Correspondence to: David F. Stern, Department of Pathology, BML 342, Yale University School of Medicine, P.O. Box 208023, New Haven, CT 06520-8023. Tel:(203) 785-4832 Fax:(203) 785-7467 E-mail:stern{at}biomed.med.yale.edu.
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Abstract |
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Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4IC) as a transgene in the mammary gland. Despite the expression of ErbB4
IC from puberty through later stages of mammary development, an ErbB4
IC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4
IC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, ß-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and
-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4
IC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.
Key Words: ErbB4, Stat5, dominant-negative mutant, transgenic mice, mammary gland development
ABERRANT signaling activity by members of the epidermal growth factor receptor (EGFR)1 family of tyrosine kinases frequently occurs in human cancer. The family consists of the EGFR (HER), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors are activated by binding of growth factors in the EGFR family, which are encoded by at least nine genes. The ligand-activated receptors can signal either through homodimerization or through heterodimerization with other EGFR family members. Each receptor/ligand combination has the potential to recruit and activate a unique set of interacting proteins, thereby initiating signaling cascades which culminate in distinct cellular responses (reviewed in
Overexpression of three of these receptors, EGFR, ErbB2, and ErbB3, is associated with carcinogenesis. For example, amplification of EGFR and ErbB2, or both, occurs in a large subset of breast carcinomas, and is associated with poor prognosis or the presence of negative prognostic indicators (reviewed in
The role of ErbB4 in mammary differentiation is further supported by patterns of receptor expression and activation during normal mouse mammary gland development. Unlike EGFR and ErbB2, which are expressed and activated during puberty ( (TGF
), induces epithelial differentiation, with expression of the milk protein ß-casein, when implanted in mammary glands of virgin mice (
Stat5, a member of the signal transducers and activators of transcription (Stat) family, also appears to play an important role in mammary differentiation. Stat5 was initially identified as mammary gland factor in nuclear extracts from lactating mice (
Although the timing of expression of ErbB4 ligands and activation of ErbB4 suggest that ErbB4 regulates differentiation, the function of ErbB4 signaling during mammary gland development is not known. To identify the role of ErbB4 signaling during breast development, we inactivated endogenous ErbB4 signaling in the mouse mammary gland through transgenic expression of a mutant ErbB4 with dominant-negative activity. The results identify a role for ErbB4 in terminal differentiation of mammary epithelium, including activation of the important mediator of mammary differentiation, Stat5.
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Materials and Methods |
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Plasmids
pMMTV-ErbB4IC contains a truncated human ErbB4 cDNA in which sequences encoding ErbB4 up to P705 are fused to sequences encoding tandem Flag epitope tags (Kodak), immediately followed by two stop codons. The coding sequences are under regulatory control of a mouse mammary tumor virus (MMTV) long terminal repeat. The vector pMMTV-SV40-Bssk (pMMTV-GAL4/236-SV40 minus the GAL4/236 gene;
IC was produced by trimolecular ligation joining the HindIII (site filled with T4 DNA polymerase)-EcoRI digested pMMTV-Aat; the 3' ~2.1 Kb SalI (site filled with T4 DNA polymerase)-SpeI fragment from pLXSN-ErbB4 (
The riboprobe template pBl-ErbB4IC was produced by subcloning the 285-bp SpeI-EcoRI digested PCR product described above into pBluescript I S/K (Stratagene). Riboprobe template pBl-ß-casein was generated by subcloning the 170-bp HindIII-StuI fragment from pFLAG1-ß-casein (generously supplied by Dr. Nancy Hynes, Friedrich Miescher Institute, Basel, Switzerland) into HindIII-SmaI digested pBluescript I S/K. The riboprobe template pBl-WAP, for quantifying mouse whey acidic protein (WAP) RNA, was generated by reverse transcriptase PCR (RT-PCR) of mouse mammary gland RNA isolated from a female at 3-d postpartum using the downstream primer 5'-CTATCTGCATTGGGCACGGCCCGG (nt 319296;
-lactalbumin, was generated by RT-PCR with the downstream primer 5'-GGGCTTCTCACAACGCCACTGTTC (nt 439416;
Generation of MMTV-ErbB4IC Transgenic Mice
Vector sequences for microinjection were separated from pMMTV-ErbB4IC by digestion at unique AatII-XhoI sites. The ~6.2-kb fragment containing the MMTV LTR, a 600-bp 5' untranslated region of c-Ha-ras, the truncated human ErbB4 cDNA with COOH-terminal tandem Flag epitope tags, and simian virus 40 3' mRNA processing signals, was purified by agarose gel electrophoresis. The purified DNA fragment was microinjected into single-cell B6SJL/F2 zygotes at a concentration of 12 µg/ml in 10 mM Tris, pH 7.5, 0.1 mM EDTA (by Ms. Carole Pelletier under the direction of Dr. David Brownstein at the Transgenic Mouse Shared Resource of the Yale University School of Medicine, New Haven, CT).
