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Mammary Gland Remodeling Depends on gp130 Signaling through Stat3 and MAPK

2004; Elsevier BV; Volume: 279; Issue: 42 Linguagem: Inglês

10.1074/jbc.m313131200

1083-351X

Ling Zhao, Stefan Hart, JrGang Cheng, J. Joseph Melenhorst, Brian Bierie, Matthias Ernst, Colin L. Stewart, Fred Schaper, Peter C. Heinrich, Axel Ullrich, Gertraud W. Robinson, Lothar Hennighausen,

NF-κB Signaling Pathways

The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Δ/Δ). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Δ/Δ mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue. The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Δ/Δ). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Δ/Δ mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue. gp130 is the common receptor subunit of IL6 family cytokines, which include interleukin-6 (IL6), 1The abbreviations used are: IL6, interleukin-6; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; DMEM, Dulbecco's modified Eagle's medium; Stat, signal transducers and activators of transcription; LIF, leukemia inhibitory factor; OSM, oncostatin M; wt, wild type.1The abbreviations used are: IL6, interleukin-6; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; DMEM, Dulbecco's modified Eagle's medium; Stat, signal transducers and activators of transcription; LIF, leukemia inhibitory factor; OSM, oncostatin M; wt, wild type. interleukin-11 (IL11), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNF), oncostatin M (OSM), cardiotrophin-1 (CT-1), interleukin-27 (IL27), neuropoietin (NP), and cardiotropin-like cytokine (CLC) (1Derouet D. 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Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar) and LIF (12Zhao L. Melenhorst J.J. Hennighausen L. Mol. Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar, 13Schere-Levy C. Buggiano V. Quaglino A. Gattelli A. Cirio M.C. Piazzon I. Vanzulli S. Kordon E.C. Exp. Cell Res. 2003; 282: 35-47Crossref PubMed Scopus (74) Google Scholar) gene expression, and the activation of the transcription factor Stat3 (14Liu X. Robinson G.W. Hennighausen L. Mol. Endocrinol. 1996; 10: 1496-1506Crossref PubMed Scopus (209) Google Scholar). While loss of Stat3 resulted in severe retardation of mammary epithelial cell death and impaired remodeling (15Chapman R.S. Lourenco P.C. Tonner E. Flint D.J. Selbert S. Takeda K. Akira S. Clarke A.R. Watson C.J. Genes Dev. 1999; 13: 2604-2616Crossref PubMed Scopus (406) Google Scholar, 16Humphreys R.C. Bierie B. Zhao L. Raz R. Levy D. Hennighausen L. Endocrinology. 2002; 143: 3641-3650Crossref PubMed Scopus (105) Google Scholar), less severe lesions were observed in IL6- (12Zhao L. Melenhorst J.J. Hennighausen L. Mol. Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar) and LIF-null (12Zhao L. Melenhorst J.J. Hennighausen L. Mol. Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar, 17Kritikou E.A. Sharkey A. Abell K. Came P.J. Anderson E. Clarkson R.W. Watson C.J. Development. 2003; 130: 3459-3468Crossref PubMed Scopus (150) Google Scholar) mice.Stat3 can be activated by several cytokines, which signal through distinct receptors, including receptor-tyrosine kinases (RTK), single-chain cytokine receptors, and receptors that share the common subunit gp130. While IL6 family cytokine-induced Stat3 activation is dependent on gp130 and Janus kinases (Jak), Jak-independent activation can be accomplished through the EGFR that possesses an endogenous tyrosine kinase and the ability to activate several other intracellular tyrosine kinases (18Olayioye M.A. Beuvink I. Horsch K. Daly J.M. Hynes N.E. J. Biol. Chem. 1999; 274: 17209-17218Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 19Muller W.J. Arteaga C.L. Muthuswamy S.K. Siegel P.M. Webster M.A. Cardiff R.D. Meise K.S. Li F. Halter S.A. Coffey R.J. Mol. Cell. Biol. 1996; 16: 5726-5736Crossref PubMed Google Scholar).Since IL6 and LIF are key inducers of Stat3 phosphorylation in mammary tissue at the onset of involution it