Estrogen Regulation of the Apolipoprotein AI Gene Promoter through Transcription Cofactor Sharing
1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês
10.1074/jbc.273.15.9270
ISSN1083-351X
AutoresDouglas C. Harnish, Mark J. Evans, Marshall S. Scicchitano, Rahmesh A. Bhat, Sotirios K. Karathanasis,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoEstrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor α (ERα), 17β-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERα plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERα DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERα binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERα plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3β and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERα coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERα and RIP140 levels. At low ratios of RIP140 to ERα, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERα and E2 but also upon the intracellular balance of ERα and coactivators utilized by ERα and the apoAI enhancer. Estrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor α (ERα), 17β-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERα plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERα DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERα binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERα plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3β and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERα coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERα and RIP140 levels. At low ratios of RIP140 to ERα, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERα and E2 but also upon the intracellular balance of ERα and coactivators utilized by ERα and the apoAI enhancer. Apolipoprotein AI (apoAI) 1The abbreviations used are: apoAI, apolipoprotein AI; HDL, high density lipoprotein; HNF, hepatocyte nuclear factor; ER, estrogen receptor; ERE, estrogen response element; CBEB, cAMP-response element binding protein; CBP, CREB-binding protein; DBD, DNA binding domain; E2, 17β-estradiol; CMV, cytomegalovirus. is the major protein constituent of plasma high density lipoproteins (HDLs), a class of lipoproteins thought to play a major role in protection against atherosclerosis (reviewed in Ref. 1Karathanasis S.K. Monogr. Hum. Genet. 1992; 14: 140-171Crossref Google Scholar). Because plasma HDL levels are correlated with plasma apoAI and liver apoAI mRNA levels (2Sorci-Thomas M. Prack M.M. Dashti N. Johnson F. Rudel L.L. Williams D.L. J. Lipid Res. 1989; 30: 1397-1403Abstract Full Text PDF PubMed Google Scholar), it is thought that factors affecting apoAI gene expression play an important role in atherosclerosis susceptibility. Although a number of pharmacological, dietary, and physiological factors affect apoAI and HDL plasma levels (3Vandenbrouck Y. Lambert G. Janvier B. Girlich D. Bereziat G. Mangeney-Andreani M. FEBS Lett. 1995; 376: 99-102Crossref PubMed Scopus (6) Google Scholar, 4Jin F.-Y. Kamanna V.S. Chuang M.-Y. Morgan K. Kashyap M.L. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1052-1062Crossref PubMed Scopus (36) Google Scholar, 5Azrolan N. Odaka H. Breslow J. Fisher E.A. J. Biol. Chem. 1995; 270: 19833-19838Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6Tang J. Srivastava R.A.K. Krul E.S. Baumann D. Pfleger B.A. Kitchens R.T. Schonfeld G. J. Lipid Res. 1991; 32: 1571-1581Abstract Full Text PDF PubMed Google Scholar, 7Ettinger W.H. Varma V.K. Sorci-Thomas M. Parks J.S. Sigmon R.C. Smith T.K. Verdery R.B. Arterioscler. Thromb. 1994; 14: 8-13Crossref PubMed Scopus (166) Google Scholar, 8Kaptein A. deWit E.C.M. Princen H.M.G. Arterioscler. 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Lipid Res. 1989; 30: 1137-1145Abstract Full Text PDF PubMed Google Scholar). Moreover, the ethinyl estradiol-induced increases in apoAI mRNA levels in these animals appears to occur via indirect dietary effects due to hormone treatment (17Staels B. Auwerx J. Chan L. van Tol A. Rosseneu M. Verhoeven G. J. Lipid Res. 1989; 30: 1137-1145Abstract Full Text PDF PubMed Google Scholar). Further, estrogen-induced increases in apoAI transcription rates in rats are dependent on the strains used (6Tang J. Srivastava R.A.K. Krul E.S. Baumann D. Pfleger B.A. Kitchens R.T. Schonfeld G. J. Lipid Res. 1991; 32: 1571-1581Abstract Full Text PDF PubMed Google Scholar). Liver-specific expression of the apoAI gene is conferred by a powerful hepatocyte-specific enhancer located in the nucleotide region −220 to −110 upstream of the apoAI transcriptional start site (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar). The activity of the enhancer depends on synergistic interactions between transcription factors bound to three distinct sites: A (−214 to −192), B (−169 to −146), and C (−134 to −119) within the enhancer (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar, 19Harnish D. Malik S. Karathanasis S.K. J. Biol. Chem. 1994; 269: 28220-28226Abstract Full Text PDF PubMed Google Scholar). Sites A and C bind various members of the nuclear receptor superfamily including the hepatocyte nuclear factor 4, HNF-4 (20Ge R. Rhee M. Malik S. Karathanasis S.K. J. Biol. Chem. 1994; 296: 13185-13192Abstract Full Text PDF Google Scholar, 21Ginsburg G.S. Ozer J. Karathanasis S.K. J. Clin. Invest. 