Identification of Transgenic Mice by PCR
Transgenic mice were identified by PCR of DNA isolated from tail biopsies. DNA purification, PCR conditions, and controls have been described previously (IC fragment were 5'-CAAGTATGCTGATCCAGATCGGGA (nt 18271850 from ErbB4 open reading frame;
IC.
RNA Isolation and RNase Protection Assay
RNA was isolated from the number 4 inguinal mammary gland by TRIzol extraction (GIBCO BRL). Riboprobe synthesis and purification, and RNA analysis using the RPA II ribonuclease protection assay kit (Ambion) were performed as described (
Tissue Preparation for Histological Analysis
For hematoxylin/eosin staining, immunohistochemistry, and in situ hybridization, a portion of the number four inguinal mammary gland was spread onto a glass microscope slide and fixed in freshly prepared 4% paraformaldehyde in PBS (15 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 3 mM KCl, pH 7.2) overnight at 4°C. The fixed tissue was embedded in paraffin and 6-µm sections were dried onto gelatin-coated slides using standard procedures.
Immunohistochemistry
Immunohistochemical detection of Flag-tagged ErbB4IC and Stat5 was performed as described elsewhere (
For immunohistochemical detection of Stat5 phosphorylated at Y694, sections were pretreated to expose phosphorylated Stat5 epitopes. Deparaffinized sections were treated with 1 mg/ml of buffered trypsin (Sigma Chemical Co.) for 20 min at 37°C. Endogenous peroxidase activity was inactivated by incubating the sections in 0.5% H2O2 in PBS for 15 min at room temperature. The sections were incubated in 2 N HCl for 1 h at room temperature followed by two washes in 100 mM borate buffer, pH 8.5, for 5 min per wash. The sections were treated with 0.2% NP-40 for 30 min at room temperature. Between each treatment the sections were washed twice in PBS for 5 min per wash. The remainder of the procedure was performed as described elsewhere (
Immunostained sections were lightly counterstained in hematoxylin (Polysciences Inc.) or methyl green (Vector Labs, Inc.) according to the manufacturer's instructions, dehydrated in EtOH, cleared in xylene, and coverslipped with Permount (Fisher Scientific Co.).
Riboprobe Synthesis and Purification
Buffers used for riboprobe synthesis and transcript purification were generally pretreated with DEPC. For in situ hybridization experiments, DNA template was linearized for sense and antisense riboprobe synthesis and contaminating ribonucleases were inactivated by proteinase treatment at 37°C for 1 h in 10 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.6% SDS, and 150 µg/ml proteinase K. In vitro transcription and subsequent DNase treatment were performed using a MAXIscript in vitro transcription kit (Ambion) with 1 µg of template DNA and 130 µCi of 35S-UTP (DuPont-NEN) exactly as described by the manufacturer. Transcripts were suspended to 200 µl in a final concentration of 10 mM DTT (Sigma Chemical Co.), 300 mM NaOAc, and 20 µg of t-RNA, and purified by EtOH precipitation with 2 M NH4OAc. The precipitated RNA was washed extensively in 70% EtOH, resuspended into 10 mM DTT, and precipitated and washed a second time. The final RNA pellet was resuspended into 10 mM DTT.