was necessary to explore the possibility that the key signaling events are mediated by their common receptor gp130. Toward this end, two mouse mutants were employed. In one mouse model, the entire gp130 gene was inactivated in mammary epithelium using Cre-loxP-mediated recombination. In the other model, a truncated mutation of the C terminus of gp130 (gp130Δ/Δ) was introduced as the Y765F, Q768A, and 769stop substitutions (20Ernst M. Inglese M. Waring P. Campbell I.K. Bao S. Clay F.J. Alexander W.S. Wicks I.P. Tarlinton D.M. Novak U. Heath J.K. Dunn A.R. J. Exp. Med. 2001; 194: 189-203Crossref PubMed Scopus (205) Google Scholar). This resulted in the loss of the four Stat3 binding sites (Tyr765,812,904,914). However, the SHP2 and Jak2 binding sites were intact, and therefore the emanating signals were functional in this mutant. The physiological lesions imposed by the loss of Stat3 signaling through the gp130Δ/Δ receptor (20Ernst M. Inglese M. Waring P. Campbell I.K. Bao S. Clay F.J. Alexander W.S. Wicks I.P. Tarlinton D.M. Novak U. Heath J.K. Dunn A.R. J. Exp. Med. 2001; 194: 189-203Crossref PubMed Scopus (205) Google Scholar) mimicked those observed in the absence of IL6 (21Kopf M. Baumann H. Freer G. Freudenberg M. Lamers M. Kishimoto T. Zinkernagel R. Bluethmann H. Kohler G. Nature. 1994; 368: 339-342Crossref PubMed Scopus (1499) Google Scholar) or LIF (22Stewart C.L. Kaspar P. Brunet L.J. Bhatt H. Gadi I. Kontgen F. Abbondanzo S.J. Nature. 1992; 359: 76-79Crossref PubMed Scopus (1752) Google Scholar). Since the MAPK pathway was unimpeded in these mice we were able to also explore its contribution to tissue remodeling in involuting mammary epithelium.EXPERIMENTAL PROCEDURESAnimals—All animals used in the course of this study were treated within published guidelines of humane animal care.Inactivation of the gp130 Gene in Mouse Mammary Epithelium—Mice were generated in which loxP sites were inserted into the promoter and intron II, 2J. Cheng and C. Stewart, unpublished data. which made it possible to specifically delete exons I and II (gp130 fl/fl). A transgene encoding Cre recombinase under the control of the WAP gene promoter (23Wagner K.U. Wall R.J. St-Onge L. Gruss P. Wynshaw-Boris A. Garrett L. Li M. Furth P.A. Hennighausen L. Nucleic Acids Res. 1997; 25: 4323-4330Crossref PubMed Scopus (414) Google Scholar, 24Wagner K.U. McAllister K. Ward T. Davis B. Wiseman R. Hennighausen L. Transgenic Res. 2001; 10: 545-553Crossref PubMed Scopus (246) Google Scholar) was bred into gp130 fl/fl mice to achieve recombination in mammary epithelium. These mice are referred to as gp130 fl/fl;WC throughout the text.Transplantation of Mammary Epithelium—LIF-/- (22Stewart C.L. Kaspar P. Brunet L.J. Bhatt H. Gadi I. Kontgen F. Abbondanzo S.J. Nature. 1992; 359: 76-79Crossref PubMed Scopus (1752) Google Scholar), wild type (wt), gp130Δ/Δ (20Ernst M. Inglese M. Waring P. Campbell I.K. Bao S. Clay F.J. Alexander W.S. Wicks I.P. Tarlinton D.M. Novak U. Heath J.K. Dunn A.R. J. Exp. Med. 2001; 194: 189-203Crossref PubMed Scopus (205) Google Scholar), gp130 fl/fl, 2J. Cheng and C. Stewart, unpublished data. gp130 fl/fl;WC, Stat3 fl/fl;WC and Stat3 fl/fl (25Raz R. Lee C.K. Cannizzaro L.A. d'Eustachio P. Levy D.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2846-2851Crossref PubMed Scopus (320) Google Scholar) mice were used as donors. 3-week-old athymic nu/nu mice (DCT, Frederick, MD) were used as recipients. All mice were anesthetized using Avertin (200 μl/10 g BW) prior to surgery. The proximal part of the number four glands (both left and right) containing the epithelium of all recipient mice were excised, and the donor mammary tissue (1 mm3) was transplanted into the cleared fat pad. Eight weeks after transplantation, the nude mice were mated, and pups were removed at birth. The number 3 endogenous gland and both transplanted glands were harvested on the day of birth of pups (L1) and days 1 (i1), 2 (i2), 3 (i3), 4 (i4), and 6 (i6) of involution.E12.5 day embryo mammary anlagen from Jak2-null and wt embryos were transplanted to nude mice as described before (26Shillingford J.M. Miyoshi K. Robinson G.W. Grimm S.L. Rosen J.M. Neubauer H. Pfeffer K. Hennighausen L. Mol. Endocrinol. 