1995; 96: 528-538Crossref PubMed Scopus (55) Google Scholar, 22Chan J. Nakabayashi H. Wong N.C.W. Nucleic Acids Res. 1993; 21: 1205-1211Crossref PubMed Scopus (64) Google Scholar), retinoid X receptor α (23Rottman J. Widom R. 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We propose that estrogen effects on apoAI gene expression vary in direction and magnitude depending upon the balance of coactivators shared by ERα and the apoAI enhancer. The −2500AI.LUC.CIII/AIV construct was created by transferring a 3.0-kilobase HindIII 5′ apoAI DNA fragment and a 7-kilobase BamHI 3′ apoAI fragment from the previously reported construct −2500AI.CAT[CIII/AIV] (21Ginsburg G.S. Ozer J. Karathanasis S.K. J. Clin. Invest. 1995; 96: 528-538Crossref PubMed Scopus (55) Google Scholar) into their respective sites in pGL2-Basic (Promega). The −2500AI.LUC construct was created by transferring only the 3.0-kilobaseHindIII fragment into pGL2-Basic. The −256AI.LUC, −220/−110AI.LUC, and −41/+397.LUC constructs were created by transferring their respective HindIII fragments from −256AI.CAT, −222[Δ−110/−41]AI.CAT, and −41AI.CAT, respectively (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar), into pGL2-Basic. Construct −220/−110ABC.LUC was generated by transferring an 110-base pair BamHI fragment from −222[Δ−110/41]AI.CAT (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar) into −41.LUC (19Harnish D. Malik S. Karathanasis S.K. J. Biol. Chem. 1994; 269: 28220-28226Abstract Full Text PDF PubMed Google Scholar). The apoAI enhancer-type mutants were created by transferring the approximately 110-base pair BamHI fragments from the corresponding chloramphenicol acetyltransferase (CAT) constructs (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar) into −41.LUC (19Harnish D. Malik S. Karathanasis S.K. J. Biol. Chem. 1994; 269: 28220-28226Abstract Full Text PDF PubMed Google Scholar). Construct TK.LUC was generated by cloning a −105/+10NheI/HindIII TK promoter fragment into pGL2-Basic. Construct −220/−110ABC/TK.LUC was generated by transferring a 110-base pair BamHI DNA fragment from −222[Δ−110/41]AI.CAT (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar) into TK.LUC. The A.LUC, B.LUC, and ERE.LUC constructs were described previously (19Harnish D. Malik S. Karathanasis S.K. J. Biol. Chem. 1994; 269: 28220-28226Abstract Full Text PDF PubMed Google Scholar, 48Bodine P.V. Green J. Harris H.A. Bhat R.A. Stein G.S. Lian J.B. Komm B.S. J. Cell. Biochem. 1997; 65: 368-387Crossref PubMed Scopus (39) Google Scholar). The pMT2-ERα construct was created by transferring anEcoRI fragment containing the coding region of ERα from the HEO plasmid (49Kumar V. Green S. Stack G. Berry M. Jin J.R. Chambon P. Cell. 1987; 51: 941-951Abstract Full Text PDF PubMed Scopus (1111) Google Scholar) into the pMT2 expression vector. The ERα mutant expression vectors AF1-DBD-X, X-DBD-AF2, and X-DBD-X were generated in pcDNA3 (Invitrogen) as described previously (50Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (615) Google Scholar). The ERα X-DBD-X construct was created by introducing three nucleotide substitutions within the ERα DNA binding domain (DBD) (51Mader S. Kumar V. deVerneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (388) Google Scholar), and the pcDNA3-ERα expression vector was described previously (48Bodine P.V. Green J. Harris H.A. Bhat R.A. Stein G.S. Lian J.B. Komm B.S. J. Cell. Biochem. 1997; 65: 368-387Crossref PubMed Scopus (39) Google Scholar). HepG2 cells stably expressing ERα (Hep89) were created by transfecting the pcDNA3-ERα expression vector into HepG2 cells by electroporation using the BTX Electro Cell Manipulator 600 according to the manufacturer's recommended settings. Stably expressing cells were selected by resistance to G418 (400 μg/ml). Distinct, well isolated colonies were picked using Bellco cloning cylinders (6 × 8 mm) and assessed for the presence of ERα. Plasmid DNAs were purified on Qiagen columns and transfected into HepG2 cells by the calcium phosphate coprecipitation method as described previously (26Harnish D. Malik S. Kilbourne E. Costa R. Karathanasis S.K. J. Biol. Chem. 1996; 271: 13621-13628Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The cells were seeded in deficient growth media (phenol