Portal de Periódicos da CAPES

The Cyclin D1 Gene Is Transcriptionally Repressed by Caveolin-1

2000; Elsevier BV; Volume: 275; Issue: 28 Linguagem: Inglês

10.1074/jbc.m000321200

1083-351X

James Hulit, Tal Bash, Maofu Fu, Ferruccio Galbiati, Chris Albanese, Daniel Sage, Amnon Schlegel, Jacob Zhurinsky, Michael Shtutman, Avri Ben-Ze′ev, Michael P. Lisanti, Richard G. Pestell,

Microtubule and mitosis dynamics

The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras,neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between −81 to −73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1. The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras,neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between −81 to −73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1. mitogen-activated protein kinase(s) extracellular signal-regulated kinase T-cell factor lymphoid enhancer factor-1 caveolin-1 Chinese hamster ovary fibroblast growth factor platelet-derived growth factor Cellular growth induced by mitogenic stimuli is coordinated by an orderly progression through sequential and distinct phases of the cell cycle (1.Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4976) Google Scholar, 2.Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4311) Google Scholar). The progression of quiescent cells from the G0 through the G1 phase of the cell cycle is orchestrated by interactions between components of the cell cycle regulatory apparatus (1.Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4976) Google Scholar, 3.Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (320) Google Scholar). The genetic program induced by serum addition includes the activation of immediate-early gene expression, which peaks within 30–60 min after serum stimulation (4.Bravo R. Semin. Cancer Biol. 1990; 1: 37-46PubMed Google Scholar, 5.Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar). The induction of immediate-early genes (for example c-fos and c-jun), is under tight control of counter-regulatory mechanisms that lead to transcriptional repression and/or rapid degradation of the target gene product. The c-fos gene is under autoregulatory trans-repression (6.Konig H. Ponta H. Rahmsdorf U. Buscher M. Schonthral A. Rahmsdorf H.J. Herrlich P. EMBO J. 1989; 8: 2559-2566Crossref PubMed Scopus (154) Google Scholar), and the JunB protein inhibits the activity and function of c-Jun (7.Chiu R. Angel P. Karin M. Cell. 1989; 59: 979-986Abstract Full Text PDF PubMed Scopus (578) Google Scholar, 8.Schutte J. Viallet J. Nau M. Segal S. Federiko J. Minna J. Cell. 1989; 59: 987-997Abstract Full Text PDF PubMed Scopus (385) Google Scholar). A second phase of serum-induced gene expression occurs 3–6 h after serum stimulation and is dependent on new protein synthesis (9.Lanahan A. Williams J.B. Sanders L.K. Nathans D. Mol. Cell. Biol. 1992; 2: 3919-3929Crossref Scopus (299) Google Scholar). The induction of the G1 phase regulatory cyclins, the cyclin-dependent kinase phosphatases, and the E2F-responsive genes contributes to the continued passage of the cell through G1 and into the S phase (10.Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar, 11.Nevins J.R. Cell Growth Differ. 1998; 9: 585-593PubMed Google Scholar). Both the cyclin D1 and cdc25A genes are induced with characteristic delayed-early gene kinetics and contribute to the induction of DNA synthesis (3.Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (320) Google Scholar, 12.Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (400) Google Scholar, 13.Chen X. Prywes R. Mol. Cell. Biol. 1999; 19: 4695-4702Crossref PubMed Scopus (69) Google Scholar). The cyclin D1 gene product encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates pRB. Immunoneutralizing antibody and antisense expression studies demonstrated that the abundance of cyclin D1 is rate-limiting in growth factor- and mitogen-induced progression through the G1 phase (14.Resnitzky D. Gossen M. Bujard H. Reed S.I. Mol. Cell. Biol. 1994; 14: 1669-1679Crossref PubMed Scopus (990) Google Scholar, 15.Xiong W. Pestell R.G. Watanabe G. Li J. Rosner M.R. Hershenson M.B. Am. J. Physiol. 1997; 272: L1205-L1210PubMed Google Scholar, 16.Lukas J. Bartkova J. Bartek J. Mol. Cell. Biol. 1996; 16: 6917-6925Crossref PubMed Scopus (294) Google Scholar, 17.Lukas J. Pagano M. Staskova Z. Draetta G. Bartek J. Oncogene. 1994; 9: 707-718PubMed Google Scholar). Mouse embryo fibroblasts derived from mice in which the cyclin D1 gene was homozygously deleted (cyclin D1−/−) displayed reduced cell proliferation (18.Brown J.R. Nigh E. Lee R.J. Ye H. Thompson M.A. Saudou F. Pestell R.G. