Prolactin Negatively Regulates Caveolin-1 Gene Expression in the Mammary Gland during Lactation, via a Ras-dependent Mechanism
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m108210200
ISSN1083-351X
AutoresDavid Park, Hyangkyu Lee, Claudia A. Riedel, James Hulit, Philipp E. Scherer, Richard G. Pestell, Michael P. Lisanti,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoCaveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant "steady-state" levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of β-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis. Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant "steady-state" levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of β-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis. epidermal growth factor mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase reverse transcription monoclonal antibody signal transducers and activators of transcription phosphate-buffered saline phenol red-free Dulbecco's modified Eagle's medium complete medium with 10% charcoal-dextran-stripped fetal bovine serum adenovirus green fluorescent protein phosphatidylinositol 3-kinase Janus kinase tetracycline-trans-activator peroxisome proliferator-activated receptor The mammary gland is one of the few organs that undergoes numerous rounds of proliferation and regression throughout adult life. Normal mammary gland development involves a complex interplay among growth factors, steroids, proto-oncogenes, and tumor suppressor genes (1Gallego M.I. Binart N. Robinson G.W. Okagaki R. Coschigano K.T. Perry J. Kopchick J.J. Oka T. Kelly P.A. Hennighausen L. Dev. Biol. 2001; 229: 163-175Crossref PubMed Scopus (192) Google Scholar, 2Wennbo H. Tornell J. Oncogene. 2000; 19: 1072-1076Crossref PubMed Scopus (96) Google Scholar, 3Wennbo H. Gebre-Medhin M. Gritli-Linde A. Ohlsson C. Isaksson O.G. Tornell J. J. Clin. Invest. 1997; 100: 2744-2751Crossref PubMed Scopus (180) Google Scholar). Development of the adult mammary gland can be divided into four distinct stages: non-pregnant, gestation, lactation, and involution. Gestation is characterized by rapid lobulo-alveolar outgrowth, whereas further proliferation and functional differentiation of the secretory epithelium are hallmarks of lactation. Finally, the end of weaning suppresses lactation and leads to involution of the lobulo-alveolar compartment, returning the mammary gland to its non-pregnant state (4Hennighausen L. Robinson G.W. Genes Dev. 1998; 12: 449-455Crossref PubMed Scopus (241) Google Scholar). Dysregulation of these constituents can lead to mammary epithelial hyperplasia and ultimately to mammary tumorigenesis (5Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). A possible role for caveolin-1 in mammary tumorigenesis was first identified using differential display and subtractive hybridization techniques. Sager and colleagues (6Sager R. Sheng S. Anisowicz A. Sotiropoulou G. Zou Z. Stenman G. Swisshelm K. Chen Z. Hendrix M.J.C. Pemberton P. Rafidi K. Ryan K. Cold Spring Harbor Sym. Quant. Biol. 1994; 59: 537-546Crossref PubMed Scopus (118) Google Scholar) identified a number of "candidate tumor suppressor genes"; these are genes whose mRNAs were down-regulated in human mammary adenocarcinoma-derived cells. In this screening approach, caveolin-1 was independently identified as one of 26 gene products down-regulated during mammary tumorigenesis. In addition, caveolin-1 expression was absent in several transformed cell lines derived from human mammary carcinomas, including MT-1, MCF-7, ZR-75–1, T47D, MDA-MB-361, and MDA-MB-474. In contrast, caveolin-1 mRNA is abundantly expressed in normal mammary epithelium (6Sager R. Sheng S. Anisowicz A. Sotiropoulou G. Zou Z. Stenman G. Swisshelm K. Chen Z. Hendrix M.J.C. Pemberton P. Rafidi K. Ryan K. Cold Spring Harbor Sym. Quant. Biol. 1994; 59: 537-546Crossref PubMed Scopus (118) Google Scholar,7Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Since these initial observations, an increasing body of evidence has accumulated that supports the idea