KLF11-mediated Repression Antagonizes Sp1/Sterol-responsive Element-binding protein-induced Transcriptional Activation of Caveolin-1 in Response to Cholesterol Signaling
2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês
10.1074/jbc.m407941200
ISSN1083-351X
AutoresSheng Cao, Martín E. Fernández-Zapico, Dongzu Jin, Vishwajeet Puri, Tiffany Cook, Lilach O. Lerman, Xiang-Yang Zhu, Raúl Urrutia, Vijay H. Shah,
Tópico(s)Lipid metabolism and disorders
ResumoCholesterol is a potent regulator of gene expression via a canonical pathway co-regulated by SREBP and Sp1. Here we establish the caveolin-1 gene promoter as a cell type-specific model for SREBP/Sp1 regulation whereby lipoprotein cholesterol depletion activates caveolin-1 transcription in endothelial type cells, but not in fibroblasts, both in vitro and in vivo. By extending this model, we describe a novel pathway distinct from the prototypical SREBP/Sp1 regulatory loop involving the Sp1-like protein, KLF11. Through a combination of RNA interference, chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and reporter assays, we demonstrate that in the presence of cholesterol, KLF11 acts as a dominant repressor of the caveolin-1 gene. Mechanistically, cholesterol depletion results in displacement of KLF11 from an Sp1 site flanking an SRE, indicating that activation by SREBP/Sp1 requires antagonism of KLF11 repression. The displacement of KLF11 results from both a down-regulation of its expression and competition by Sp1 for DNA binding. Therefore, these studies identify a novel pathway whereby KLF11 repression is coordinated with Sp1/SREBP activation of cholesterol-dependent gene expression in a cell type-specific manner and outline the mechanisms by which these functions are achieved. Cholesterol is a potent regulator of gene expression via a canonical pathway co-regulated by SREBP and Sp1. Here we establish the caveolin-1 gene promoter as a cell type-specific model for SREBP/Sp1 regulation whereby lipoprotein cholesterol depletion activates caveolin-1 transcription in endothelial type cells, but not in fibroblasts, both in vitro and in vivo. By extending this model, we describe a novel pathway distinct from the prototypical SREBP/Sp1 regulatory loop involving the Sp1-like protein, KLF11. Through a combination of RNA interference, chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and reporter assays, we demonstrate that in the presence of cholesterol, KLF11 acts as a dominant repressor of the caveolin-1 gene. Mechanistically, cholesterol depletion results in displacement of KLF11 from an Sp1 site flanking an SRE, indicating that activation by SREBP/Sp1 requires antagonism of KLF11 repression. The displacement of KLF11 results from both a down-regulation of its expression and competition by Sp1 for DNA binding. Therefore, these studies identify a novel pathway whereby KLF11 repression is coordinated with Sp1/SREBP activation of cholesterol-dependent gene expression in a cell type-specific manner and outline the mechanisms by which these functions are achieved. Sterol-responsive element (SRE) 1The abbreviations used are: SRE, sterol-responsive element; SREBP, SRE-binding protein; Sp, stimulatory protein; LDL, low density lipoprotein; KLF, Kruppel-like factor; BAEC, bovine aortic endothelial cell(s); NHS, normal human serum; LPDS, lipoprotein-depleted serum; siRNA, short interfering RNA; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation. -binding proteins (SREBP) and the sequence-specific enhancer factor Sp1 function synergistically to activate transcription of target genes in response to cholesterol depletion (1Naar A. Beaurang P. Robinson K. Oliner J. Avizonis D. Scheek S. Zwicker J. Kadonaga J. Tjian R. Genes Dev. 1998; 12: 3020-3031Crossref PubMed Scopus (173) Google Scholar, 2Van der Sudhof T. Westhuyzen D. Goldstein J. Brown M. Russell D. J. Biol. Chem. 1987; 262: 10773-10779Abstract Full Text PDF PubMed Google Scholar, 3Brown M. Goldstein J. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3090) Google Scholar). The prototypical target of this canonical pathway is the key gene for cholesterol uptake, the low density lipoprotein (LDL) receptor gene, in which cholesterol depletion activates LDL receptor gene transcription through binding of SREBP with a specific cognate DNA element flanked by a DNA sequence that recognizes Sp1 (2Van der Sudhof T. Westhuyzen D. Goldstein J. Brown M. Russell D. J. Biol. Chem. 1987; 262: 10773-10779Abstract Full Text PDF PubMed Google Scholar, 4Dawson P. van der Hofmann S. Westhuyzen D. Sudhof T. Brown M. Goldstein J. J. Biol. Chem. 1988; 263: 3372-3379Abstract Full Text PDF PubMed Google Scholar). More recently, complimentary and alternative co-regulatory proteins have been identified that influence SREBP/Sp1 cooperation, suggesting additional complexities to this transcriptional paradigm (5Bennett M. Ngo T. Athanikar J. Rosenfeld J. Osborne T. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Several target promoters have been utilized as models to enhance our understanding of these pathways including LDL receptor, fatty acid synthase, and others (5Bennett M. Ngo T. Athanikar J. Rosenfeld J. Osborne T. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Magana M. Koo S. Towle H. Osborne T. J. Biol. Chem. 2000; 275: 4726-4733Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). A number of proteins that have zinc finger motifs similar to Sp1, including the Kruppel-like factor (KLF) proteins, have recently been identified as regulators of gene transcription (7Fernandez Zapico M. Mladek A. Ellenrieder V. Folch-Puy E. Miller L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (89) Google Scholar). The structure of these proteins is defined by the presence of three homologous, DNA-binding zinc finger domains linked to an amino-terminal region containing transcriptional regulatory motifs (7Fernandez Zapico M. Mladek A. Ellenrieder V. Folch-Puy E. Miller L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (89) Google Scholar, 8Black A. Black J. Azizkha-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (911) Google Scholar, 9Zhang X. Zhang D. Michel F. Blum J. Simmen F. Simmen R. J. Biol. Chem. 2003; 278: 21474-21482Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Nandan M. Yoon H. Zhao W. Ouko L. Chanchevalap S. Yang V. Oncogene. 2004; 23: 3404-3413Crossref PubMed Scopus (120) Google Scholar, 12Schrick J. Hughes M. Anderson K. Croyle M. Lingrel J. Gene (Amst.). 1999; 236: 185-195Crossref PubMed Scopus (17) Google Scholar). KLF proteins bind GC-rich DNA sequences, thereby functioning as activators or repressors depending on the specific KLF protein and the specific target gene (7Fernandez Zapico M. Mladek A. Ellenrieder V. Folch-Puy E. Miller L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (89) Google Scholar, 8Black A. Black J. Azizkha-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (911) Google Scholar, 9Zhang X. Zhang D. Michel F. Blum J. Simmen F. Simmen R. J. Biol. Chem. 2003; 278: 21474-21482Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Liu Y. Sinha S. Owens G. J. Biol. Chem. 2003; 278: 48004-48011Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Ou X. Chen K. Shih J. J. Biol. Chem. 2004; 279: 21021-21028Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, the role for KLF proteins in coordination with SREBP and Sp1 in the context of cholesterol-dependent target genes is unexplored. Caveolins are a family of 22-kDa proteins that are implicated in cholesterol homeostasis, signal transduction, and vesicle trafficking (14Smart E. Graf G. McNiven M. Sessa W. Engelman J. Scherer P. Okamoto T. Lisanti M. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar, 15Liu P. Rudick M. Anderson R. J. Biol. Chem. 2002; 277: 41295-41298Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). Experimental evidence indicates that the caveolin-1 gene is a cholesterol target gene particularly in fibroblast cells (16Fielding C. Bist A. Fielding P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3753-3758Crossref PubMed Scopus (221) Google Scholar, 17Bist A. Fielding P. Fielding C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10693-10698Crossref PubMed Scopus (233) Google Scholar, 18Fra A. Pasqualetto E. Mancini M. Sitia R. Gene (Amst.). 