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Peroxisome Proliferator-activated Receptor γ Suppresses Proximal α1(I) Collagen Promoter via Inhibition of p300-facilitated NF-I Binding to DNA in Hepatic Stellate Cells

2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês

10.1074/jbc.m510094200

1083-351X

Sharon Yavrom, Li Chen, Shigang Xiong, Jiaohong Wang, Richard A. Rippe, Hidekazu Tsukamoto,

Liver Disease and Transplantation

Depletion of peroxisome proliferator-activated receptor γ (PPARγ) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells in the genesis of liver fibrosis (Miyahara, T., Schrum, L., Rippe, R., Xiong, S., Yee, H. F., Jr., Motomura, K., Anania, F. A., Willson, T. M., and Tsukamoto, H. (2000) J. Biol. Chem. 275, 35715-35722; Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., and Tsukamoto, H. (2004) J. Biol. Chem. 279, 11392-11401). In support of this notion, ectopic expression of PPARγ suppresses hepatic stellate cells activation markers, most notably expression of α1(I) procollagen. However, the mechanisms underlying this antifibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2-kb α1(I) procollagen promoter to demonstrate that a region proximal to -133 bp is where PPARγ exerts its inhibitory effect. Within this region, two DNase footprints with Sp1 and reverse CCAAT box sites exist. NF-I, but not CCAAT DNA-binding factor/NF-Y, binds to the proximal CCAAT box in hepatic stellate cells. A mutation of this site almost completely abrogates the promoter activity. NF-I mildly but independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARγ inhibits NF-I binding to the most proximal footprint (-97/-85 bp) and inhibits its transactivity. The former effect is mediated by the ability of PPARγ to inhibit p300-facilitated NF-I binding to DNA as demonstrated by chromatin immunoprecipitation assay. Depletion of peroxisome proliferator-activated receptor γ (PPARγ) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells in the genesis of liver fibrosis (Miyahara, T., Schrum, L., Rippe, R., Xiong, S., Yee, H. F., Jr., Motomura, K., Anania, F. A., Willson, T. M., and Tsukamoto, H. (2000) J. Biol. Chem. 275, 35715-35722; Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., and Tsukamoto, H. (2004) J. Biol. Chem. 279, 11392-11401). In support of this notion, ectopic expression of PPARγ suppresses hepatic stellate cells activation markers, most notably expression of α1(I) procollagen. However, the mechanisms underlying this antifibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2-kb α1(I) procollagen promoter to demonstrate that a region proximal to -133 bp is where PPARγ exerts its inhibitory effect. Within this region, two DNase footprints with Sp1 and reverse CCAAT box sites exist. NF-I, but not CCAAT DNA-binding factor/NF-Y, binds to the proximal CCAAT box in hepatic stellate cells. A mutation of this site almost completely abrogates the promoter activity. NF-I mildly but independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARγ inhibits NF-I binding to the most proximal footprint (-97/-85 bp) and inhibits its transactivity. The former effect is mediated by the ability of PPARγ to inhibit p300-facilitated NF-I binding to DNA as demonstrated by chromatin immunoprecipitation assay. Cirrhosis, the advanced stage of liver fibrosis, is the 12th leading cause of medial mortality in 2002 with 27,257 annual deaths according to a report by the Center for Disease Control. This mortality is even higher among those with productive ages between 45 and 54, ranking it as the fourth leading cause of death, highlighting medical and socioeconomic significance of the disease (1Kochanek K.D. Murphy S.L. Anderson R.N. Scott C. Natl. Vital Stat. Rep. 2004; 53: 1-115PubMed Google Scholar). Currently, there is no medical treatment for the disease other than liver transplantation. Therefore, the understanding of cellular and molecular mechanisms of liver fibrogenesis is of primary importance for the development of new treatments. The effector cell type for liver fibrosis is the hepatic stellate cell (HSC). 