that caveolin-1 may function as a tumor suppressor in the mammary gland. For example, Lee et al. (8Lee S.W. Reimer C.L. Oh P. Campbel l.D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (399) Google Scholar) demonstrated that exogenous expression of caveolin-1 in human breast cancer-derived cells (T-47D) leads to a 50% reduction in cell proliferation and a 15-fold reduction in anchorage-independent growth in soft agar. Furthermore, p53-null fibroblasts express virtually undetectable levels of caveolin-1 (8Lee S.W. Reimer C.L. Oh P. Campbel l.D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (399) Google Scholar). Mutation and/or amplification of the NEU proto-oncogene occurs in a significant number of human mammary tumors (up to 30%). Mutational activation of the NEU proto-oncogene (NEU T) was shown to have a reciprocal relationship with caveolin-1 expression. Activated mutants of the c-Neu protein expressed in NIH-3T3 and Rat1 cells caused a dramatic down-regulation of caveolin-1 protein expression. In addition, mammary tumors derived from Neu transgenic mice displayed a dramatic reduction in caveolin-1 expression. Conversely, recombinant expression of caveolin-1 is sufficient to block Neu-mediated signal transduction, via the caveolin-1 scaffolding domain (5Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Caveolin-1 expression also diminishes the metastatic potential of the mammary tumor cell line, MTLn3. MTLn3 cells were originally derived from a metastatic rat mammary adenocarcinoma. In response to EGF,1 MTLn3 cells undergo lamellipodia extension and increased chemotaxis. However, recombinant expression of caveolin-1 in MTLn3 cells blocks EGF-induced lamellipodia extension and cell migration. Thus, expression of caveolin-1 in MTLn3 cells induces a non-motile phenotype (9Zhang W. Razani B. Altschuler Y. Bouzahzah B. Mostov K.E. Pestell R.G. Lisanti M.P. J. Biol. Chem. 2000; 275: 20717-20725Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). To further investigate the possible clinical significance of caveolin-1 in mammary tumorigenesis, Hayashi et al. (10Hayashi K. Matsuda S. Machida K. Yamamoto T. Fukuda Y. Nimura Y. Hayakawa T. Hamaguchi M. Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar) screened 92 human breast cancer samples for a mutation in the caveolin-1 gene. Their results identified a mutation at residue 132 (P132L) in 16% of the samples tested. The mutation most closely correlated with invasive scirrhous carcinomas. NIH-3T3 cells, stably expressing the caveolin-1 (P132L) mutant, demonstrated increased growth in soft agar, hyperactivation of the Ras-p42/44 MAPK cascade, and an altered cellular morphology due to disruption of the actin cytoskeleton. These results emphasize the importance of wild-type caveolin-1 gene expression in the normal regulation of mammary epithelial cell proliferation (10Hayashi K. Matsuda S. Machida K. Yamamoto T. Fukuda Y. Nimura Y. Hayakawa T. Hamaguchi M. Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar). From the above studies, it is clear that caveolin-1 assumes a dynamic role in regulating mammary epithelial cell proliferation. However, the normal physiological regulators of caveolin-1 expression in vivo remain obscure. To identify these in vivo regulators, we analyzed the expression levels of caveolin-1 in the adult mouse mammary gland during different stages of its development. Through this analysis, we expected to find a stage of development in which caveolin-1 levels are normally up-regulated or down-regulated. This would allow us to then identify the hormonal regulators that control caveolin-1 expression. Here, we demonstrate that caveolin-1 expression is significantly down-regulated in the mammary gland during late pregnancy and lactation. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Furthermore, we show that prolactin negatively regulates caveolin-1 expression via a Ras-p42/44-MAPK-dependent mechanism. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of bothβ-casein promoter activity and β-casein synthesis, as revealed by quantitative RT-PCR. β-Casein is an established marker of differentiation and milk production. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis. Caveolin-1 mouse mAb 2297 and caveolin-2 mouse mAb 65 (used for immunoblotting (11Scherer P.E. Tang Z.-L. Chun M.C. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 12Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar)) were the gifts of Dr. Roberto Campos-Gonzalez, Transduction Laboratories, Inc. Anti-PPAR-γ antibodies were purchased from Santa Cruz Biotechnology; the anti-β-actin mAb (clone AC-15) was purchased from Sigma. hTERT-HME1 cells, a telomerase immortalized, human mammary epithelial cell line, was purchased from CLONTECH (13Clontech, Inc. CLONTECHniques. 2000; 15: 1-2Google Scholar, 14Clontech, Inc. CLONTECHniques. 2000; 14: 2-3Google Scholar). HC11 cells, derived from the COMMA-D cell line, were the generous gift of Dr. J. M. Rosen, Baylor College of Medicine (Houston, TX), with the permission of Dr. B. Groner, at The Friedrich Miescher Institute (Basel, Switzerland); COMMA-D cells were first isolated from the mammary glands of mice in mid-pregnancy. A variety of other reagents were purchased commercially. Cell culture reagents were from Life Technologies, Inc. Ovine prolactin (o-prolactin), dexamethasone, and insulin were purchased from Sigma Chemical Co. Recombinant human EGF was purchased from Upstate Biotechnology, Inc. The MEK inhibitor, PD98059, was purchased from Calbiochem. hTERT-HME1 cells were grown in complete growth media consisting of MCDB 170 medium supplemented with 52 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 10 ng/ml human EGF, 5 μg/ml insulin, and 50 μg/ml gentamicin (Clonetics). Cells were maintained in growth medium at 37 °C and 5% CO2. Prior to hormone treatment, cells were grown to ∼ 80% confluency, washed with PBS, and incubated in phenol red-free Dulbecco's modified Eagle's medium complete medium with 10% charcoal-dextran-stripped fetal bovine serum (PRF-CDS DMEM) for 12 h. Cells were then treated with increasing concentrations of oxytocin (0–1000 nm) or o-prolactin (0–100 μg/ml) for 72 h. To assess MEK involvement, PD98059 (50 μm) was added 24 h prior to o-prolactin treatment. HC11 cells were grown to confluency in RPMI 1640 medium supplemented with 10% donor calf serum, insulin (5 μg/ml), and epidermal growth factor (EGF, 10 ng/ml). For lactogenic hormone induction, the cells were maintained at confluency for 3 days in growth medium. HC11 cells were then primed in RPMI 1640 medium supplemented with 10% charcoal-dextran stripped horse serum and insulin (5 μg/ml) for 24 h. For hormonal induction, the following hormones were added to priming medium: dexamethasone (1 μg/ml) and o-prolactin (5 μg/ml) (15Wartmann M. Cella N. Hofer P. Groner B. Liu X. Hennighausen L. Hynes N.E. J. Biol. Chem. 1996; 271: 31863-31868Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Ali S. J. Biol. Chem. 1998; 273: 7709-7716Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). C57Bl/6 mice (10–12 weeks old, Jackson Laboratories) were treated once a day with subcutaneous injections of 1 IU of oxytocin or 10 IU of prolactin for three consecutive days. All hormones were dissolved in 200 μl of PBS. On the fourth day, mice were given a final injection and sacrificed 2 h later (17Tazebay U.H. Wapnir I.L. Levy O. Dohan O. Zuckier L.S. Zhao Q.H. Deng H.F. Amenta P.S. Fineberg S. Pestell R.G. Carrasco N. Nat. Med. 2000; 6: 871-878Crossref PubMed Scopus (416) Google Scholar). Mammary glands were excised and subjected to immunoblot and/or Northern blot analysis as detailed below. The cDNA encoding caveolin-1 was subcloned into the multiple cloning site (Hin dIII/Bam HI) of the cytomegalovirus-driven pCB7 vector, as described previously (18Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (347) Google Scholar). The β-casein promoter-luciferase reporter was as characterized previously (19Matsumura I. Kitamura T. Wakao H. Tanaka H. Hashimoto K. Albanese C. Downward J. Pestell R.G. Kanakura Y. EMBO J. 1999; 18: 