2000; 243: 75-83Crossref PubMed Scopus (28) Google Scholar), although the mechanism of regulation remains incompletely understood. The vascular endothelium is particularly enriched in the caveolin-1 member of the caveolin protein family and because of the prominent endothelium-specific effects of cholesterol modulation (19Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J. Hansen S. Nishimoto I. Lisanti M. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 20Garcia-Cardena G. Martasek P. Masters B.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar), the regulation of caveolin-1 expression in endothelial cells is of significant interest (21Minshall R. Sessa W. Stan R. Anderson R. Malik A. Am. J. Physiol. 2003; 285: L1179-L1183Crossref PubMed Scopus (272) Google Scholar). The present studies uncover several novel findings that expand our understanding of the cell type-specific, molecular mechanisms of cholesterol target gene regulation through SREBP and Sp1 by demonstrating antagonistic interactions between SREBP/Sp1, and the Sp1-like protein, KLF11, using the caveolin-1 promoter as a model. We describe a model in which lipoprotein depletion activates caveolin-1 gene expression in vitro and in vivo in vascular endothelium but not in fibroblasts. Extending this model, we demonstrate that a SRE/Sp1-dependent mechanism is responsible for these divergent effects of lipoprotein depletion in different cell types and that KLF11 acts as a dominant repressor of the caveolin-1 gene in the presence of cholesterol. Mechanistically, cholesterol depletion results in displacement of KLF11 from this Sp1 site through down-regulation of KLF11 expression and competition by Sp1 for DNA binding. Thus, these studies delineate a novel co-regulatory role of KLF11 in the canonical SREBP/Sp1 pathway of cholesterol gene regulation by demonstrating that activation is not only achieved by the presence of a transcriptional activator but rather requires antagonism of a pre-existing repressed state. Cell Culture and Plasmid Construction—Bovine aortic endothelial cells (BAEC; VecTec, P3-P6), ECV 304 cells, HEK 293 cells, and human skin fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, and 100 units/ml of penicillin/streptomycin. For cholesterol depletion experiments, the cells were serum-starved for 24 h and then transferred to varying volume ratios of normal human serum (NHS; Sigma) and human lipoprotein-depleted serum (LPDS; Sigma) for 16 h or as indicated in individual experiments. In some analyses, LPDS was supplemented with human LDL (Sigma) at concentrations between 0.05 and 0.2 mg/ml during the full duration of LPDS incubation. Using genomic DNA from Hep G2 cells as template and synthetic oligomers as primers (5′-GGGGTACCCCATTGTTCCAGAAAATATCGG-3′ and 5′-CCAAGCTTCCCTGGGCTGTGCTTTAAGGG-3′), the 5′-flanking region (bp –737 to –37) previously identified as the caveolin-1 promoter region (17Bist A. Fielding P. Fielding C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10693-10698Crossref PubMed Scopus (233) Google Scholar, 22Engelman J. Zhang X. Razani B. Pestell R. Lisanti M. J. Biol. Chem. 1999; 274: 32333-32341Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 23Hurlstone A. Reid G. Reeves J. Fraser J. Strathdee G. Rahilly M. Parkinson E. Black D. Oncogene. 1999; 18: 1881-1890Crossref PubMed Scopus (123) Google Scholar) was obtained by PCR. The PCR product was purified by agarose gel electrophoresis, digested with KpnI and HindIII, and cloned into the promoterless luciferase reporter vector pGL3. The deletion constructs were generated using standard molecular biology techniques and cloned into pGL3. All of the constructs were verified by sequencing. RNA Expression Analysis—For Northern blotting, total RNA was isolated from cells using TRIzol reagent (Invitrogen). 