2002; 16: 563-570Crossref PubMed Scopus (64) Google Scholar). The transplanted mammary tissue was allowed to grow for 8 weeks before harvest.Histology and Immunohistochemistry/Immunofluorescence—Tissues were fixed in 10% neutral buffered formalin overnight at 4 °C, dehydrated, and embedded in paraffin. Tissue blocks were sectioned at 5 μm and stained with hematoxylin and eosin. For immunostaining, tissue sections on poly-l-lysine-coated slides were deparaffinized in xylene and rehydrated in decreasing ethanol concentrations. All tissue sections were treated with antigen unmasking reagent (Vector Laboratories) by boiling for 10 min following the manufacturer's protocol. The Stat5a antibody (14Liu X. Robinson G.W. Hennighausen L. Mol. Endocrinol. 1996; 10: 1496-1506Crossref PubMed Scopus (209) Google Scholar) was used at a 1:600 dilution. The Stat3 antibody (Santa Cruz Biotechnology) was diluted 1:200. Antibodies for gp130 (Santa Cruz Biotechnology), β-catenin (Transduction Laboratories) and Npt2b (a gift from Dr. Jürg Biber, Department of Physiology, University of Zürich, Zürich, Switzerland) (27Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (312) Google Scholar) were used at a 1:100 dilution. All primary antibodies were incubated with sections overnight at 4 °C. Immunoperoxidase staining was performed according to the manufacturer's protocol (Vector ABC kit, DAB substrate kit, Vector Laboratories). For immunofluorescence, Alexafluor-conjugated secondary antibodies (Molecular Probes) were incubated with sections for 30 min at room temperature in the dark, then mounted in Vectashield (Vector Laboratories) and visualized with a microscope (Olympus) using a mercury bulb for excitation of the fluorescent conjugates.Western Blots—Protein was extracted from frozen tissues (1 g/10 ml lysis buffer), homogenized in lysis buffer (10 mm Tris-HCl, pH 8.0; 5 mm EDTA; 50 mm NaCl; 30 mm Na4P2O7; 50 mm NaF; 200 μm Na3VO4; 1% Triton-X 100; 1 mm phenylmethylsulfonyl fluoride; 5 μg/ml aprotinin; 1 μg/ml pepstatin A, and 2 μg/ml leupeptin) using a Polytron. The homogenate was incubated on a vertical rotator at 4 °C for 1 h, and fat was cleared from extracts by spinning twice at 14,000 rpm at 4 °C for 20 min. 100 μg of protein from each sample was fractionated in 8 and 14% Tris-glycine gels (Invitrogen) and transferred onto polyvinylidene difluoride (Invitrogen) membrane using a Novex Western blot apparatus. After transfer and blocking (5% bovine serum albumin and 2% nonfat milk, 20 mm Tris-HCl, pH 7.6/137 mm NaCl) at room temperature for 1 h, the membranes were incubated with primary antibodies (1:1000 diluted) as follows. Stat5a (14Liu X. Robinson G.W. Hennighausen L. Mol. Endocrinol. 1996; 10: 1496-1506Crossref PubMed Scopus (209) Google Scholar), WAP (28Shamay A. Solinas S. Pursel V.G. McKnight R.A. Alexander L. Beattie C. Hennighausen L. Wall R.J. J. Anim. Sci. 1991; 69: 4552-4562Crossref PubMed Scopus (39) Google Scholar), Stat3, gp130, Stat1, Bax, SGP2, EGFR (Santa Cruz Biotechnology), P-Stat3-Tyr (705), P-Stat3-Ser (727), Stat3, P-p44/42 (Thr202/Tyr204) MAPK, p44/42 MAPK, P-p38 (Thr180/Tyr182) MAPK, p38 MAPK, P-Akt (Ser473), Akt (Cell Signaling), and actin (1:4000, Chemicon). All primary antibodies were applied for 1 h at room temperature or at 4 °C overnight. The membranes were incubated with secondary horseradish peroxidase-conjugated antibodies (Transduction Laboratories, 1:5000) for 30 min at room temperature. Proteins were visualized using the ECL detection system (Amersham Biosciences). Blots were stripped using a Western stripping buffer (Alpha Diagnostic) for 20 min at room temperature.Involution Experiments—At day 10 of lactation, pups were removed from both gp130-null and wild-type mice. The number 4 gland from the left side was collected by biopsy. Mice were sacrificed at days 