2The abbreviations used are: HSChepatic stellate cellPPARγperoxisome proliferator-activated receptor γPPREperoxisome proliferator-activated receptor response elementCBPCREB-binding proteinCREBcAMP-response element-binding proteinFPfootprintGFPgreen fluorescent proteinEMSAelectrophoretic mobility shift assayChIPchromatin immunoprecipitationBSCbiliary fibrosis-derived stellate cell.2The abbreviations used are: HSChepatic stellate cellPPARγperoxisome proliferator-activated receptor γPPREperoxisome proliferator-activated receptor response elementCBPCREB-binding proteinCREBcAMP-response element-binding proteinFPfootprintGFPgreen fluorescent proteinEMSAelectrophoretic mobility shift assayChIPchromatin immunoprecipitationBSCbiliary fibrosis-derived stellate cell. HSCs are liver mesenchymal cells that are believed to function as pericytes for the liver microcirculatory system called sinusoids. They are located in the perisinusoidal space, an anatomical area nestled between the nonluminal surface of the sinusoidal endothelial cell and the microvilli surface of the hepatocyte (2Friedman S.L. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). HSCs constitute 7-10% of the liver cell population and it stores 85% of the body's total vitamin A content (3Rojkind M. Giambrone M.A. Biempica L. Gastroenterology. 1979; 76: 710-719Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 4Friedman S.L. J. Gastroenterol. 1997; 32: 424-430Crossref PubMed Scopus (126) Google Scholar). They also produce and maintain the normal matrix milieu (basement membrane components) of the perisinusoidal space. In addition, HSC provides direct and indirect homeostatic control over hepatocytes through communication via gap junctions (5Rojkind M. Novikoff P.M. Greenwel P. Rubin J. Rojas-Valencia L. de Carvalho A.C. Stockert R. Spray D. Hertzberg E.L. Wolkoff A.W. Am. J. Pathol. 1995; 146: 1508-1520PubMed Google Scholar) and the release of soluble factors such as hepatocyte growth factor (6Skrtic S. Wallenius V. Ekberg S. Brenzel A. Gressner A.M. Jansson J.O. J. Hepatol. 1999; 30: 115-124Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), epimorphin (7Miura K. Nagai H. Ueno Y. Goto T. Mikami K. Nakane K. Yoneyama K. Watanabe D. Terada K. Sugiyama T. Imai K. Senoo H. Watanabe S. Biochem. Biophys. Res. Commun. 2003; 311: 415-423Crossref PubMed Scopus (34) Google Scholar), and pleiotrophin (8Asahina K. Sato H. Yamasaki C. Kataoka M. Shiokawa M. Katayama S. Tateno C. Yoshizato K. Am. J. Pathol. 2002; 160: 2191-2205Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, these cells are also responsible for a severalfold increase in the production of extracellular matrix components in the genesis of liver fibrosis (4Friedman S.L. J. Gastroenterol. 1997; 32: 424-430Crossref PubMed Scopus (126) Google Scholar).Upon fibrogenic stimulation, quiescent HSCs transdifferentiate to myofibroblastic cells to produce excessive extracellular matrix. This cellular transition process is characterized by the loss of vitamin A storage; cellular proliferation and migration; acquisition of a myofibroblastic phenotype, such as expression of α smooth muscle actin, induction of fibrogenic extracellular matrix genes (collagen type I and III), expression of autocrine cytokines, such as platelet-derived growth factor (9Kinnman N. Goria O. Wendum D. Gendron M.C. Rey C. Poupon R. Housset C. Lab. Invest. 2001; 81: 1709-1716Crossref PubMed Scopus (94) Google Scholar), transforming growth factor α, transforming growth factor β (10Suzuki K. Fukutomi Y. Matsuoka M. Torii K. 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In search of the molecular basis of this unique HSC transdifferentiation phenomenon, we and others recently disclosed that this process is accompanied by reduced levels of a member of the nuclear hormone receptor family, peroxisome proliferator-activated receptor γ (PPARγ) (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Further, our subsequent studies demonstrated that this molecular "defect" in transdifferentiated HSC is part of the loss of adipogenic transcriptional program required for the maintenance of HSC quiescence (17She H. Xiong S. Hazra S. Tsukamoto H. J. Biol. Chem. 2005; 280: 4959-4967Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). In fact, this loss of the adipogenic program and the transdifferentiation process are coordinately reversed by ectopic expression of PPARγ (18Hazra S. Xiong S. Wang J. Rippe R.A. Krishna V. Chatterjee K. Tsukamoto H. J. Biol. Chem. 2004; 279: 11392-11401Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) or sterol regulatory element-binding protein 1c (17She H. Xiong S. Hazra S. Tsukamoto H. J. Biol. Chem. 2005; 280: 4959-4967Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), another key adipogenic transcription factor.One of the most pivotal antifibrotic effects of PPARγ is its ability to inhibit type I collagen expression at the level of the transcription (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Type I collagen makes up 40-50% of the total collagen proteins in the normal liver and is increased to 60-70% in the cirrhotic liver (3Rojkind M. Giambrone M.A. Biempica L. Gastroenterology. 