In Situ Hybridization
In situ hybridization was performed on 6-µm paraffin sections of mammary glands from female mice at 1- and 12-d postpartum using 35S-UTP labeled riboprobes. Sections were deparaffinized in xylene, washed in 100% EtOH, and defatted by incubating with chloroform for 5 min. The sections were hydrated through a descending EtOH series and washed in PBS for 5 min. The tissue was etched in 2 µg/ml of protease K in PBS for 10 min at 37°C and rinsed in PBS. Tissue sections were postfixed in 4% paraformaldehyde in PBS for 10 min, quenched with 0.2% glycine in PBS for 5 min, and washed in PBS for 5 min. Nonspecific binding sites were blocked by incubating the sections for 10 min in 100 mM triethanolamine (Sigma Chemical Co.), pH 8.0, 0.9% NaCl, containing 0.25% acetic anhydride (Sigma Chemical Co.). The slides were washed in 2x SSC (20x SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.0) for 5 min, dehydrated through an ascending ethanol series, treated with chloroform for 5 min, washed two times with 100% ethanol for 2 min per wash, and air dried.
The hybridization mixture contained 10 mM Tris, pH 7.5, 600 mM NaCl, 2 mM EDTA, 10 mM DTT, 1x Denhardt's (Sigma Chemical Co.), 500 µg/ml total yeast RNA (Ambion), 100 µg/ml poly-A (Pharmacia Biotech, Inc.), 100 mg/ml dextran sulfate (Sigma Chemical Co.), 50% deionized formamide (Ambion), and 4 x 104 cpm/µl of 35S-UTPlabeled riboprobe, and was heated at 80°C for 10 min immediately before use. To each section, 50 µl of hybridization mixture was applied, the sections were overlaid with parafilm coverslips, and hybridized at 50°C for 16 h in a humid chamber containing 10 mM Tris, pH 7.5, 600 mM NaCl, 2 mM EDTA, and 50% formamide (Sigma Chemical Co.). After hybridization, the parafilm coverslips were removed and the slides were washed twice at low stringency for 15 min per wash at 50°C in 2x SSC, 50% formamide, and 0.1% ß-mercaptoethanol. Nonhybridized probe was digested by placing the slides in 10 mM Tris, pH 8.0, 500 mM NaCl, containing 20 µg/ml of RNase A (Sigma Chemical Co.) for 30 min at 37°C. The low stringency washes were repeated and the slides were washed an additional two times in 0.1x SSC and 1% ß-mercaptoethanol at 50°C for 15 min per wash. The slides were dehydrated through an ascending ethanol series, with a final concentration of 600 mM NaCl included in ethanol solutions under 80%, and air dried. Dried slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1:1 with ddH2O at 45°C, and were exposed at 4°C in light-tight slide boxes containing silica gel desiccant packets (Sigma Chemical Co.). Before developing, the slides were warmed to room temperature and developed in Kodak D-19 developer for 2.5 min, washed in ddH2O for 30 s, fixed in Kodak fixer for 3 min, and washed in running tap water for 15 min. The sections were lightly counterstained with hematoxylin using the same procedure described for immunohistochemistry.