1, 2, 3 of involution. Two weeks after weaning the right number 4 glands were collected for protein and histological analyses. The litter sizes were 7-10 pups. Three mice were used for each time point.Cell Culture and Transfection—Primary mammary epithelial cells were obtained from wild type, gp130Δ/Δ and Jak2-null transplanted mammary tissues from 3-6 virgin mice. The mammary transplants were cut into fine pieces and subjected to collagenase III (2 mg/ml, Invitrogen) digestion at 37 °C for an hour. The digested tissues were put through 20-gauge needles and centrifuged at 1000 rpm for 3 min. The cell pellets were washed once with Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum. Subsequently mammary tissue (normally 3-5 cells in a cluster) was suspended in complete DMEM with insulin (10 μg/ml) and EGF (10 ng/ml), and seeded into 6-well tissue culture plates. Cells were left undisturbed for a week. The medium was changed to serum-free DMEM for 24 h, and the cells were treated with IL6 (10 ng/ml, 100 ng/ml, PeproTech), IL6 with U0126 (10 μm, Biomol), and AG1478 (3 μm, Biomol) for 15 min before harvesting for protein extraction. Cells were lysed using the lysis buffer described previously, and immunoprecipitation and Western blot were performed using standard procedures.COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum. Transfections of COS-7 cells with cDNA expression vectors (1.0 μg each) encoding wt gp130 (wt), gp130YYFFFF (YY are tyrosine residues 767, 814, 905, and 915 (which correspond to the tyrosine residues 765, 812, 904, and 914 in murine gp130, Ref. 29Schmitz J. Dahmen H. Grimm C. Gendo C. Muller-Newen G. Heinrich P.C. Schaper F. J. Immunol. 2000; 164: 848-854Crossref PubMed Scopus (73) Google Scholar) were mutated to phenylalanine residues), IL6R (gp80) and control empty vector (EV) were carried out using LipofectAMINE (Invitrogen) according to the manufacturer's protocol. Briefly, for transient transfections in 6-cm dishes the cells were incubated for 24 h with the transfection mix containing 15 μl of Polyfect and 2 μg of total plasmid DNA per dish. Cells were washed and cultured in serum-free medium for another 24 h prior to IL6 stimulation. Cells were lysed for 10 min on ice in 200 μl of lysis buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol, 10 mm Na4P2O7, 2 mm Na3VO4,10mm NaF, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin (HNTG). Lysates were precleared by centrifugation at 13,000 rpm for 10 min at 4 °C. 100 μl of lysates were immunoprecipitated using EGFR antibodies (108.1) (30Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1491) Google Scholar) and 20 μl of protein A-Sepharose (Sigma) for 4 h at 4 °C. Precipitates were washed three times with 0.5 ml of lysis buffer, suspended in 2× SDS sample buffer, boiled for 3 min, and subjected to gel electrophoresis and Western blotting as described above. Phosphotyrosine was detected using the 4G10 monoclonal antibody (Upstate). Polyclonal anti-phospho-p44/p42 (Thr202/Tyr204) MAPK antibody, anti-phospho-Stat3, and Stat3 antibodies were purchased from New England Biolabs (Beverly, MA). Polyclonal anti-p44/42 MAPK antibody was from Santa Cruz Biotechnology.Statistics—Western blot bands were quantified using Alpha image 2000 v4 software. Three independent experiments were used to quantitate the data for analysis. All bands were normalized to actin or unphosphorylated total protein. Data are shown as mean ± S.E., and a Student's t test was used to compare two groups of samples.RESULTSLIF Contributes to Mammary Epithelial Cell Death during Involution but Does Not Influence Mammary Development—The level of LIF expression increases sharply at the onset of involution (12Zhao L. Melenhorst J.J. Hennighausen L. Mol. Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar), and its role in mammary epithelial cell death and tissue remodeling was therefore investigated. Since LIF-null mice are infertile, mammary tissue was transplanted into wild-type hosts and analyzed after puberty, during pregnancy and involution. In virgin mice, no histological differences were detected between wild type (Fig. 1A, panel a) and LIF-null (Fig. 1A, panel d) mammary tissue 8 weeks after transplantation. A ductal tree filled the entire fat pad and exhibited extensive branching. Analysis of bona fide LIF-null mice during puberty revealed an abnormal branching (Fig. 1A, panels b and e), and the effect was abolished upon transplantation (Fig. 1A, panels a and d). Moreover, similar morphology was observed during pregnancy in wild type and null transplanted tissue (Fig. 1A, panels c and f). This suggests that the lesion resulted from systemic or stromal effects in LIF-null mice.To explore the contribution of LIF in the remodeling process, tissue was analyzed at parturition and days 1-6 of involution. No obvious differences were noted at parturition (Fig. 1B, panels a and e) and days 1 and 2 of involution (data not shown). However, from day 3-6 LIF-null tissue (Fig. 1B, panels f-h) retained more alveoli, and the reappearance of the stroma was delayed. Histological differences between LIF-null and wild-type mammary tissues were observed until day 6 of involution (Fig. 1B, panels d and h). Less nuclear Stat3 staining was observed in LIF-null tissue (Fig. 1C).In addition to LIF and IL6, expression of OSM, another IL6 family cytokine, was also detected in mammary tissue. OSM expression in involuting glands peaked at 24 h and was absent in lactating glands (data not shown). This suggests that OSM may also be involved in mammary tissue remodeling.Inactivation of the gp130 Gene in Mammary Epithelium—To examine the contribution of the receptor subunit gp130 in the remodeling of mammary epithelium and in the activation of Stat3, we analyzed mice with two distinct mutations in the gp130 gene. One strain expressed a gp130 with a truncated C-terminal region, and in the other strain the entire gene was inactivated.Deletion of Stat3 Binding Sites in gp130—The gp130 gene in gp130Δ/Δ mice carries a knockin mutation (20Ernst M. Inglese M. Waring P. Campbell I.K. Bao S. Clay F.J. Alexander W.S. Wicks I.P. Tarlinton D.M. Novak U. Heath J.K. Dunn A.R. J. Exp. Med. 2001; 194: 189-203Crossref PubMed Scopus (205) Google Scholar) that results in a C-terminal truncation at amino acid 769 and a loss of all proposed Stat3 binding sites (31Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Since these mice are infertile mammary tissue was transplanted into wild-type recipients, and the ability of the cells to develop during pregnancy and subsequently engage in remodeling was examined. While pregnancy-induced mammary development was normal (data not shown), involution was delayed (Fig. 2A). Wild-type tissue at day 3 of involution had undergone extensive remodeling (Fig. 2A, panel b) but mutant mammary tissue continued to display distended alveoli with evidence of milk secretion (Fig. 2A, panel e). The levels of milk protein WAP and apoptosis-related protein SGP2 also confirmed the delayed involution (Fig. 2B). However, this delay of remodeling was transient and full involution was eventually achieved. The levels of Stat3 and MAPK activation in gp130Δ/Δ tissue was examined by Western blot analyses (Fig. 2B). Phosphorylated Stat3 was still present in mutant tissue, while phosphorylated p44/42 MAPK appeared to be lower (Fig. 2B). This suggests that Stat3 can be activated in mammary tissue through pathways that are independent of the known Stat3 binding sites in gp130. This possibly occurs through activation by the LIFR, which dimerizes with gp130Δ/Δ.Fig. 2A, histology of mammary transplants from wt (panels a-c) and gp130Δ/Δ (panels d-f) mammary tissues at day 1 (i1-a, d), day 3 (i3-b, e), and day 4 (i4-c, f) of involution. Bar, 50 μm. B, Western blot of p-Stat3, Stat3, p-p44/42 MAPK, p44/42 MAPK, WAP, SGP2, and E-cadherin from wt and gp130Δ/Δ mammary tissues at day 18 of pregnancy (p18), at parturition (L1), and day 1 of involution (i1). p18 serves as a control for the non-involuting gland because at L1, the transplanted tissue has started to involute.View Large Image Figure ViewerDownload (PPT)Complete Inactivation of gp130—The gp130Δ/Δ mutation resulted in the loss of Stat3 binding sites but retained the SHP2 and Jak2 binding sites, and therefore intact MAPK signaling, which contributes to the involution process (12Zhao L. Melenhorst J.J. Hennighausen L. Mol. Endocrinol. 