1367-1377Crossref PubMed Scopus (292) Google Scholar). Adenoviral vectors (Ad-Cav-1, Ad-GFP, and Ad-tTA) were as we described previously (9Zhang W. Razani B. Altschuler Y. Bouzahzah B. Mostov K.E. Pestell R.G. Lisanti M.P. J. Biol. Chem. 2000; 275: 20717-20725Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Cells were cultured in their respective media and allowed to reach ∼80–90% confluency. Subsequently, they were washed with PBS and treated with lysis buffer (10 mmTris, pH 7.5, 50 mm NaCl, 1% Triton X-100, 60 mm octyl glucoside) containing protease inhibitors (Roche Molecular Biochemicals). For protein isolation from tissue, 100 mg of the excised mammary gland was homogenized in lysis buffer. Cell and tissue lysates were then centrifuged at 12,000 × g for 10 min to remove insoluble debris. Protein concentrations were quantified using the BCA reagent (Pierce) and the volume required for 10 μg of protein determined. Samples were then separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands) followed by immunoblot analysis. All subsequent wash buffers contained 10 mm Tris, pH 8.0, 150 mmNaCl, 0.05% Tween 20, which was supplemented with 1% bovine serum albumin and 2% nonfat dry milk (Carnation) for the blocking solution and 1% bovine serum albumin for the antibody diluent. Primary antibodies were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution, Pierce) were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce). Total RNA was extracted from 100 mg of tissue from each sample using the TRIzol reagent protocol (Life Technologies, Inc.). Twenty micrograms of total RNA for each sample was separated using a 1.2% agarose gel under RNase-free conditions and transferred to nitrocellulose. The filters were hybridized using the ExpressHyb solution (CLONTECH). The blots were probed with the radiolabeled caveolin-1 cDNA. A 13-kb DNA segment containing thecaveolin-1 exons 1 and 2 was identified by screening a mouse genomic DNA library as previously described (20Engelman J.A. Zhang X.L. Razani B. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1999; 274: 32333-32341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). A portion ofcaveolin-1 exon 1 in addition to intron 1 and the 3-kb upstream promoter were derived from this segment and subcloned into the vector pA3LUC, a promoter-less vector containing the luciferase cDNA as a reporter (21Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar, 22Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). In this way, the affect of various signal transduction pathways on the regulation of caveolin-1 at the transcriptional level could be assessed. Transient transfections were performed using LipofectAMINE Plus (Life Technologies, Inc.). Briefly, 150,000 hTERT-HME1 or HC11 cells were seeded in 6-well plates 12–24 h prior to transfection. Each well was then transfected with 1.0 μg of the indicated luciferase reporter and 0.2 μg of pSV-β-gal (Promega). The pSV-β-gal, an SV40-driven vector expressing β-galactosidase, was used as a control for transfection efficiency. 0.5 μg of pCB7 or pCB7-caveolin-1 was co-transfected where indicated. For hTERT-HME1 cells: At 12 h post-transfection, the cells were rinsed once with PBS and incubated in PRF-CDS DMEM with 10 μg/ml prolactin for 48 h. The cells were lysed in 200 μl of extraction buffer, 100 μl of which was used to measure luciferase activity, as described previously (23Pestell R.G. Albanese C. Hollenberg A. Jameson J.L. J. Biol. Chem. 1994; 269: 31090-31096Abstract Full Text PDF PubMed Google Scholar). Another 50 μl of the lysate was used to conduct a β-galactosidase assay, as described previously (24Subramaniam A. Gulick J. Robbins J. J. Biol. Chem. 1990; 265: 13986-13994Abstract Full Text PDF PubMed Google Scholar). Each experimental value has been normalized using its respective β-galactosidase activity and represents the average of two separate transfections performed in parallel; the error bars in the figures represent the observed standard deviation. All experiments were performed at least three times independently and yielded virtually identical results. For HC11 cells: see above for hormone treatment. Conditions for adenoviral transduction of cells were optimized by immunofluorescence and immunoblot analysis so that relatively high protein expression was achieved without toxicity to the cells. 