5.0 μg of RNA was separated on a 1% agarose gel containing 6% formaldehyde. RNA was then transferred to a Nylon membrane and immobilized by UV cross-linking. After UV cross-linking, the membranes were pre-hybridized and hybridized with cDNA encoding full-length caveolin-1 or 18 S rRNA, each labeled using RadPrime DNA labeling system (Invitrogen). After washing, the blots were then exposed to a phosphorus imaging plate and analyzed using the Storm 840 image analysis system (Molecular Dynamics). Band density was normalized to the intensity of 18 S. Reverse transcription-PCR analysis was performed as per our previously published protocol (7Fernandez Zapico M. Mladek A. Ellenrieder V. Folch-Puy E. Miller L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (89) Google Scholar). Western Blot Analysis and Immunoprecipitation—Protein lysates were prepared from cultured cells and from aortic endothelial cell monolayers scraped from pigs that were fed either normal chow diet or a hypercholesterolemic diet (2% cholesterol, 15% lard; Harland Teklad, Madison, WI) for 12 weeks as per our previous studies with this model (24Herrmann J. Gulati R. Napoli C. Woodrum J. Lerman L. Rodriguez-Porcel M. Sica V. Simari R. Ciechanover A. Lerman A. FASEB J. 2003; 17: 1730-1732Crossref PubMed Scopus (52) Google Scholar). The cells were washed three times with ice-cold phosphate-buffered saline and lysed with 1 ml of lysis buffer (50 mm Tris-HCl, 0.1 mm EDTA, 0.1 mm EGTA, pH 7.5) containing 0.1% SDS, 0.1% deoxycholate (Sigma), 1% Nonidet P-40, and protease inhibitors (1 mg/ml aprotinin, 150 mmol/liter phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin) as previously described (25Cao S. Yao Y. McCabe T. Yao Q. Katusic Z. Sessa W. Shah V. J. Biol. Chem. 2001; 276: 14249-14256Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Extracted protein was quantified by the Lowry assay, and the proteins were separated by 12% SDS-PAGE and electroblotted onto nitrocellulose membranes. Caveolin-1 detection was performed using a polyclonal antibody (Transduction Laboratories), whereas SREBP detection was performed using a monoclonal antibody (BD Biosciences Pharmingen), followed by respective peroxidase-conjugated secondary antibodies (Jackson Laboratories) and enhanced chemiluminescence (ECL®; Amersham Biosciences). The protein levels of the housekeeping gene actin were assayed for internal control of protein loading. Thin Layer Chromatography—After incubation of cells with NHS and LPDS for 16 h, BAEC were scraped in 1 ml of phosphate-buffered saline, and an aliquot of this suspension was used for protein assay, whereas the remainder of the cell suspension was utilized for cholesterol extraction as previously described (26Puri V. Jefferson J. Singh R. Wheatley C. Marks D. Pagano R. J. Biol. Chem. 2003; 278: 20961-20970Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The lower chloroform layer was removed and dried under a N2 jet. A solution of 19:1 CHCl3/CH3OH was added and then loaded on thin layer chromatography using 65:15:1 of CHCl3/C2H5O C2H5/CH3COOH as the developing solvent. After drying and I2 staining, scanning densitometry was used to measure the concentration of cholesterol based on standards run in parallel and normalized for total cellular protein. Promoter-Reporter Assay—Because BAEC are not amenable to transfection, promoter-reporter experiments were performed in ECV 304 cells, which are a transformed cell line that maintain some phenotypic characteristics typical of endothelial cells (27Paxinou E. Weisse M. Chen Q. Souza J. Hertkorn C. Selak M. Daikhin E. Yudkoff M. Sowa G. Sessa W. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11575-11580Crossref PubMed Scopus (78) Google Scholar, 28Sowa G. Liu J. Papapetropoulos A. Rex-Haffner M. Hughes T. Sessa W. J. Biol. Chem. 1999; 274: 22524-22531Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Some transfection experiments were also performed in HEK 293 cells with identical results. The cells were cultured in 24-well plates and transfected with the luciferase reporter plasmid, pGL3, encoding full-length caveolin-1 promoter DNA or indicated deletion constructs using the calcium phosphate precipitation method. The cells were also co-transfected with a Rous sarcoma virus-Renilla luciferase reporter gene. In some experiments, cDNA encoding Sp1 or KLF 9, 10, 11, 13, or 16 was also transfected into cells. Next day, the cells were serum-starved, incubated with varying volume concentrations of NHS and LPDS for 16 h, and then lysed. Luciferase activity was determined from cell lysates with a dual luciferase assay system (Promega) in which firefly luciferase activity was measured using a luminometer, and the data were normalized to Renilla luciferase activity to control for transfection efficiency (29Cao S. Yao J. Shah V. J. Biol. Chem. 2003; 278: 5894-5901Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To assay promoter-reporter activity in fibroblast cells, the experiments were performed with human skin fibroblasts with other experimental conditions remaining identical to that described above. Sp1 RNA interference was achieved using a pool of four siRNA duplexes obtained from Dharmacon Research (SMARTpool, Lafayette, CO) (30Reynolds A. Leake D. Boese Q. Scaringe S. Marshall W. Khvorova A. Nat. Biotechnol. 