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 5609-5619Crossref PubMed Scopus (211) Google Scholar). Several lines of evidence suggest that c-Fos and c-Jun may induce the cyclin D1 gene and thereby enhance S phase entry. Thus, c-Fos was shown to induce the cyclin D1 gene (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar), and the low levels of cyclin D1 in mouse embryo fibroblasts derived fromc-fos/FOSB−/− mice were rescued by c-Fos overexpression (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar). In addition,c-jun −/− mouse embryo fibroblasts display a proliferative defect in response to serum and a reduction in cyclin D1 abundance (20.Wisdom R. Johnson R.S. Moore C. EMBO J. 1999; 18: 188-197Crossref PubMed Scopus (530) Google Scholar). Serum and growth factor signaling to discrete transcription factor targets is coordinated by evolutionarily conserved modular intracellular signaling kinase cascades (21.Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1163) Google Scholar). Mitogen-activated protein kinases (MAPKs),1 which relay these signals, are proline-directed serine/threonine kinases and include extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase, and p38 MAPKs (22.Lin A. Smeal T. Binetruy B. Deng T. Chambard J.C. Karin M. Adv. Second Messenger Phosphoprotein Res. 1993; 28: 255-260PubMed Google Scholar). The modularity and specificity in these signal transduction cascades are coordinated by several mechanisms, including selective phosphorylation of downstream kinases (23.Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2256) Google Scholar), targeting by specific MAPK phosphatases, subcellular localization of the kinases (24.Wilkinson M.G. Milar J.B.A. Genes Dev. 1998; 12: 1391-1397Crossref PubMed Scopus (72) Google Scholar), MAPK isoform-selective targeting of specific transcription factors (25.Gupta S. Barrett T. Whitmarsh A. Cavanagh J. Derijard B. Davis R. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1180) Google Scholar), and the interaction with scaffolding proteins that mediate the interactions between components of the MAPK module (26.Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (628) Google Scholar, 27.Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar). The ERK/MAPK cascade is also regulated by the relative abundance of the caveolin-1 protein (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar, 29.Engelman 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). Caveolin-1 is an important component of caveolar membranes, invaginations of the plasma membrane thought to participate in vesicular trafficking and signal transduction events (30.Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar). Caveolins are most abundant in differentiated cells, and caveolin-1 levels have been shown to be reduced in fibroblasts transformed by oncogenic Ha-ras (G12V) or v-abl (31.Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (472) Google Scholar) and in mammary adenocarcinoma cells induced by overexpression of ErbB2 (32.Engelman 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). Furthermore, caveolin-1 was identified as one of 26 genes whose mRNA was down-regulated in human breast cancer cell lines (33.Sager, R., Sheng, S., Anisowicz, A., Sotiropoulou, G., Zou, Z., Stenman, G., Swisshelm, K., Chen, Z., Hendrix, M. J., and Pemberton, P. E. A. (1994) Cold Spring Harbor Symp. Quant. Biol. 537–546Google Scholar). Overexpression of caveolin-1 in v-abl- and Ha-ras-transformed NIH-3T3 cells abrogated their anchorage-independent growth (34.Engelman J.A. Wykoff 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), and transfection with antisense caveolin-1 was sufficient to induce cellular transformation and ERK activity (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). Despite these studies, the molecular mechanisms by which caveolin-1 regulates cellular transformation are largely unknown. In this study, we assessed whether caveolin-1 can directly regulate cyclin D1 expression. We show that the cyclin D1 gene is inhibited during overexpression of caveolin-1 as a result of repression of the cyclin D1 promoter and that the DNA sequences required contain the T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF-1)-binding site. We conclude that repression of the cyclin D1 gene by caveolin-1 may contribute to the inhibition of cellular transformation. The abundance of cyclin D1, JunB, and caveolin-1 was determined by Western blot analysis as described previously using antibodies to cyclin D1 (DCS-6, NeoMarkers, Fremont, CA), JunB (N-17, Santa Cruz Biotechnology Inc.), c-Fos, and Rho-GTPase guanine-nucleotide dissociation inhibitor (35.Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 36.Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines G.K.I. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar); anti-caveolin-1 IgG (monoclonal antibody 2297, a gift of Dr. Roberto Campos-Gonzalez, Transduction Laboratories Inc.) (37.Scherer P.S. Tang Z. Chun M. 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); and an anti-caveolin-1 rabbit anti-peptide antibody directed against residues 2–21 (Santa Cruz Biotechnology Inc.) (38.Scherer P.E. Lewis R.Y. Volonté 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). The human cyclin D1 promoter-reporter constructions (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar, 36.Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines G.K.I. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 39.Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar, 40.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1918) Google Scholar) and the c-jun promoter-luciferase reporter from −225 to +150 (41.Clarke N. Arenzana N. Hai T. Minden A. Prywes R. Mol. Cell. Biol. 1998; 18: 1065-1073Crossref PubMed Scopus (95) Google Scholar) were previously described. The reporter c-fosLUC (35.Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) contains the human c-fos promoter from −361 to +157 in the pA3LUC reporter (42.Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). The junB promoter was cloned by polymerase chain reaction using oligonucleotides to the published sequences (5′-GGT ACC CGC GAG CCG CCT CCT CCC and 3′-AAG CTT CCG GGC GGC CCA GGC GGT) and was subcloned into the pA3LUC reporter to create the junBLUC reporter. The pALUC reporter, which contains 7 kilobases of the human cyclin A promoter (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar, 36.Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines G.K.I. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 39.Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar) and the cdc25ALUC reporter (43.Iavarone A. Massague J. Mol. Cell. Biol. 1999; 19: 916-922Crossref PubMed Scopus (113) Google Scholar) were previously described. The serum response element from the c-fos promoter from −332 to −277 was linked to the minimal TATA region of the E4 promoter and cloned into the reporter pA3LUC. The expression vectors encoding caveolin-1 (44.Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar) and the caveolin-1 mutants Cav-1-(1–81) and Cav-1-(Δ61–101) (45.Schlegel A. Schwab R.B. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1999; 274: 22660-22667Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar); Cav-1ΔN, Cav-1ΔC, and caveolin-3 (46.Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar); and caveolin-1β (37.Scherer P.S. Tang Z. Chun M. 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) were previously described. The expression of the caveolin-1 mutant expression plasmids was confirmed in cultured cells. Cell culture, transfections, and luciferase assays were performed as described (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar). CHO cells (GRC+ LR-73; a generous gift from Dr. J. Pollard (47.Pollard J.W. Stanners C.P. J. Cell. Physiol. 1979; 98: 571-585Crossref PubMed Scopus (75) Google Scholar)) were maintained in α-minimum Eagle's medium with 10% (v/v) calf serum and 1% penicillin/streptomycin. The NIH-3T3 cells stably expressing antisense caveolin-1 and revertants of such NIH-3T3 lines have been described previously (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). Fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) were from Upstate Biotechnology, Inc. In transient expression studies, cells were transfected using calcium phosphate precipitation; the medium was changed after 6 h; and luciferase activity was determined after another 24 h. The effect of an expression vector was compared with that of an equal amount of empty vector. Luciferase content was measured during the initial 10 s of the reaction using an AutoLumat LB953 (EG&G Berthold), and the values are expressed in arbitrary light units (35.Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Statistical analyses were performed using the Mann-Whitney U test with significant differences established as p < 0.05. Previous studies have demonstrated that cyclin D1 levels are increased (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar) and that caveolin-1 levels are decreased in Ha-ras(G12V)-transformed fibroblast cells (31.Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (472) Google Scholar). We have therefore examined the abundance of cyclin D1 and caveolin-1 in tumors derived from murine mammary tumor virus ras-transformed cells. Cyclin D1 levels were increased in each of the tumors examined (Fig.1 A) and were associated with reduced or undetectable caveolin-1 levels. In previous studies, we showed that cyclin D1 levels were increased in mammary tumors from murine mammary tumor virus src transgenic mice (36.Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines G.K.I. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), whereas caveolin-1 levels were undetectable in these tumors (32.Engelman 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). To examine the effect of serum and growth factors on caveolin-1 and cyclin D1 levels, NIH-3T3 cells were serum-starved or treated with serum, FGF, or PDGF for 16 h. Serum starvation was associated with a reduction in cyclin D1 levels and an increase in caveolin-1 abundance. The addition of serum, FGF (5 ng/ml), or PDGF (50 ng/ml) induced cyclin D1 levels and reduced caveolin-1 abundance (Fig. 1 B). To determine whether caveolin-1 overexpression can directly regulate cyclin D1 levels, cell lines stably overexpressing antisense caveolin-1 (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar) were examined for the abundance of cyclin D1. Revertants of 3T3 cells that have lost antisense caveolin expression (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar), similar to the parental NIH-3T3 cell line (data not shown), showed a 4-fold increase in caveolin-1 protein levels (Fig. 1 C) compared with the antisense caveolin-1-expressing clone. Cyclin D1 levels were increased by 60% in the antisense caveolin-1 stable cell line compared with the revertant (Fig. 1 C). Caveolin-1 and cyclin D1 expression levels therefore appear to be inversely related in 3T3 cells and mammary tumor tissue. In contrast with the reduction in cyclin D1 protein levels by caveolin-1, the JunB protein was increased in association with increased caveolin-1 levels (Fig. 1 C). To determine whether caveolin-1 overexpression can regulate the activity of the cyclin D1 gene promoter, transient expression studies were performed using a caveolin-1 expression plasmid and the empty expression vector (pCB7). The results summarized in Fig.2 B show that overexpression of caveolin-1 repressed the activity of the cyclin D1 promoter in a dose-dependent manner. In contrast, overexpression of caveolin-3 (Fig. 2 A, Cav-3) did not inhibit the activity of the cyclin D1 promoter (Fig. 2 B). Cyclin D1 promoter-luciferase construct was repressed by 70% using a reporter/expression vector ratio of 4:1 (Fig. 2 B). Previous studies have shown that caveolin-1 can inhibit the function of the serum-responsive transcription factor Elk-1 in a heterologous luciferase reporter assay (32.Engelman 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). We therefore examined the effect of caveolin-1 overexpression on the native c-fos gene promoter. Comparison was made with the effect of caveolin-1 on the cyclin D1 promoter. The data are shown as mean luciferase activity in Fig.3 A. In contrast with the −1745CD1LUC reporter, which was repressed by caveolin-1, the c-fos promoter was not significantly repressed using a reporter/expression vector ratio of 4:1. As recent studies identified a serum response element in the cdc25A gene that is distinct from the one in the c-fos gene (13.Chen X. Prywes R. Mol. Cell. Biol. 1999; 19: 4695-4702Crossref PubMed Scopus (69) Google Scholar), the activities of thecdc25A promoter and those of the immediate-early genes c-jun and junB were determined in cells transfected with caveolin-1. The results shown in Fig. 3 Cdemonstrate that caveolin-1 inhibited the activity of thecdc25A promoter by 80% and that of the c-jungene reporter by 70%. The effect of caveolin-1 on the cyclin D1 and c-fos promoters was not significantly changed by serum concentrations (data not shown). In contrast, the junBpromoter was induced by caveolin-1 by 15–30-fold (Fig. 3 D). Together, these studies suggest that caveolin-1 repression of cyclin D1 promoter activity is inhibited by a serum-independent mechanism. To examine the DNA sequences in the cyclin D1 promoter required for repression by caveolin-1, the promoter activities in a series of cyclin D1 promoter constructs containing truncations and point mutations were assayed. Repression of the cyclin D1 promoter by caveolin-1 was maintained when the sequences between −1745 and −163 base pairs of the promoter were deleted (Fig.4 A). Since the cAMP response element site of the cyclin D1 promoter was previously shown to convey serum responsiveness in fibroblasts (18.Brown J.R. Nigh E. Lee R.J. Ye H. Thompson M.A. Saudou F. Pestell R.G. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 5609-5619Crossref PubMed Scopus (211) Google Scholar) and the AP-1 site is involved in mitogenic responses to angiotensin II and Ras (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar, 35.Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), we used point mutants in these sites and found that the inhibitory effect of caveolin-1 on the cyclin D1 promoter was not affected in these mutants (Fig. 4, B and C). Within the proximal −163 base pairs that are still responsive to caveolin-1 overexpression, a binding site for the β-catenin· TCF complex was recently identified (40.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1918) Google Scholar) (Fig. 4 A). Mutation of the β-catenin/TCF element (−163FOP) reduced the ability of caveolin-1 to inhibit the cyclin D1 promoter from 80 to <50% (Fig. 4 D). Additional experiments were conducted comparing the effect of caveolin-1 with equal amounts of empty expression vector cassette (pCB7) at a reporter/expression vector ratio of 1:4 (n = 12). When normalized as paired experiments with the effect of the expression vector normalized to 100%, further experiments confirmed the trend of reduced repression by mutation of the TCF site (Fig. 4 D). These findings suggest that the TCF site is required for full repression of the cyclin D1 promoter by caveolin-1 and that additional elements may contribute to full repression. The TCF/LEF sequence in the cyclin D1 promoter is identical to the consensus TCF/LEF-1 site (40.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1918) Google Scholar) and was sufficient for repression by caveolin-1 when it was linked to a minimal promoter (Fig.4 E). To determine the domains in caveolin-1 that are required for inhibition of the cyclin D1 promoter, various caveolin-1 mutants were assayed for their ability to inhibit a full-length cyclin D1 promoter-luciferase construct. These mutants have previously been shown to be expressed at equivalent levels to the wild-type caveolin-1 in cultured cells (37.Scherer P.S. Tang Z. Chun M. 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, 45.Schlegel A. Schwab R.B. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1999; 274: 22660-22667Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 48.Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 49.Ikezu T. Ueda H. Trapp B.D. Nishiyama K. Sha J.F. Volonte D. Galbiati F. Byrd A.L. Bassell G. Serizawa H. Lane W.S. Lisanti M.P. Okamoto T. Brain Res. 1998; 804: 177-192Crossref PubMed Scopus (162) Google Scholar). Cav-1β-(32–178) repressed the cyclin D1 promoter to a similar extent as the full-length α-isoform (residues 1–178) (Fig. 5 A). Deletion of the caveolin-1 carboxyl terminus did not affect repression. In contrast, deletion of the N-terminal 95 residues (positions 96–178) not only abolished repression, but caused a modest induction of the cyclin D1 promoter. The abundance of the transfected ΔC and ΔN mutants was identical by Western blotting of cultured cells (48.Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), suggesting that loss of expression is not responsible for the failure of the ΔN mutant to repress the cyclin D1 promoter. Since deletion of the N-terminal residues 61–101 prevents caveolin-1 oligomerizationin vivo (48.Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and this domain is sufficient as a glutathioneS-transferase fusion for multimerization in vitro(44.Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar), we therefore determined whether oligomerization of caveolin-1 was required for repression of cyclin D1 promoter activity. Using the caveolin-1 N-terminal mutant Cav-1-(Δ61–101), we found that this mutant was capable of repressing cyclin D1 promoter activity to a similar extent as the α-isoform (Fig. 5 A). The C-terminal half of the oligomerization domain of caveolin binds to and regulates the activity of several signaling molecules in the Ras/ERK pathway (29.Engelman 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,32.Engelman 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, 50.Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). To determine further the possibility of whether ERK signaling is involved in the repression of the cyclin D1 promoter, we used a mutant caveolin-1 that is completely defective in inhibiting p42/p44 MAPK signaling (Cav-1-(1–81) (45.Schlegel A. Schwab R.B. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1999; 274: 22660-22667Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar)) and found that repression of the cyclin D1 promoter activity by this mutant was minimally affected (Fig.5 A). These results suggest that caveolin-1 oligomerization and inhibition of the ERK signaling pathway are not required for repression of the cyclin D1 promoter activity and that the region responsible for this activity resides between amino acids 32 and 60; thus, a unique domain is required for full repression of cyclin D1. Interestingly, this region is not conserved among the various caveolins (Fig. 5 B). The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRB protein, thereby promoting entry into the DNA synthetic phase of the cell cycle (2.Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4311) Google Scholar). Antisense cyclin D1 inhibits S phase entry induced by serum, growth factors, or steroids and inhibits transformation by Ha-ras,src, and neu (3.Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (320) Google Scholar, 51.Liu J.-J. Chao J.-R. Jiang M.-C. Ng S.-Y. Yen J.J.-Y. Yang-Yen H.-F. Mol. Cell. Biol. 1995; 15: 3654-3663Crossref PubMed Scopus (263) Google Scholar, 52.Lee R.J. Albanese C. Fu M. D'Amico M. Lin B. Watanabe G. Haines G.K.I. Siegel P.M. Hung M.C. Yarden Y. Horowitz J.M. Muller W.J. Pestell R.G. Mol. Cell. Biol. 2000; 20: 672-683Crossref PubMed Scopus (310) Google Scholar). Caveolin-1 levels are reduced in a variety of tumor types (32.Engelman 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), whereas increasing the level of caveolin-1 levels can suppress the transformed phenotype (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). In the present study, we found that cyclin D1 protein abundance and promoter activity were inhibited by overexpression of caveolin-1 protein. Caveolin-1 also inhibited the activity of thecdc25A promoter. The Cdc25A phosphatase dephosphorylates inhibitory phosphorylation sites on cyclin-dependent kinases (12.Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (400) Google Scholar, 53.Galaktionov K. Lee A.K. Eckstein J. Draetta G. Meckler J. Loda M. Beach D. Science. 1995; 269: 1575-1577Crossref PubMed Scopus (501) Google Scholar), and overexpression of Cdc25A enhances transformation by oncogenic ras (53.Galaktionov K. Lee A.K. Eckstein J. Draetta G. Meckler J. Loda M. Beach D. Science. 1995; 269: 1575-1577Crossref PubMed Scopus (501) Google Scholar). Taken together, these studies demonstrate that the transcriptional activity of two major components of the cell cycle regulatory apparatus that governs DNA synthesis and cell transformation may be regulated by caveolin-1. Caveolin-interacting proteins include G-protein α-subunits, Ha-Ras, Src family tyrosine kinases, endothelial nitric-oxide synthase, epidermal growth factor receptor, and other related tyrosine kinases and protein kinase C isoforms (54.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). The caveolin-1 mutants used in the present study suggest that repression of the cyclin D1 promoter activity most probably involves different domains of the caveolin-1 molecule than those required for regulation of epidermal growth factor receptor signaling and formation of caveola structures. Three distinct caveolin genes have so far been identified (caveolin-1, -2, and -3), which can form homo- or hetero-oligomers (54.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). Interestingly, the loss of only caveolin-1, but not the other family members, was observed in tumors; and selective reduction of caveolin-1 levels, without affecting caveolin-2, was sufficient to drive transformation of NIH-3T3 cells (28.Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar, 30.Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar). The structural conservation is high among the three caveolar proteins, but divergence is displayed at the N terminus of these molecules (Fig. 5 B) (30.Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar). In agreement with this observation, caveolin-1, but not caveolin-3, was found to repress the cyclin D1 promoter. Caveolin oligomers directly bind cholesterol and interact with glycosphingolipids, enhancing the formation of the caveolar structures (55.Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K. Sanders M. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (478) Google Scholar). A central hydrophobic domain (residues 102–134) forms a hairpin-like structure within the membrane, which positions both the N- and C-terminal domains of the molecule in the cytoplasm. Deletion of the C-terminal domain abrogates the interaction of homo-oligomers; this interaction contributes to the formation of the caveolin-rich scaffold (48.Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Similar to the effect observed by the α-isoform of caveolin-1, the C-terminal mutant (Cav-1ΔC) repressed the cyclin D1 promoter, suggesting that interaction of caveolin-1 homo-oligomers is apparently not required for cyclin D1 promoter inhibition. Deletion of the N-terminal 95 residues of the molecule abolished this repression of the cyclin D1 promoter by caveolin-1. The N terminus of caveolin-1 is involved in homo-oligomerization and interaction with the ERK signaling pathway (50.Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar), but its deletion (Cav-1-(Δ61–101)) did not affect the magnitude of cyclin D1 promoter inhibition, supporting the view that formation of caveolin-containing structures is not necessary for cyclin D1 promoter repression. This study links, for the first time, the caveolin-1 protein with inhibition of the cell cycle regulatory apparatus involved in tumorigenesis. Cyclin D1 overexpression is known to induce mammary tumors in transgenic mice (56.Wang T.C. Cardiff R.D. Zukerberg L. Lees E. Arnold A. Schmidt E.V. Nature. 1994; 369: 669-671Crossref PubMed Scopus (892) Google Scholar) and cooperates in oncogenic transformation with several oncogenes, including ras,myc, and E1A (57.Bodrug S.E. Warner B.J. Bath M.L. Lindeman G.J. Harris A.W. Adams J.M. EMBO J. 1994; 13: 2124-2130Crossref PubMed Scopus (406) Google Scholar, 58.Hinds P.W. Dowdy S.F. Eaton E.N. Arnold A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 709-713Crossref PubMed Scopus (454) Google Scholar, 59.Zwicker J. Brusselbach S. Jooss K.U. Sewing A. Behn M. Lucibello F.C. Muller R. Oncogene. 1999; 18: 19-25Crossref PubMed Scopus (40) Google Scholar). Cdc25A also cooperates in cell transformation with ras (53.Galaktionov K. Lee A.K. Eckstein J. Draetta G. Meckler J. Loda M. Beach D. Science. 1995; 269: 1575-1577Crossref PubMed Scopus (501) Google Scholar). Since cyclin D1 is frequently overexpressed in a variety of human tumors (3.Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (320) Google Scholar) and caveolin-1 abundance is reduced in many tumors (54.Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar), our studies point to the possibility that loss of caveolin-1 expression during tumorigenesis may lead to cellular proliferation through induction of the cyclin D1 gene. The cyclin D1 gene is induced by several signaling pathways implicated in cellular transformation, including the phosphatidylinositol 3-kinase, β-catenin/TCF/LEF, ERK, and nuclear factor-κB signaling pathways (40.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1918) Google Scholar, 60.Guttridge D.C. Albanese C. Reuther J.Y. Pestell R.G. Baldwin A.S. Mol. Cell. Biol. 1999; 19: 5785-5799Crossref PubMed Google Scholar, 61.Gille H. Downward J. J. Biol. Chem. 1999; 274: 22033-22040Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Examining the effect of caveolin-1 mutants on cyclin D1 promoter activity, we found that repression of the cyclin D1 promoter apparently does not involve the ERK pathway since an N-terminal caveolin-1 mutant incapable of inhibiting signaling by the Ras/ERK pathway (45.Schlegel A. Schwab R.B. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1999; 274: 22660-22667Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) could still repress cyclin D1 promoter activity. Thus, although the ERK pathway can activate cyclin D1 (19.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar, 35.Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), a different pathway is most probably affected by caveolin-1. A mutation in the TCF/LEF site of the cyclin D1 promoter that abolished binding to TCF proteins in electrophoretic mobility shift assays (40.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1918) Google Scholar) reduced repression by caveolin-1. Interestingly, we found that caveolin-1 also inhibited the activity of the c-junpromoter, a gene that is also activated by β-catenin/TCF signaling and that contains a TCF site in its promoter (62.Mann B. Gelos M. Siedow A. Hanski M.L. Gratchev A. Ilyas M. Bodmer W.F. Moyer M.P. Riecken E.O. Buhr H.J. Hanski C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1603-1608Crossref PubMed Scopus (723) Google Scholar). In contrast, the c-fos promoter, which is induced by ERK, was not significantly repressed by caveolin-1, further supporting the view that caveolin-1 repression of promoters in mammary epithelial cells involves a pathway that is distinct from the ERK pathway. Future studies will have to address the molecular mechanisms involved in the role of caveolin-1 in the regulation of the β-catenin/TCF/LEF signaling pathway.