (Fig. 7I, ERBB4/ERBB4). Western blot analysis of anti-STAT5 immune complexes, probed with a STAT5 antibody, revealed equivalent amounts of STAT5 protein in control and Erbb4Flox/FloxWap-Cre mammary glands (Fig. 7I, STAT5/STAT5). In addition, phosphorylation of STAT5 immunoprecipitated from control lysates was demonstrated by western blot analysis using both a phosphotyrosine antibody (Fig. 7I, STAT5/P-tyr) and a specific antibody that recognizes STAT5 phosphorylated at Y694 (Fig. 7I, STAT5/P-STAT5). Consistent with immunohistochemical results, STAT5 protein present in STAT5 immune complexes prepared from Erbb4Flox/FloxWap-Cre mammary tissue lacked detectable tyrosine phosphorylation when probed with the phosphotyrosine antibody (Fig. 7I, STAT5/P-tyr) or the antibody specific for STAT5 phosphorylated at Y694 (Fig. 7I, STAT5/P-STAT5). The relative epithelial cell populations in Erbb4+/+Wap-Cre control and Erbb4Flox/FloxWap-Cre mammary gland lysates was compared by probing 50 µg of total lysate with an antibody specific for keratin 18 (Fig. 7I, NA/K18). Taken together, the immunohistochemical and western blot data implicates ERBB4 as a crucial mediator of STAT5 activation during late pregnancy and at parturition.
ERBB4-null mammary glands fail to express STAT5 regulated milk
genes
STAT5 transactivates the expression of several milk genes, including
casein beta (csnb) and Wap, which harbor canonical
STAT5 binding -interferon activation sites (GAS) within their promoters
(Rosen et al., 1999
). STAT5
function in mammary glands from biparous
Erbb4Flox/FloxWap-Cre mice was assessed by northern blot
analysis of milk-gene expression. As predicted high levels of ß-casein
and WAP expression, with lower levels of
-lactalbumin expression, were
detected in mammary glands from biparous
Erbb4+/+Wap-Cre mice at L1
(Fig. 8; lanes 1,2). By
contrast, expression of ß-casein and WAP was drastically reduced in
mammary tissue from biparous Erbb4Flox/FloxWap-Cre mice at
L1, whereas expression of
-lactalbumin appeared unaffected by the
absence of ERBB4 (Fig. 8; lanes
3,4). Detection of GAPDH (GAPD - Mouse Genome Informatics) expression
confirmed equal RNA loading in each lane
(Fig. 8; lanes 1-4). Impaired
expression of csnb and Wap, two genes directly regulated by
STAT5, demonstrates that STAT5 function is impaired in biparous
Erbb4Flox/FloxWap-Cre mice at L1. Although the
-lactalbumin gene also harbors a GAS element
(Rosen et al., 1999
),
consistent with our observations, direct regulation of
-lactalbumin by
STAT5 lacks experimental confirmation. Taken together, these results indicate
that the inability of Erbb4Flox/FloxWap-Cre dams to
support their young is, in part, caused by the impaired expression of
STAT5-regulated genes that encode essential milk proteins.
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Discussion |
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Our results demonstrating the essential contribution of ERBB4 signaling to
breast development are corroborated by recent experiments from Martin Gassmann
and colleagues (Tidcombe et al.,
2003). Heart defects associated with embryonic lethality of mice
with deletions of both Erbb4 alleles were rescued by expressing ERBB4
under the control of a cardiac-specific promoter
(ERBB4-/-HER4heart). The
ERBB4-/-HER4heart mice survived to adulthood; however,
they failed to lactate at parturition
(Tidcombe et al., 2003
).
Severe lactational defects in uniparous
ERBB4-/-HER4heart mice underscores the essential
contribution of ERBB4 signaling to pregnancy-induced mammary development and
supports the conclusion that the absence of a lactation phenotype in uniparous
Erbb4Flox/FloxWap-Cre mice results from incomplete WAP-CRE
mediated deletion of Erbb4 during the first pregnancy.
There is limited phenotypic overlap between the biparous
Erbb4Flox/FloxWap-Cre mice described herein and our
previous description of mammary gland defects in mice overexpressing a
dominant-negative ERBB4 transgene (ERBB4IC). Indeed, significant
differences in the severity and temporal presentation of lobuloalveolar
defects were observed. Most noteworthy is that condensed lobuloalveoli at
mid-lactation was a distinct feature of ERBB4
IC-expressing mammary
glands (Jones et al., 1999
).
However, this phenotype was present in less than 5% of the mammary gland and
ERBB4
IC failed to impact mammary gland function. Interpretation of
results from ERBB4
IC mice is complicated because dominant-negative ERBB
proteins suffer from non-specific pan-dominant effects, potentially
attenuating signaling through all four ERBB receptors. Indeed, despite
complete ablation of epithelial ERBB4 expression during the first lactation
(see Fig. 2D), a mid-lactation
phenotype was not observed in uniparous
Erbb4Flox/FloxWap-Cre mice. The lack of phenotypic overlap
between ERBB4
IC and uniparous Erbb4Flox/FloxWap-Cre
mice at mid-lactation underscores the fact that ERBB4
IC harbors
non-specific activity and directly impacts mammary developmental pathways in
addition to ERBB4. Our current genetic experiments clearly indicate that the
essential contribution of ERBB4 signaling to breast development and lactation
occurs during pregnancy and at parturition.