2004; 22: 326-330Crossref PubMed Scopus (1678) Google Scholar). The nonsilencing control siRNA targeted a DNA sequence (AATTCTCCGAACGTGTCACGT) with no matches upon BLAST search and was obtained from Qiagen. Transfection with siRNA was performed using Oligofectamine reagent (Invitrogen) with the final concentration of siRNA being 100 nm. The cells were then transfected with caveolin-1 promoter plasmid, and luciferase activity was determined as described above. Aliquots of cell lysates were used for Western blotting to confirm the specificity and level of Sp1 protein knockdown. Electrophoretic Mobility Shift Assay—5.0 μg of BAEC nuclear extract was incubated in a binding buffer containing 50 mm HEPES, pH 7.9, 1.0 mm EDTA, 100 mm KCl, 20% glycerol, 2.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1.5 μg of poly(dI-dC) for 15 min on ice. Wild type and mutant 32P-labeled probes encoding three GC-rich 10-bp regions, located at –646 to –637 and –395 to –386 and at –287 to –278 from the caveolin-1 translation initiation site (termed SRE1, SRE2, and SRE3), were added, and incubated at room temperature for 10 min. In some experiments, an excess of cold probe, at the indicated dilutions, was added concomitant with the addition of the radiolabeled probe. The mixture was electrophoresed at 12.5 V/cm on 5% nondenaturing polyacrylamide gel in 0.25× TBE. The gel was vacuum-dried, and the autoradiographs were obtained. Electrophoretic mobility shift assay experiments to examine competition between Sp1 and KLF11 were performed in a similar manner except that the labeled probe was a GC box sequence, ATTCGATCGGGGCCGGGGCGAGC or a Sp1-binding site sequence from the caveolin-1 promoter, ACCTTTGGCGGGCGGCCAGG. Probes were incubated with cell extracts derived from Panc1 cells or with recombinant GST-KLF11 zinc finger protein. Oligonucleotide Capture Assay—Nucleoprotein identification was performed using a modified oligonucleotide capture assay (Roche Applied Science) (31Teale B. Singh S. Khanna K. Findik D. Lavin M. J. Biol. Chem. 1992; 267: 10295-10301Abstract Full Text PDF PubMed Google Scholar). Copies of oligonucleotides containing the sequence for SRE3 (GCACCCCA) were directly synthesized. These oligonucleotides underwent a self-priming reaction during the PCR, resulting in long concatamers that were ligated to the tethered oligonucleotide on the streptavidin magnetic particles. Bound protein was separated from the crude nuclear extract with a magnetic separator and washed extensively. Bound proteins were eluted from the magnetic particles and separated by SDS-PAGE followed by silver stain or alternatively by Western blot analysis using an SREBP-1 monoclonal antibody. A random sequence was generated and used as a negative control. Chromatin Immunoprecipitation Assay—Chromatin immunoprecipitation (ChIP) was performed as we previously described (7Fernandez Zapico M. Mladek A. Ellenrieder V. Folch-Puy E. Miller L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (89) Google Scholar). In brief, BAEC and fibroblasts were cross-linked with formaldehyde for 20 min at 25 °C, harvested in SDS-lysis buffer (Upstate Biotechnology, Inc., Lake Placid, NY), and sheared to fragment DNA. The samples were then immunoprecipitated using agarose-conjugated antibodies to Sp1 (Santa Cruz Biotechnology, Santa Cruz, CA), KLF11 (Transduction Laboratories, Lexington, KY), irrelevant control antibody (actin monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA), or protein A-agarose beads alone at 4 °C overnight. Following immunoprecipitation, the samples were washed and eluted using a chromatin immunoprecipitation kit (Upstate