2D. S. Park, H. Lee, C. Riedel, J. Hulit, P. E. Scherer, R. G. Pestell, and M. P. Lisanti, unpublished observations. Twenty-four hours prior to infection, ∼3 × 106 HC11 cells were plated in 10-cm dishes. At the time of infection, cells were washed once with PBS and incubated for 1 h with serum-free media containing either Ad-Cav-1 + Ad-tTA (100 + 100 plaque-forming units/cell, respectively) or Ad-GFP + Ad-tTA (100 + 100 plaque-forming units/cell, respectively). Cells were then washed with PBS and maintained in HC11 growth media. Total cellular RNA was extracted from HC11 cells using the TRIzol reagent (Life Technologies, Inc., Rockville, MD). Total RNA (2 μg) was reverse-transcribed with Moloney murine leukemia virus-RT (Life Technologies, Inc., Life-Technologies, Inc.) oligo(dT) primers at 37 °C for 1 h. β-Casein-specific primers for RT-PCR were as described previously (1Gallego M.I. Binart N. Robinson G.W. Okagaki R. Coschigano K.T. Perry J. Kopchick J.J. Oka T. Kelly P.A. Hennighausen L. Dev. Biol. 2001; 229: 163-175Crossref PubMed Scopus (192) Google Scholar): CasYP (ACT ACA TTT ACT GTA TCC TCT GAC), nucleotides 107–130, and CasYM (GTG CTA CTT GCT GCA GAA AGT ACA G), nucleotides 620–644. cDNAs were amplified under the following conditions: initially 2 min at 94 °C; 28 or 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, and a final extension for 5 min at 72 °C (1Gallego M.I. Binart N. Robinson G.W. Okagaki R. Coschigano K.T. Perry J. Kopchick J.J. Oka T. Kelly P.A. Hennighausen L. Dev. Biol. 2001; 229: 163-175Crossref PubMed Scopus (192) Google Scholar). The glyceraldehyde-3-phosphate dehydrogenase cDNA was used as a positive internal control, with RGAPH-1 (GTG AAG GTC GGT GTG AAC GGA TTT GGC CGT), nucleotides 50–76, and RGAPH-2 (CCA CCA CCC TGT TGC TGT AG), nucleotides 997–1016. As a negative internal control, equal amounts of RNA were amplified for 28 or 35 cycles using CasYP and CasYM without reverse transcription (1Gallego M.I. Binart N. Robinson G.W. Okagaki R. Coschigano K.T. Perry J. Kopchick J.J. Oka T. Kelly P.A. Hennighausen L. Dev. Biol. 2001; 229: 163-175Crossref PubMed Scopus (192) Google Scholar). Mammary epithelial cells and adipocytes were separated as described elsewhere (25Nedergaard J. Ailhaud G. Adipose Tissue Protocols. Methods in Molecular Biology. Humana Press, Totowa, NJ2001: 203-204Google Scholar). Briefly, mammary glands (4 and 5) were excised from 10- to 12-week-old virgin, day 15 pregnant, day 10 lactating, and day 4 post-weaning C57Bl/6 mice. Samples were then immersed in Hanks' balanced saline solution supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), and gentamicin (50 μg/ml). Tissue samples were minced and digested using type I collagenase (1 mg/ml). Digestions were performed in a 50-cc conical tube on a shaker at 37 °C for 30–60 min. After collagenase treatment, the slurry was passed through a 350-μm nylon mesh, and samples were centrifuged at 500 × g for 1 min to separate the parenchymal fraction from the floating adipocyte fraction. The pelleted samples and the floating adipocytes were washed three times in Hanks' balanced saline solution (25Nedergaard J. Ailhaud G. Adipose Tissue Protocols. Methods in Molecular Biology. Humana Press, Totowa, NJ2001: 203-204Google Scholar). The samples were then treated with lysis buffer (see above) and examined by immunoblot analysis. Because caveolin-1 has been shown to act as an inhibitor of mitogenesis in cultured cells, we examined the expression of caveolin-1 during mammary gland development. Rapid expansion of the lobulo-alveolar compartment occurs during pregnancy and continues throughout lactation. Therefore, we initially examined caveolin-1 and caveolin-2 protein levels during these developmental stages