1979; 76: 710-719Abstract Full Text PDF PubMed Scopus (450) Google Scholar). Type I collagen is a heterotrimeric protein composed of two α1(I) and one α2(I) collagen polypeptides encoded by two different genes that are coordinately up-regulated in liver fibrogenesis (19Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar). Treatment of activated HSC with a ligand for PPARγ (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) or transduction of these cells with a PPARγ plasmid (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) represses basal α1(I) procollagen promoter activity that is largely dependent on a proximal 2.2-kb 5′-flanking region (19Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 20Rippe R.A. Lorenzen S.I. Brenner D.A. Breindl M. Mol. Cell. Biol. 1989; 9: 2224-2227Crossref PubMed Scopus (80) Google Scholar). However, the mechanism by which PPARγ inhibits type I collagen promoter activity is currently unknown. The present study investigated where in the proximal 2.2 kb α1(I) procollagen promoter PPARγ renders its inhibition and how it achieves this effect. Our results demonstrate that the 5′-flanking α1(I) procollagen promoter proximal to -133 bp is where PPARγ renders its inhibitory effect. This inhibition is mediated by the ability of PPARγ to suppress NF-I binding and transactivity via inhibition of p300-facilitated NF-I binding.MATERIALS AND METHODSHSC Isolation and Cell Culture—HSCs were isolated from normal male Wistar rats as previously described (21Tsukamoto H. Cheng S. Blaner W.S. Am. J. Physiol. 1996; 270: G581-G586PubMed Google Scholar). Briefly, nonparenchymal cells were isolated via sequential digestion with Pronase and type IV collagenase, followed by differential low speed centrifugation. A pure fraction of HSC was isolated by arabinogalactan gradient ultracentrifugation and collecting the cells at the interface between the medium and a 1.035 density gradient. Cell purity was determined using phase-contrast microscopy and UV-excited fluorescence microscopy. Cell viability was determined by trypan blue exclusion. Cells were cultured on a 100-mm dish in low glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 mg/ml streptomycin, 10,000 units/ml penicillin, and 25 μg/ml amphotericin B. Cells were maintained in the culture medium for 7 days, at which time adenoviral vectors were added. Spontaneously immortalized, activated HSCs were established from a rat with cholestatic liver fibrosis and termed as biliary fibrosis-derived stellate cells (BSCs) (22Sung C.K. She H. Xiong S. Tsukamoto H. Am. J. Physiol. 2004; 286: G722-G729Crossref PubMed Scopus (64) Google Scholar). These cells have the phenotype similar to activated HSC. BSCs were cultured in 10% low glucose medium and primarily used in transient transfection experiments.Adenoviral Vector Infection—Full-length PPARγ1 cDNA was cloned from pCMX-PPARγ1 into the transfer vector, subsequently allowing homologous recombination with the pAdEasy-1 adenoviral plasmid containing a green fluorescent protein (GFP) reporter (Stratagene, La Jolla, CA) as previously described (18Hazra S. Xiong S. Wang J. Rippe R.A. Krishna V. Chatterjee K. Tsukamoto H. J. Biol. Chem. 2004; 279: 11392-11401Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). A control vector containing cytomegalovirus-driven GFP reporter gene was also constructed. On the seventh day of primary rat HSC culture, a vector was applied to HSCs at a multiplicity of infection of 100 to infect and transduce PPARγ or GFP. The following day, the medium was changed, and the cells were cultured for an additional four days to observe the effects on HSC as described (18Hazra S. Xiong S. Wang J. Rippe R.A. Krishna V. Chatterjee K. Tsukamoto H. J. Biol. Chem. 2004; 279: 11392-11401Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).RNA Isolation and Quantitative Real Time PCR—Total RNA was extracted from isolated HSC transduced with