red-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with heat-inactivated 10% fetal bovine serum, 1% Glutamax, 1% minimum essential medium nonessential amino acids, 100 units/ml penicillin and 100 μg/ml streptomycin) at 2.5 × 105 cells/well in a 12-well dish (Falcon) before transfection. Different amounts of the expression vectors pMT2-ERα, pMT2-HNF-4 (52Mietus-Snyder M. Sladek F. Ginsburg G. Kuo C.F. Ladias J. Darnell J. Karathanasis S.K. Mol. Cell. Biol. 1992; 12: 1708-1718Crossref PubMed Scopus (241) Google Scholar), pCMV.HNF-3β (53Pani L. Overdier D.G. Porcella A. Qian X. Lai E. Costa R.H. Mol. Cell. Biol. 1992; 12: 3723-3732Crossref PubMed Google Scholar), pEFRIP (33Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. EMBO J. 1995; 14: 3741-3751Crossref PubMed Scopus (674) Google Scholar), or RSV-CBP-HA (38Kwok R.P. Lundblad J.R. Chrivia J.D. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1296) Google Scholar) were cotransfected as indicated. Luciferase and β-galactosidase activity was determined as described previously (26Harnish D. Malik S. Kilbourne E. Costa R. Karathanasis S.K. J. Biol. Chem. 1996; 271: 13621-13628Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The data shown represent the mean ± S.E. from at least three independent experiments, each in duplicate. Statistical analysis of the data was carried out using the Dunnett's method (54Dunnett C. J. Am. Stat. Assoc. 1955; 50: 1096-1121Crossref Scopus (5197) Google Scholar) to compare treated versus control samples. HepG2 and Hep89 cells were seeded in deficient growth media and treated over 72 h in the presence or absence of 1 μm E2. Total RNA was isolated (Biotecx Labs), subjected to electrophoresis, and hybridized with32P-labeled apoAI PstI cDNA fragment (55Breslow J.L. Ross D. McPherson J. Williams H.W. Kurnit D. Nussbaum A.L. Karathanasis S.K. Zannis V.I. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6861-6865Crossref PubMed Scopus (103) Google Scholar) or32P-labeled human glyceraldehyde-3-phosphate dehydrogenase cDNA (Stratagene). The relative intensities of the hybridized signals were quantitated by phosphoimaging (Molecular Dynamics). Protein-DNA complexes were analyzed by incubation of bacterially expressed HNF-4, HepG2 nuclear extracts (26Harnish D. Malik S. Kilbourne E. Costa R. Karathanasis S.K. J. Biol. Chem. 1996; 271: 13621-13628Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), or baculovirus-expressed human ERα (Panvera) with 32P-labeled DNA probes corresponding to either the 110-base pair apoAI enhancer or the vitellogenin ERE followed by electrophoresis in low ionic strength polyacrylamide gels as described previously (26Harnish D. Malik S. Kilbourne E. Costa R. Karathanasis S.K. J. Biol. Chem. 1996; 271: 13621-13628Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Nuclear extracts were prepared as described previously (26Harnish D. Malik S. Kilbourne E. Costa R. Karathanasis S.K. J. Biol. Chem. 1996; 271: 13621-13628Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Protein concentrations were determined by the BCA method. Proteins were transferred from a 4, 20% SDS-polyacrylamide gel to nitrocellulose and blotted using affinity-purified human monoclonal ER antibody (Stress Gen), rabbit anti-HNF-3β antibody (gift of R. Costa), or rabbit anti-HNF4 serum (from F. Sladek) as the primary antibodies followed by peroxidase-conjugated goat anti-rabbit IgG antibody (Zymed Laboratories Inc.). Detection was performed using the Enhanced Chemiluminescence Western blotting Detection System (Amersham Pharmacia Biotech). Human hepatoma HepG2 cells retain many liver-specific functions; however, they no longer express ERα. Therefore, HepG2 cells stably expressing ERα were created to monitor the regulation of the apoAI gene by estrogen. The resulting cell line, Hep89, expressed ERα by Western blot (Fig. 1 A). Further, the activity of a synthetic vitellogenin estrogen response element luciferase reporter (ERE.LUC) was stimulated 25-fold by 100 nm 17β-estradiol (E2) in Hep89 cells, whereas no E2-dependent promoter activity was observed in the parental HepG2 cells (Fig. 1 B). Northern blotting demonstrated a 2–3-fold decrease in apoAI mRNA steady state levels in Hep89 cells after a 72-h treatment with 1 μm E2, whereas apoAI mRNA levels remained unchanged in HepG2 cells (Fig. 1, C and D). Therefore, apoAI mRNA steady state levels are regulated by E2 in a receptor-dependent manner. The ERα effects on apoAI mRNA levels could be due to changes in mRNA stability or apoAI transcription. To determine the potential of ERα to regulate apoAI gene transcription, a luciferase reporter under the control of both 5′- and 3′-flanking regulatory sequences of the human apoAI gene (reporter-2500 AI.LUC.CIII/AIV, Ref. 21Ginsburg G.S. Ozer J. Karathanasis S.K. J. Clin. Invest. 