Cell Transfections
293T cells were transfected using FuGENE6 transfection reagent (Boehringer Mannheim Corp.) according to the manufacturer's instructions. In brief, cells 25% confluent in 100-mm tissue culture dishes were transfected with 500 µl of growth medium without serum, containing 10 µl of FuGENE6 and 2 µg of each plasmid, for a total of 4 µg. The cells were incubated with transfection mixture for 48 h in a humidified incubator at 37°C with 5% CO2. Plasmid pLXSN (
Immunoprecipitation from Cell Extracts and Western Blot Analysis
Transfected 293T cells were lysed in 2.0 ml of ice-cold EBC buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% NP-40, with 1x Complete protease inhibitor cocktail [Boehringer Mannheim Corp.], 1 mM phenylmethylsulfonyl fluoride, and 1 mM pervanadate) on ice for 10 min. The cell lysates were cleared by centrifugation in a SS-34 rotor at 5,000 rpm for 15 min at 4°C. Immunoprecipitation of ErbB4 and Stat5a from 500 µl of lysate was performed by adding 50 µl of preswollen protein ASepharose (Pharmacia Biotech, Inc.) and 2 µg of anti-ErbB4 (C-18; Santa Cruz) or 2 µg of anti-Stat5b (C-17; Santa Cruz;
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Results |
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Transgenic Mice Derived from MMTV-ErbB4IC
Since the embryonic lethality of ErbB4 gene disruption precludes characterization of postnatal mammary development (IC encodes a protein of ~120 kD, as determined by anti-Flag Western blot analysis of stably transfected FR3T3 cells (data not shown). Expression of pMMTV-ErbB4
IC inhibits EGF-stimulated tyrosine phosphorylation of the endogenous EGFR, verifying that, like cytoplasmic deletion mutants of other receptor tyrosine kinases, ErbB4
IC has dominant-negative activity (data not shown). The in vivo specificity of pMMTV-ErbB4
IC dominant-negative activity is described in the Discussion.
To determine the effect of dominant-negative ErbB4 activity within the developing mammary gland, transgenic mice were derived by injecting MMTV-ErbB4IC DNA into the pronuclei of fertilized one-cell zygotes from B6SJL/F2 mice. 19 founders with transgene integration (identified by PCR) were crossed into an FVB strain, and transgene expression by the F2 female offspring was determined by RNase protection analysis. Transgene expression was detected in mammary glands of five week-old female offspring from six different founders (data not shown). The highest levels of transgene expression were observed in the offspring of founders 5963 and 5997. Phenotypic analysis of mammary glands from mice expressing ErbB4
IC was performed on F3 females derived by crossing founder line 5963 F2 mice with FVB strain mice. The phenotype of line 5963 was confirmed by analysis of the second founder line, 5997.
Expression of the MMTV-ErbB4IC Transgene in the Mammary Gland
The temporal expression pattern of ErbB4IC RNA in the mammary gland was determined by RNase protection assay (Fig 1). The riboprobe hybridizes to the extreme 3' end of the ErbB4
IC transgene, including unique sequences encoding the tandem Flag epitope tags, resulting in a protected fragment of 285 bp (Fig 1, lanes 416). Transgene expression was first detected in prepubescent females at 3 wk and expression levels increased slightly with age, reaching maximal expression in the mature nulliparous mammary gland at 10 wk (Fig 1, lanes 46). The apparent decrease in expression at 19 wk (Fig 1, lane 7) was not observed in other experiments. Expression levels were similar from early to mid-pregnancy (Fig 1, lanes 8 and 9), increased at late pregnancy (lane 10), were highest at 1- and 3-d postpartum (Fig 1, lanes 11 and 12), and were reduced from 12-d postpartum (Fig 1, lane 13) through weaning (Fig 1, lanes 1416).