Biotechnology, Inc.) according to the manufacturer's instructions. Cross-links were removed at 65 °C for 4 h, and immunoprecipitated DNA was purified using phenol/chloroform extraction (500 μl) and ethanol precipitation. Sp1- and KLF11-bound DNA was detected by visualizing PCR products on a 2% agarose gel. Statistical Analysis—All of the experiments were performed at least three times, each in duplicate. The data are expressed as the means ± S.E. Cholesterol Diminishes Endothelial Caveolin-1 mRNA and Protein Levels in Vitro and in Vivo—Caveolin-1 expression is important for cholesterol-mediated endothelial cell signaling. Interestingly, caveolin-1 mRNA levels were increased 4-fold in BAEC exposed to lipoprotein depletion achieved by incubating cells for 16 h with 30% LPDS, as compared with 30% NHS, as depicted in the representative Northern blot (Fig. 1A, left panel). Concentration dependence of the stimulatory effect of lipoprotein depletion on caveolin-1 mRNA levels in endothelial cells, achieved by altering the volume ratio of NHS and LPDS in the culture medium, thereby varying the degree of cholesterol depletion, revealed that increasing the volume ratio of LPDS compared with NHS was associated with incremental increases in caveolin-1 mRNA levels as depicted in the densitometric calculations compiled from Northern blot analysis (Fig. 1A, right panel). To further establish the specific role of lipoproteins in the aforementioned Northern blot analyses, we next sought to determine whether supplementation of exogenous LDL could reverse the increase in caveolin-1 mRNA levels detected in response to lipoprotein depletion. As seen in Fig. 1B (left panel), supplementation of 0.1 mg/ml of LDL to LPDS entirely negated the increase in caveolin-1 mRNA levels, which occurred in response to cholesterol depletion in BAEC. To confirm that our experimental conditions did indeed modulate intracellular cholesterol levels, we measured total cellular cholesterol from cells incubated with NHS, LPDS, and LPDS plus LDL. As seen in Fig. 1C, cholesterol levels, as assayed by thin layer chromatography and normalized for cellular protein, were reduced by incubation of cells with LPDS, and this reduction was negated by supplementation of exogenous LDL (0.05 mg/ml). These results were corroborated by studies utilizing the cholesterol-binding probe, filipin, which demonstrated prominent filipin staining in cells incubated with NHS but not highly detected in cells incubated with LPDS (data not shown). We next established the biologic relevance of the aforementioned findings by examining caveolin-1 protein levels in an animal model of hypercholesterolemia. To examine this paradigm in vivo, we examined caveolin-1 protein levels from aortic endothelial cell lysates scraped from pigs fed a high cholesterol diet in the context of a well established 12-week feeding protocol aimed at inducing hypercholesterolemia (24Herrmann J. Gulati R. Napoli C. Woodrum J. Lerman L. Rodriguez-Porcel M. Sica V. Simari R. Ciechanover A. Lerman A. FASEB J. 2003; 17: 1730-1732Crossref PubMed Scopus (52) Google Scholar). In these analyses (Fig. 1D; representative blot in the left panel and densitometric analysis from three experiments in the right panel), endothelial caveolin-1 protein levels were diminished in pigs with higher levels of serum cholesterol (serum total cholesterol and LDL cholesterol, 9.5 ± 2.1 and 6.8 ± 1.7 mmol/liter) as compared with pigs with lower levels of serum cholesterol (serum total cholesterol and LDL cholesterol, 2.2 ± 0.1 and 0.9 ± 0.2 mmol/liter; p < 0.05 compared with hypercholesterolemic pig levels of total cholesterol and LDL cholesterol). These observations are qualitatively consistent with results obtained in BAEC exposed to varying levels of cholesterol. Sp1 Is Required for the Enhancer Effect of SREBP in Response to Cholesterol Depletion—Based on the prominent regulatory effect of cholesterol modulation on endogenous caveolin-1 expression, we used the caveolin-1 promoter as a model to characterize the role of SREBP, Sp1, and Sp1-like proteins in cholesterol-dependent gene regulation in endothelial type cells. For this purpose, ECV cells transfected with the caveolin-1 promoter