1995; 96: 528-538Crossref PubMed Scopus (55) Google Scholar) was cotransfected with an ERα expression vector into HepG2 cells. After transfection, the cells were treated with estrogen agonists and antagonists, and the reporter activity in cell extracts was determined. Dose-response experiments with E2 resulted in a 75% maximal repression of apoAI promoter activity with an EC50 value of approximately 12 nm (Fig. 2 A). The repression was both ligand- and receptor-dependent, since it did not occur with either ERα or E2 alone (Fig. 2 B). The estrogen receptor antagonist ICI 182,780 (1 μm) also repressed promoter activity but to a lesser extent (30% repression). A 10-fold molar excess of ICI (1 μm) over E2 (100 nm) effectively competed the E2-mediated repression to the level seen with ICI alone. ERE.LUC reporter activity was also regulated by ERα in a ligand-dependent fashion, except that in contrast to the apoAI promoter, ICI acted as a pure antagonist for ERE activation. The mechanism of ERα repression on the apoAI promoter may be distinct from those involved in ERα activation of an ERE. A collection of apoAI promoter reporters (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar) was used to delineate elements involved in ERα-induced repression of the apoAI promoter. Deletion of the entire 3′-flanking region of the apoAI gene (reporter −2500 AI.LUC) or deletion of both the 3′ region and approximately 2.25 kilobases of the 5′ region (reporter −256AI.LUC) did not affect ERα and E2-induced repression (Fig. 3 A). A reporter containing only the apoAI hepatocyte-specific enhancer driving the expression of the apoAI basal promoter (reporter −220/−110AI.LUC) was also repressed approximately 60% by ERα plus E2. The activity of the apoAI enhancer in a heterologous reporter containing the thymidine kinase promoter was also repressed by ERα plus E2, whereas the thymidine kinase basal promoter reporter activity remained unaffected (Fig. 3 B), demonstrating that ERα regulation occurs directly on the apoAI enhancer. ApoAI enhancer activity in hepatocytes depends on synergistic interactions between transcription factors bound to three distinct sites designated A (−214 to −192), B (−169 to −146), and C (−134 to −119) (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar). To determine the contribution of each of these sites to the ERα and E2-induced repression, nucleotide mutations (denoted byX, Fig. 3 B) were introduced into sites A, B, or C, rendering them incapable of binding their cognate transcription factors (18Widom R.L. Ladias J.A. Kouidou S. Karathanasis S.K. Mol. Cell. Biol. 1991; 11: 677-687Crossref PubMed Scopus (104) Google Scholar). Although the basal activity of these reporters was reduced compared with the intact enhancer, the range of repression was 48–69% in response to ERα and E2. These results suggest that repression occurs at a level distinct from inhibition of individual transcription factors bound to the enhancer. To further probe the mechanism by which ERα and E2 repressed the apoAI enhancer, vectors expressing ERα with a deletion in the N-terminal transcription activation function (AF1) or point mutations in the C-terminal transcription activation function (AF2) or in the DBD were cotransfected with either the −220/−110 ABC.LUC or ERE.LUC reporter into HepG2 cells (Fig. 4). Deletion of AF1 (mutant X-DBD-AF2) had no effect on apoAI enhancer repression but inhibited ERE activation by 80%. In contrast, inactivation of AF2 (mutant AF1-DBD-X) diminished both apoAI repression and ERE activation. Inactivation of both AF1 and AF2 completely abolished both apoAI repression and ERE activation. Finally, point mutations within the DNA binding domain that converts its binding selectivity from an ERE to a glucocorticoid response element (mutant AF1-X-AF2) eliminated repression of the apoAI enhancer. As shown previously (51Mader S. Kumar V. deVerneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (388) Google Scholar), this ERα DNA binding domain mutant activated a reporter driven by a glucocorticoid response element (data not shown). These data suggest that the DNA binding domain of ERα cooperates with either AF-1 or AF-2 to repress the activity of the apoAI enhancer. Moreover, the observation that the X-DBD-AF2 mutant is as efficacious as wild type ERα in apoAI repression but not ERE reporter activation suggests that different mechanisms underlie ERα activation of an ERE reporter and repression of the apoAI reporter. The requirement of an intact DNA binding domain for ERα repression of the apoAI enhancer suggested that ERα could bind directly to the enhancer and interfere with the synergistic interactions between bound transcripti
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