of the mammary gland. C57Bl/6 mice between the ages of 10 and 12 weeks were sacrificed on day 16 of pregnancy and day 10 of lactation, with virgin mice being sacrificed as controls. The mammary glands were then excised, and caveolin protein levels were assessed by Western blotting. As shown in Fig. 1, initial screening revealed that caveolin-1 protein levels are significantly down-regulated during lactation, whereas a more modest reduction of caveolin-1 expression is seen during pregnancy. Although caveolin-2 levels are also mildly down-regulated, repression of caveolin-2 is much less dramatic than that of caveolin-1. Additional time points, spanning pregnancy to involution, were added to characterize the initiation, duration, and termination of caveolin-1 repression. As shown in Fig.2 A, the first and second trimesters of pregnancy (days 1–14) are characterized by a gradual decrease of caveolin-1 levels, whereas the third trimester (days 15–21) displays a more pronounced decrease of caveolin-1 expression. This pronounced down-regulation of caveolin-1 protein levels continues throughout lactation (Fig. 2 B). With the termination of suckling and the onset of involution, caveolin-1 expression briskly returns to non-pregnant levels. In addition, caveolin-3 expression (a muscle-specific caveolin gene) is up-regulated during lactation, presumably due to terminal differentiation of the myo-epithelial cells. To determine at what molecular level lactation regulates caveolin-1 expression, Northern blot analyses were performed on the same mammary gland samples. As Fig. 3 illustrates, caveolin-1 mRNA transcript levels fall dramatically with the onset of lactation. On the third day of weaning, transcript levels begin to re-emerge and then fully return to non-pregnant levels by day 6 of weaning. To examine if the caveolin-1 protein is secreted into milk, samples were collected and examined for the presence of caveolin-1. However, no detectable levels of caveolin-1 were found in milk samples (not shown). During pregnancy, a complex array of ovarian and pituitary hormones, such as progesterone, prolactin, and oxytocin, stimulate the development of the lobulo-alveolar compartment within the mammary gland. Upon parturition, progesterone levels fall sharply, while prolactin and oxytocin levels remain elevated; this is a hallmark of the beginning of lactation. Suckling maintains high levels of prolactin and oxytocin secretion throughout lactation (26Higuchi T. Honda K. Fukuoka T. Negoro H. Hosono Y. Nishida E. Endocrinol. Jpn. 1983; 30: 353-359Crossref PubMed Scopus (33) Google Scholar). To dissect the hormonal regulation of caveolin-1 expression observed in the lactating mammary gland, 10- to 12-week-old virgin mice were treated with subcutaneous injections of prolactin, oxytocin, or PBS alone. Fig.4 demonstrates that, although oxytocin treatment moderately decreases caveolin-1 expression in comparison with PBS-treated mice, prolactin injection markedly reduced the caveolin-1 protein to nearly undetectable levels. To further characterize the effects of prolactin and oxytocin on mammary epithelial cells, hTERT-HME1 cells were utilized. hTERT-HME1 cells are derived from primary human mammary epithelial cells that have been immortalized by stable transfection with human telomerase. This particular cell line has been well characterized and displays expression patterns and behaviors similar to non-immortalized primary mammary epithelial cells (13Clontech, Inc. CLONTECHniques. 2000; 15: 1-2Google Scholar, 14Clontech, Inc. CLONTECHniques. 2000; 14: 2-3Google Scholar). Consistent with the idea that hTERT-HME1 cells are immortalized but not oncogenically transformed, these cells abundantly express caveolin-1. There is a distinct lack of caveolin-1 expression in all previously studied breast cancer-derived cell lines. Therefore, this cell line is ideal for studying the effects of various hormones on caveolin-1 expression in culture. hTERT-HME1 cells were treated with increasing concentrations of oxytocin (0–10
Referência(s)