were incubated with varying volume ratios of NHS and LPDS. As depicted in Fig. 2A, an increase in the volume ratio of LPDS to NHS incrementally induces caveolin-1 promoter activity. The cells incubated with 25% LPDS alone display a 3-fold up-regulation in the activity of this promoter. The specificity of the influence of cholesterol depletion on the caveolin-1 promoter was supported by demonstrating the lack of influence of cholesterol depletion on the empty pGL3 vector (Fig. 2B, left columns), as well as on a promoter not known to be regulated by cholesterol, Osf2/Cbfa1 (Fig. 2B, middle columns) (32Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar). Furthermore, supplementation of LDL cholesterol (0.05 and 0.1 mg/ml) to the LPDS cholesterol depletion medium abrogates the stimulatory influence of cholesterol depletion on caveolin-1 promoter activity, thereby resulting in activity levels similar to that observed with NHS (Fig. 2B, right columns). Similar to other genes regulated by cholesterol, the caveolin-1 promoter contains SRE-binding sites (17Bist A. Fielding P. Fielding C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10693-10698Crossref PubMed Scopus (233) Google Scholar). We performed deletion analysis of the caveolin-1 promoter to dissect the importance of three SRE sites for cholesterol depletion-dependent activation of the caveolin-1 promoter. Constructs included SRE1/2 and SRE3, the former of which contains the first two SRE-like sites, at –646 to –637 and –395 to –386, and the latter of which contains the third SRE-like site at –287 to –278 (Fig. 3A, left panel). The cells were transfected for reporter assay with vectors encoding deletion constructs, SRE1/2 (pGL3-SRE1/2) or SRE3 (pGL3-SRE3), or the full-length promoter (pGL3-cavFL), or empty vector (pGL3) and incubated with NHS, LPDS, or LPDS with LDL supplementation (0.1 mg/ml). As depicted in Fig. 3A (right panel), cholesterol depletion conditions activated pGL3-SRE3 by 3-fold, a magnitude identical to the effect of cholesterol depletion on pGL3-cavFL, and LDL supplementation inhibited the activation event observed with these constructs. pGL3-SRE1/2 demonstrated minimal activity, indicating that the stimulatory influence of cholesterol depletion on caveolin-1 gene transcription is mediated through elements within the promoter region encoded within the pGL3-SRE3 promoter fragment. Subsequently, we performed gel shift assays using oligonucleotides encoding SRE1, SRE2, and SRE3 and nuclear extracts derived from BAEC. As seen in Fig. 3B, prominent nucleoprotein binding was detected with the oligonucleotide probe encoding SRE3 (arrow). Binding was reduced with excess cold probe (see lanes labeled 25×) and furthermore, mutation of the SRE3 (SRE3m) resulted in loss of binding of this nucleoprotein. A small amount of nucleoprotein binding was also detected with an oligonucleotide encoding SRE2. Conversely, neither an oligonucleotide probe encoding SRE1 nor the mutated oligonucleotide probe encoding this sequence (SRE1m) binds nucleoproteins in a specific manner under these conditions. Additionally, we purified the detected SRE3-binding protein by employing a modified oligonucleotide capture assay, using a streptavidin-bound SRE3 sequence combined with Western blot analysis. As seen in Fig. 3C (top panel), the SRE3-bound nucleoprotein was detected on silver stain as a doublet at ∼68 kDa, whereas no protein binding was detected with a control probe. Western blot analysis of bound proteins (Fig. 3C, bottom panel) recognized SREBP-1, in its transcriptionally active cleaved molecular weight form, as the protein that binds to the SRE3 site. Thus, the SRE3 site on the caveolin-1 promoter binds specifically to SREBP-1, suggesting that the reversible binding of this protein to SRE3 constitutes an important regulatory step in the expression of the caveolin-1 gene in endothelial cells. Thus, these studies establish that cholesterol depletion increases caveolin-1 promoter activity in endothelial type cells via the binding of SREBP-1 to distinct GC-rich regions and serve as a base line for subsequent experiments shown below aimed at characteriz
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