Ad.GFP and Ad.PPARγ using Trizol reagent (Invitrogen). Two nanograms of total RNA was used in a 20-μl reaction with reverse transcriptase for 30 min followed by 40 cycles of PCR to produce products using TaqMan Gold One Step PCR Kit (Applied Biosystems, Foster City, CA) and the ABI 7700 SDS thermocycler (Applied Biosystems). Synthesized cDNA was amplified using specific primers for α1(I) collagen (5′-TCGATTCACCTACAGCACGC, 5′-CATTAGCATCCGTGGGAACA), glyceraldehyde-3-phosphate dehydrogenase (5′-TGCACCACCAACTGCTTAG, 5′-GGATGCAGGGATGATGTTC). Probes were 5,6-carboxylfluorescein amidite labeled at the 5′-end and black hole quencher-1 labeled at the 3′-end (Biosearch Technologies Inc., Novoato, CA).Plasmids and Transient Transfection—pCMX and pCMX-PPARγ were gifts from Ron Evans (The Salk Institute, La Jolla, CA). pSG5 and p300 were gifts from Michael Stallcup (University of Southern California). Mouse collagen promoter deletion constructs were as follows: pCol2-lucif, pCol3-lucif, pCol6-lucif, pCol7-lucif, and pUC-Cat (-220, -133, -120, and -92 bp/+115 bp) were used as previously described (19Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 20Rippe R.A. Lorenzen S.I. Brenner D.A. Breindl M. Mol. Cell. Biol. 1989; 9: 2224-2227Crossref PubMed Scopus (80) Google Scholar). pPac, pPac-NF-I and pPac-Sp1 were used to assess the effects of PPARγ on NF-I- or Sp1-driven promoter activity. Renilla pRL-TK was purchased from Promega. Luciferase promoter deletion constructs were created via restriction enzyme digestion (XbaI and XhoI) of the pUC-Cat constructs and insertion into the pGL3-luciferase (Promega) backbone. Briefly, BSC or NIH3T3 cells were seeded in 6- or 24-well plates and incubated overnight. A collagen promoter construct, an expression plasmid (pCMX or PPARγ), and F2 reagent (Targeting System, San Diego, CA) were mixed and added to serum-free, high glucose Dulbecco's modified Eagle's medium and incubated for 25 min at 37 °C and then placed onto the cells. Two hours later, the cells were supplemented with 10% high glucose Dulbecco's modified Eagle's medium. The following day, the medium was changed, and the cells were incubated for an additional 8 h. The cell lysates were collected using 5× passive lysis buffer (Promega) and dual luciferase assay (Promega) was performed using a luminometer (E&G Berthold). Mutations of three nucleotides (TGG to CAA) in the most proximal reverse CCAAT box was created by site-directed mutagenesis according to the QuikChange™ protocol (Stratagene). Primers were designed to introduce 3-nucleotide mutations into the wild type CCAAT of the FP-1 region of the luciferase promoter deletion construct (-133 bp/+115 bp). The DNA sequence of each construct was verified using an ABI Prism 377 sequencer (PerkinElmer Life Sciences). The following primers were used: FP-1-mutant NF-I (forward), 5′-gggccaggcagttctgatCAActgggggccgggctgctggctc-3′; FP-1-mutant NF-I (reverse), 5′-gagccagcagcccggcccccagTTGatcagaactgcctggccc-3′.Electrophoretic Mobility Shift Assay—Nuclear proteins were extracted from HSC infected with Ad.GFP or Ad.PPARγ using Dignam A and C reagents (23Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3907) Google Scholar). Extracts (5-10 μg) were incubated in a reaction mixture (20 mm HEPES, pH 7.6, 100 mm MgCl2, 0.2 mm EDTA, 2 mm dithiothreitol, 20% glycerol, 200 μg/ml poly(dI-dC)) on ice for 10 min followed by an additional 20-min incubation on ice with 2 ng of α-32P-labeled double-stranded oligonucleotides as described below: ARE-7, 5′-GCTTACTGGATCAGAGTTCACAGAT; FP-1, 5′-GATTGGCTGGGGGCCGGGCTGCT; FP-2, 5′-GGTTCCAAATTGGGGGCCGGGCCAG; Sp1, 5′-GATCAATGGGGCGGGGCAAT; NF-I, 5′-GGTTTTGGATTGAAGCCAATATGAG.The reaction mixture was resolved on a 6% nondenaturing polyacrylamide gel (Bio-Rad) in 0.5× TBE (45 mm Tris, 45 mm boric acid, 1 mm EDTA). The gel was dried and subjected to phosphorimaging for detection of shifted bands. For a supershift analysis, polyclonal antibodies against NF-I, Sp1, or Sp3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added and incubated on ice for an additional 30 min. For competition analysis, a 200-fold molar excess of a cold probe was added to the reaction mixture just prior to the addition of α-32P-labeled probe.Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was performed using the ChIP assay kit according to the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY). In brief, ChIP assay was performed on HSC cultured on plastic for 7 days or NIH3T3 cells without or with transfection with NF-I, PPARγ, and/or p300 expression plasmids or respective empty vectors. After a 48-h incubation, ∼4 ×106 cells/ChIP assay, were cross-linked with 1% formaldehyde at 37 °C for 10 min and rinsed twice with ice-cold phosphate-buffered saline. The cells were harvested by brief centrifugation and lysed in SDS-lysis buffer (50 mmol/liter Tris-HCl, pH 8.1, 10 mmol/liter EDTA, 1% SDS, protease inhibitors). The lysates were sonicated on ice with two pulses at 15 s each to achieve chromatin fragments ranging between 200 and 1000 bp in size followed by centrifugation at 15,000 rpm for 10 min at 4 °C. Supernatants were collected and diluted 10-fold in a ChIP dilution buffer (a 20-μl aliquot was removed to serve as an input sample) followed by preimmunoprecipitation clearing with 80 μl of a mixture of salmon sperm DNA/Protein A at 4 °C with rotation for 30 min. Immunoprecipitation was carried out with 1 μg of antibodies (anti-NF-I, anti-p300, and anti-CCAAT DNA-binding factor (CBF)/NF-Y antibodies from Santa Cruz Biotechnology) at 4 °C overnight with rotation. After immunoprecipitation, 60 μl of a mixture of salmon sperm DNA and Protein A was added and incubated at 4 °C with rotation for 30 min and followed by brief centrifugation. The precipitates were washed twice with low salt buffer, once with high salt buffer, and once with LiCl buffer. Then the precipitates were washed again with the TE buffer. The immune complexes were extracted twice with 250 μl of elution buffer. The extracted complexes and the input were heated at 65 °C for 4 h after the addition of 20 μl of 5 mol/liter NaCl to reverse cross-link. Following proteinase K treatment, DNA was extracted by phenol/chloroform solution and precipitated with 20 μg of glycogen. The recovered DNA was resuspended and subjected to 35 cycles of PCR using the following primers: α1(I) procollagen promoter FP-1 region, 5′-TGGACTCCTTTCCCTTCCTTTCCCTCCT-3′ and 5′-TGGGCCCCTTTTATACCATC-3′; aP2 gene PPRE region, 5′-TGCACATTTCACCCAGAGAG-3′ and 5′-TGTTTGGGCTGTGACACTTC-3′. The PCR products were analyzed on 1.5% agarose gel.RESULTSPPARγ Inhibits α1(I) Procollagen mRNA Expression and Promoter Activity—PPARγ is depleted in activated HSC, whereas activation markers including the α1(I) collagen gene are induced (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Ectopic expression of PPARγ in culture-activated HSC by an adenoviral vector reduces the level of α1(I) procollagen mRNA by half as compared with HSC transduced with a control GFP vector as determined by real time PCR (Fig. 1A). Transient transfection experiment using the HSC cell line (BSC cells) reveals that PPARγ expression also decreases by 50% the activity of a proximal 2.2-kb α1(I) procollagen promoter known to encompass the highest basal activity in fibroblasts (19Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 20Rippe R.A. Lorenzen S.I. Brenner D.A. Breindl M. Mol. Cell. Biol. 1989; 9: 2224-2227Crossref PubMed Scopus (80) Google Scholar) (Fig. 1B). These results confirm the previous finding (15Miyahara T. Schrum L. Rippe R. Xiong S. Yee Jr., H.F. Motomura K. Anania F.A. Willson T.M. Tsukamoto H. J. Biol. Chem. 2000; 275: 35715-35722Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 16Galli A. Crabb D.W. Ceni E. Salzano R. Mello T. Svegliati-Baroni G. Ridolfi F. Trozzi L. Surrenti C. Casini A. Gastroenterology. 2002; 122: 1924-1940Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 18Hazra S. Xiong S. Wang J. Rippe R.A. Krishna V. Chatterjee K. Tsukamoto H. J. Biol. Chem. 2004; 279: 11392-11401Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) and further support that the inhibitory effect of PPARγ is at the level of the proximal promoter.A Site of the Inhibitory Effect of PPARγ Is Located within the Most Proximal -220 bp α1(I) Collagen Promoter Region—In order to assess the region within the 2.2-kb promoter that is subjected to PPARγ-mediated inhibition, we performed transient transfection experiments using four deletion constructs of the promoter (Fig. 2A). The relative activity of each of the deletion constructs was tested first by transfection experiments in BSCs (Fig. 2B). Our results reveal that two repressor elements: one between -2.2 and -1.8 kb and another between -1.1 and -220 bp. An enhancer element is also