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ErbB4IC Protein Expression Is Associated with Condensed Lobuloalveoli during Lactation
To determine the effects of ErbB4IC expression on female mammary gland development, wholemounts and histological sections were examined from virgin mice at 3, 5, 6, 8, 10, and 19 wk of age; during early (12 d), mid- (16 d), and late (19 d) pregnancy; after parturition at days 3, 6, 9, 12, 15, or 18; and 24 d after weaning. At least three mice were analyzed at each time point. Despite the extensive time frame for transgene expression, and the fact that expression was highest shortly after parturition (Fig 1), the only identifiable phenotypes were detected on day 12 postpartum. The fat pad of a nontransgenic mouse at 12-d postpartum is completely invested with engorged lobuloalveoli displacing stromal adipose cells. Secretory activity is demonstrated by lumens lined with protruding secretory epithelium (Fig 2 A, arrow). Engorged active secretory lobuloalveoli were also observed in ErbB4
IC-expressing mice at 12-d postpartum (Fig 2 B, arrow). In some transgenic mice (3 out of 5 examined), however, a subpopulation of lobuloalveoli failed to expand and contained an unusually high level of lumenal secretory lipids (Fig 2 B, asterisk). Adipose cells were still abundant in this region of the mammary gland fat pad. The condensed lobuloalveoli resembled undifferentiated lobuloalveoli that are normally predominant during late pregnancy. We next used anti-Flag immunohistochemistry to determine if the condensed lobuloalveoli expressed the Flag-tagged ErbB4
IC transgene. Intense cytoplasmic immunostaining of epithelium within condensed lobuloalveoli was observed (Fig 2 D, asterisks). Anti-Flag immunostaining was not observed in distended lobuloalveoli in the same tissue sections (Fig 2 D, arrow). The lack of detectable transgene expression in this subpopulation of lobuloalveoli may be a result of variegated transgene expression. Variegated promoter expression within the mouse mammary gland has been reported for several mammary specific promoters, including the MMTV LTR promoter used in this study (
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Although the alveolar condensation associated with high ErbB4IC expression might be caused by selective growth inhibition or apoptosis, neither BrdU incorporation experiments, nor TUNEL analysis revealed differences between the phenotypically normal and condensed lobuloalveolar populations in ErbB4
IC animals (data not shown). These results suggest instead that ErbB4
IC expression inhibits normal lobuloalveolar development and function at 12-d postpartum.
ErbB4IC Expression Inhibits Milk Gene Expression
ErbB4IC expression at 12-d postpartum impaired lobuloalveolar development, resulting in condensed alveolar structures with pronounced lipid secretory activity. These structures resembled normal undifferentiated lobuloalveoli observed at late pregnancy and parturition. To determine if the ErbB4
IC-expressing lobuloalveoli were lactationally active, we performed in situ hybridization using antisense riboprobes specific for the milk genes ß-casein, WAP, and
-lactalbumin. Serial paraffin sections containing both normal expanded lobuloalveolar structures and condensed lobuloalveoli were examined (Fig 3 A, arrow and asterisks, respectively). ErbB4
IC expression within condensed lobuloalveoli was confirmed by anti-Flag immunohistochemistry (Fig 3 B, asterisks). The sense probes for ß-casein, WAP, and
-lactalbumin yielded similar levels of background hybridization in both expanded and condensed lobuloalveoli (Fig 3C, Fig E, and Fig G, arrows and asterisks, respectively). With antisense probe, equivalent high levels of ß-casein RNA expression was observed in both the normal and ErbB4
IC-expressing lobuloalveoli (Fig 3 D, arrow and asterisks, respectively). However, the ErbB4
IC-expressing lobuloalveoli showed a moderate diminution in WAP expression (Fig 3 F). Strikingly,
-lactalbumin expression was reduced to sense probe background levels in condensed areas, but not in normal areas of the same section (Fig 3 H). The decrease in WAP and the absence of
-lactalbumin expression suggests that terminal differentiation in ErbB4
IC-expressing lobuloalveolar epithelium has been disrupted. Similar in situ hybridization analysis performed on mammary glands from female mice at 1-d postpartum yielded equivalent levels of expression of these genes in transgenic and nontransgenic sisters (data not shown).
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Stat5 Localized to the Nucleus of ErbB4IC-expressing Mammary Epithelium Is Not Phosphorylated at Y694
The condensed lobuloalveoli and pattern of impaired milk gene expression observed in ErbB4IC-expressing mammary tissue resembles mammary defects observed in mice with Stat5 gene disruptions (
IC-expressing lobuloalveoli (Fig 4 D). Strong immunostaining was detected in the nuclei of both normal (compare Fig 4B and Fig C) and ErbB4
IC-expressing lobuloalveoli (compare Fig 4E and Fig F).