Multiple ERBB4 activities in the mammary gland
Our current results indicate that ERBB4 has essential functions during
pregnancy-induced epithelial proliferation and during the differentiation of
secretory epithelium at parturition. This functional dichotomy suggests that
activated ERBB4 couples to divergent signaling pathways during breast
development. Ligand-induced ERBB heterodimerization represents an important
mechanism driving ERBB signal diversification (reviewed by
Alroy and Yarden, 1997).
However, it remains unclear whether ERBB4 regulates development as a signaling
homodimer or through heterodimerization with other ERBB family members.
Indeed, all four ERBB receptors are highly phosphorylated within the mammary
gland at parturition (Schroeder and Lee,
1998
), and each may therefore contribute to ERBB4 signaling at
this developmental stage. For example, waved 2 mice, harboring a
mutant EGFR with reduced tyrosine kinase activity
(Luetteke et al., 1994
),
exhibit impaired alveolar development and lactational defects
(Fowler et al., 1995
) similar
to those seen in ERBB4-deficient mammary glands.
Experimental evidence implicates ERBB2 as the central mediator of
ligand-induced signaling through the ERBB family
(Graus-Porta et al., 1997) and,
as such, it may contribute to ERBB4 function in the developing mammary gland.
In support of this, we have previously reported alveolar developmental defects
at parturition in transgenic mice expressing a dominant-negative ERBB2
receptor (ERBB2
IC) (Jones and
Stern, 1999
). Analysis of milk-gene expression by in situ
hybridization revealed reduced levels of ß-casein and WAP transcripts in
alveolar epithelium expressing ERBB2
IC (F.E.J. and D. Stern,
unpublished). In addition, mice lacking the ERBB4 ligand HRG
exhibit
impaired epithelial proliferation during pregnancy and reduced ß-casein
expression at parturition (Li et al.,
2002
). Phenotypic overlap between Hrg
-null,
ERBB2
IC-expressing and ERBB4-deficient mammary glands at parturition
underscores a possible role for HRG
-driven ERBB2/ERBB4 signaling during
pregnancy-induced epithelial proliferation and functional differentiation.
ERBB4 regulates STAT5 function
The mammary gland phenotype observed in
Erbb4Flox/FloxWap-Cre mice was reminiscent of observations
reported for Stat5a-null mice
(Liu et al., 1997;
Miyoshi et al., 2001
;
Teglund et al., 1998
). Loss of
ERBB4 or STAT5A expression results in the accumulation of histologically
identical lobuloalveolar defects during pregnancy, and a failure to lactate at
parturition. The extent of phenotypic overlap observed in
Erbb4Flox/FloxWap-Cre and Stat5a-null mice
suggests that the ERBB4 and STAT5A signaling pathways are directly coupled
during functional differentiation of breast epithelium. In support of this, we
demonstrate by both immunohistochemistry and western blot analysis complete
ablation of STAT5 activation in mammary epithelium from
Erbb4Flox/FloxWap-Cre mice at late pregnancy and
parturition. Similar to STAT5A-null mice, ERBB4-deficient mammary epithelium
fails to express the differentiation marker NPT2B and exhibits a dramatic
reduction in the expression of the STAT5-regulated milk genes csnb
and Wap. Our suggestion that ERBB4 directly activates STAT5 in the
pregnant mammary gland is further supported by our previous results
demonstrating a physical interaction between ERBB4 and STAT5, which resulted
in phosphorylation of the STAT5 protein at the regulatory amino acid Y694 and
at additional novel tyrosine residue(s)
(Jones et al., 1999
).
Pregnancy-induced functional differentiation of mammary epithelium requires
both ERBB4 and PRLR (Ormandy et al.,
1997). Interestingly, loss of either ERBB4 or PRLR leads to
ablation of STAT5 activation (Gallego et
al., 2001
; Miyoshi et al.,
2001
), which suggests that these two pathways cooperate to
activate STAT5. Our previous results identified an early role for PRLR
signaling in cell fate determination during the pregnancy-induced transition
from ductal to secretory alveolar epithelia. PRLR- and STAT5-null epithelium
retained expression of the ductal epithelial marker NKCC1
(Miyoshi et al., 2001
;
Shillingford et al., 2002
). By
contrast, dramatically reduced expression of NKCC1 indicates that
ERBB4-deficient mammary epithelium successfully undergoes pregnancy-induced
cell specification (see Fig.
6). However, despite evidence of intact PRLR signaling (see
Fig. 9), ERBB4-null epithelium
lacks functional STAT5 and fails to express the epithelial differentiation
marker NPT2B. Based upon our current understanding, we propose a novel
mechanism for STAT5 regulation that first requires PRLR signaling at early
pregnancy during STAT5-regulated cellular specification. Then at late
pregnancy, ERBB4 supplants PRLR and functions as the obligate mediator of
STAT5-induced epithelial differentiation and lactation. This model is
supported by our results demonstrating STAT5 activity in mammary glands of
Erbb4Flox/FloxWap-Cre mice at early pregnancy, and by
evidence that PRLR is dispensable for STAT5 activation at late pregnancy. The
molecular switch between PRLR and ERBB4 as the obligate STAT5-regulating
receptor may be driven by enhanced ERBB4-ligand expression at late pregnancy,
and/or by altered STAT5 function mediated by novel ERBB4-induced STAT5
phosphorylation events (Jones et al.,
1999
).
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Conclusions |
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ACKNOWLEDGMENTS |
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