2002; 16: 2902-2912Crossref PubMed Scopus (50) Google Scholar). To fully understand the gp130-activated signals in mammary tissue remodeling it was therefore necessary to inactivate the entire protein. Mice were generated that carried loxP sites in the promoter of the gp130 gene and in intron II. 2J. Cheng and C. Stewart, unpublished data. This permitted the complete inactivation of the gene through Cre-mediated recombination. The gp130 gene was specifically inactivated in differentiating mammary epithelium during pregnancy using the WAP-Cre transgene (23Wagner K.U. Wall R.J. St-Onge L. Gruss P. Wynshaw-Boris A. Garrett L. Li M. Furth P.A. Hennighausen L. Nucleic Acids Res. 1997; 25: 4323-4330Crossref PubMed Scopus (414) Google Scholar, 24Wagner K.U. McAllister K. Ward T. Davis B. Wiseman R. Hennighausen L. Transgenic Res. 2001; 10: 545-553Crossref PubMed Scopus (246) Google Scholar). Mammary tissue was analyzed from gp130 fl/fl;WC mice after one and several pregnancies by Western blots and histology analysis. Greatly reduced levels of gp130 were observed in gp130 fl/fl;WC mice (p < 0.05) (Fig. 3A). The residual signal was due to the stromal compartment of the gland in which the WAP-Cre transgene is not active. The levels of phosphorylated p44/42 MAPK were lower after two pregnancies, and after five pregnancies both the total amount and phosphorylated form of p44/42 MAPK were reduced (Fig. 3A, p < 0.05) compared with fl/fl mice.Fig. 3A, Western blot analysis of gp130 protein after 1 (P1), 2 (P2), and 5 (P5) pregnancies in gp130 fl/fl (fl/fl) and gp130 fl/fl;WC (fl/fl;WC) mammary tissues. The level of gp130 protein is lower in all fl/fl;WC samples compared with fl/fl tissue (p < 0.05). p-p44/42 MAPK level is also lower after two pregnancies in fl/fl;WC tissue compared with that of the fl/fl tissue. *, p < 0.05. B, histology of L10 (panels a and b) and day 3 involution mammary glands (panels c and d) from gp130 fl/fl (panels a and c) and gp130 fl/fl;WC (panels b and d) mice. Enlarged images are embedded in panels c and d. Bar, 100 μm. C, morphology of mammary glands harvested 2 weeks after weaning from fl/fl (panels a and c) and fl/fl;WC (panels b and d) mice. Bar, 100 μm (panels a and b) and 50 μm (panels c and d).View Large Image Figure ViewerDownload (PPT)Cre activity in WAP-Cre transgenic mice is activated strongly during the first pregnancy and leads to gene deletion such that all cells in subsequent pregnancies derive from progenitor cells in which recombination occurred (24Wagner K.U. McAllister K. Ward T. Davis B. Wiseman R. Hennighausen L. Transgenic Res. 2001; 10: 545-553Crossref PubMed Scopus (246) Google Scholar). In order to obtain extensive inactivation of the gp130 gene, gp130 fl/fl;WC mice were taken through two pregnancies, followed by the analysis of mammary tissue during involution. In these mice involution and the associated tissue remodeling was greatly impaired (Fig. 3B), and the glands had a secretory appearance even 2 weeks after weaning (Fig. 3C, panels b and d). Immunofluorescence confirmed the absence of gp130 in gp130 fl/fl;WC (Fig. 4A) tissue and its presence in fl/fl (Fig. 4D) mammary tissue. The sodium phosphate transporter Npt2b at the apical membrane of mammary alveolar epithelium, which reflects its functional status (32Shillingford J.M. Miyoshi K. Robinson G.W. Bierie B. Cao Y. Karin M. Hennighausen L. J. Histochem. Cytochem. 2003; 51: 555-566Crossref PubMed Scopus (51) Google Scholar), was present in mammary epithelium from gp130 fl/fl; WC mice 2 days after weaning (Fig. 4C), similar to that seen in tissue from wild-type lactating mice (Fig. 4E). In contrast, greatly reduced levels wer