found between -1.8 and -1.1 kb. Of all four deletion constructs, the region proximal to -220 bp is shown to have the highest promoter activity in consistent with the previous findings (19Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 20Rippe R.A. Lorenzen S.I. Brenner D.A. Breindl M. Mol. Cell. Biol. 1989; 9: 2224-2227Crossref PubMed Scopus (80) Google Scholar). Co-transfection of each deletion construct with a PPARγ or empty vector reveals that PPARγ inhibits all of the deletion constructs by ∼50% (Fig. 2C). Since the -220 bp region has the highest activity and PPARγ expression equally reduces the activity of each promoter construct, we concluded that PPARγ primarily exerts its inhibitory effect within the -220 bp proximal region and +115 bp of the first exon. Our review of the -220 bp proximal promoter sequence fails to reveal a PPRE. Thus, PPARγ must mediate the effect not via direct repression but via its interaction with other trans-acting factor(s) and/or cis-regulatory element(s).FIGURE 2PPARγ suppresses the collagen promoter within the -220 bp proximal promoter region. A, a schematic diagram of four deletion constructs within the proximal 2.2 kb α1(I) collagen promoter. B, relative promoter activities of the collagen promoter deletion constructs as compared with the highest activity achieved by the -220/+115 bp promoter. *, p < 0.05 as compared with the activity of pCOL3 (n = 4). C, PPARγ equally inhibits each deletion construct by 50%, suggesting that the primary site of the inhibitory effect of PPARγ is located within the -220 bp collagen promoter region. *, p < 0.05 as compared with pCMX-transfected cells (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition of PPARγ Is Confined to DNase Footprint (FP)-1 and -2 Regions within the -133 bp α1(I) Collagen Promoter—To further define the site of PPARγ-mediated inhibition, we designed and created an additional set of deletion constructs within the most proximal -220 bp region. This region is known to contain four protected footprints as determined by DNase footprinting analysis of activated HSC (24Rippe R.A. Almounajed G. Brenner D.A. Hepatology. 1995; 22: 241-251PubMed Google Scholar) (Fig. 3A), and newly created deletions are designed to test these FP regions: -133/+115 (FP-3 and FP-4 deleted but intact FP-2 and -1); -120/+115 (distal region of FP-2 deleted but intact FP-1); and -92/+115 bp (only proximal region of FP-1). Transient transfection experiments reveal that the two distal footprints (FP-3 and FP-4) contribute minimally to basal collagen promoter activity (a statistically significant change in the promoter activity is not attained by this deletion: pCOL-133) (Fig. 3B). However, an additional deletion, including the distal half of FP-2 reduces the basal promoter activity to 50%, and a further deletion within FP-1 reduces the promoter activity by another 50%. Overexpression of PPARγ expression vector results in a 50% inhibition on the pCOL-133 promoter (Fig. 3C). This effect is attenuated when FP-2 is disrupted (pCOL-120). PPARγ also retains a modest inhibitory effect on the promoter activity rendered only by the most proximal portion of the FP-1 (pCOL-92). However, the absolute magnitude of the activity inhibited by PPARγ accounts only for 25% of the inhibition seen with pCOL-220 or pCOL-133. These results suggest that both FP-1 and FP-2 contain the sites via which PPARγ renders a major inhibitory effect on the promoter.FIGURE 3PPARγ inhibits the proximal 220 bp collagen promoter at the FP-1 and FP-2 sites. A, a schematic diagram of deletion constructs within the -220 bp α1(I) collagen promoter in reference to the known four DNase footprints. B, relative activities of the collagen promoter deletion constructs transfected in BSCs. *, p < 0.05 as compared with pCOL-220 (n = 5). C, co-transfection experiments using deletion constructs and a PPARγ expression vector reveals that PPARγ inhibits the -220 and -133 bp promoters by 45-50%, but this inhibition is attenuated when FP-2 is disrupted in pCOL-120. PPARγ still suppresses pCOL-92 containing the proximal portion of FP-1. *, p < 0.05 as compared with pCMX-transfected cells (n = 5-7).View Large Image Figure ViewerDownload Hi-res image Download (PPT)PPARγ Inhibits NF-I Binding to FP-1 but Has No Effects on Sp1 Binding to FP-2—Since FP-1 and FP-2 are shown to be the most likely sites of the inhibitory action of PPA