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Since functional Stat5 is phosphorylated at Y694 (reviewed in IC (Fig 5 F). The lack of Y694 phosphorylation of nuclear Stat5 in ErbB4
IC-expressing lobuloalveolar epithelium suggests that it is functionally inactive.
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ErbB4 and Stat5 Interaction and ErbB4 Mediated Phosphorylation of Stat5 at Y694 Requires a Functional Stat5 SH2 Domain
Since expression of ErbB4IC appears to inhibit phosphorylation of Stat5 at the regulatory site Y694, it is possible that ErbB4 normally regulates this effector protein during mammary development. To determine if ErbB4 can induce phosphorylation of Stat5a at this site, the proteins were ectopically expressed at high levels in human embryonic kidney 293T cells (Fig 6). Despite high levels of Stat5a expression in transfected cell lysates (Fig 6 H, lanes 9 and 10, open circle), significant Stat5a tyrosine phosphorylation was observed only when Stat5a was coexpressed with ErbB4 (Fig 6 E, lane 10, open circle). This phosphorylation included Y694, since it was detected by the anti-Stat5 phospho-Y694 antibody (Fig 6 G, lane 10). When Stat5a and ErbB4 were coexpressed in 293T cells, they could be cross-coimmunoprecipitated (Fig 6 D, lane 4, E and F, lane 10). Stat5a coimmunoprecipitated with anti-ErbB4 (Fig 6 D, lane 4) and was not phosphorylated at Y694 (Fig 6 C, lane 4), suggesting that phosphorylation of Stat5a results in rapid release of Stat5a from an ErbB4/Stat5a complex.
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To determine the specificity of ErbB4/Stat5a interaction and ErbB4-mediated phosphorylation of Stat5a on Y694, 293T cells were transfected with mutant STAT5a alleles, with inactivating mutations in the SH2 domain (R618 to V) or at Y694 (Y to F). The two Stat5a mutants were expressed at levels comparable to wild-type Stat5a (Fig 6 H, compare lanes 9 and 10 to 11 and 12), but the Stat5a mutants were not phosphorylated at Y694 when coexpressed with ErbB4 (Fig 6 G, lanes 11 and 12). Interestingly, the Stat5a Y694F mutant was tyrosine phosphorylated at sites other than Y694 when coexpressed with ErbB4 (Fig 6 E, lane 12). Alternative tyrosine phosphorylation of the Stat5a Y694F mutant also has been observed in 293T cells when cotransfected with a T cell receptor and Lck tyrosine kinase (Welte, T., and X.-Y. Fu, unpublished observations), and with activation of the EGFR (
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Discussion |
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Members of the EGFR family have important functions during several stages of mammary gland development. Stromal expression of EGFR is required for ductal morphogenesis (
To elucidate the function of ErbB4 during mouse mammary gland development, we inactivated ErbB4 signaling in the developing mouse mammary gland through the directed expression of dominant-negative ErbB4 as a transgene. Despite significant levels of transgene expression throughout pregnancy and even greater levels of expression early postpartum, an ErbB4IC-specific phenotype was not observed until mid-lactation at 12-d postpartum. Lobuloalveoli expressing ErbB4
IC at 12-d postpartum were condensed, with lumens predominantly filled with secretory lipids, a phenotype resembling normal tissue at late pregnancy. Furthermore, the ErbB4
IC-expressing lobuloalveoli failed to terminally differentiate, as evidenced by a lack of
-lactalbumin expression. ErbB4
IC also inhibited Stat5 phosphorylation at Y694, suggesting that Stat5 is an important downstream mediator of ErbB4 signaling during lactation.
The ErbB4IC phenotype is significantly different from the phenotypes observed in transgenic mice harboring MMTV-driven dominant-negative EGFR or ErbB2 (
IC did not affect virgin mammary gland development, but did inhibit lobuloalveolar development at parturition (
IC phenotype described here, and is not accompanied by suppression of mRNA for WAP, or
-lactalbumin (Jones, F., unpublished data). Although the dominant-negative receptors have some ability to inactivate heterologous dimerization partners in vitro, the nonoverlapping phenotypes obtained with dominant-negative EGFR, ErbB2, and ErbB4 suggests that each of these dominant-negative receptors does not act as a pandominant-negative.
Corroborative evidence supporting a role for ErbB4 signaling during mid-lactation comes from the timing of ErbB4 activation during mouse mammary gland development, since ErbB4 tyrosine phosphorylation is dramatically enhanced at 14-d postpartum (
Additional members of the EGFR family and their ligands have been implicated in lobuloalveolar development and lactation. Our in vivo experiments identified a role for ErbB2 signaling in lobuloalveolar development at parturition ( aggravates this defect (
The ErbB4IC-expressing mammary epithelium resembles the phenotype observed in mice with a disrupted Stat5a gene (
IC-expressing lobuloalveoli was not phosphorylated on the regulatory Y694. Phosphorylation of this residue is essential for some Stat5 functions including dimerization and DNA binding (
IC expressing lobuloalveolar epithelium. The current paradigm is that Stat5 phosphorylation at Y694 and subsequent dimerization are essential for Stat5 nuclear localization (
receptor (
The coupling of ErbB4 to Stat5 regulation is reinforced by a survey of the ability of ErbB family receptor combinations to regulate Stats (
In transient transfection assays, ErbB4 induced phosphorylation of Stat5a on Y694, and the two proteins could be coprecipitated in a Stat5 SH2-dependent manner. This suggests that Stat5 is a direct substrate for ErbB4, although we cannot rule out the possible recruitment of a second tyrosine kinase into the complex. Indeed, c-src is an important mediator of Stat5a activation by ErbB family members, and Janus kinases (JAKs) can associate stably with these receptors (
Activation of Stat5 during lactation is thought to be mediated by prolactin receptor (PrlR) signaling (reviewed in
In contrast to expression of the other EGFR family members, expression of ErbB4 in breast cancer is associated with favorable prognosis (
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Footnotes |
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Frank E. Jones' current address is University of Scranton, Institute of Molecular Biology and Medicine, Scranton, PA 18510.
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Acknowledgements |
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We are grateful to Carole Pelletier and David Brownstein at the Transgenic Mouse Shared Resource of the Yale University School of Medicine for producing transgenic animals. We thank Joe Jerry and John Wysolmerski for advice on in situ hybridization. We thank Lothar Hennighausen for supplying Stat5 antiserum. We thank Nancy Hynes for supplying mouse ß-casein cDNA. We thank JoAnn Falato for exceptional administrative assistance. We thank Marc Schwartz, Dhara Amin, Jonathan McMenamin-Balano, and Amy Jackson-Fisher for critical reading of this manuscript. Finally, we thank Rajani Ramabhadran for excellent technical support and other members of the Stern lab for advice and critical insights. This work is dedicated to June Allison, a courageous survivor of breast cancer.
This work was supported by PHS grants R01CA45708 (to D.F. Stern and F. Jones), R01GM55590 (to X.-Y. Fu and T. Welte), the United States Army Medical Research and Material Command grant DAMD17-96-1-6158 (to F. Jones) and Austrian APART Fellowship (to T. Welte).
Submitted: 7 July 1999
Revised: 19 August 1999
Accepted: 30 August 1999
1.used in this paper: EGFR, epidermal growth factor receptor; LPrlR, long prolactin receptor isoform; MMTV, mouse mammary tumor virus; NRG1, neuregulin-1; PrlR, prolactin receptor; SPrlR, short prolactin receptor isoform; Stat, signal transducers and activators of transcription; TGF, transforming growth factor
; WAP, whey acidic protein
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References |
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