Title: Adipogenic Transcriptional Regulation of Hepatic Stellate Cells
Abstract: Hepatic stellate cells (HSC) undergo transdifferentiation (activation) from lipid-storing pericytes to myofibroblastic cells to participate in liver fibrogenesis. Our recent work demonstrates that depletion of peroxisome proliferator-activated receptor γ (PPARγ) constitutes one of the key molecular events for HSC activation and that ectopic expression of this nuclear receptor achieves the phenotypic reversal of activated HSC to the quiescent cells. The present study extends these findings to test a novel hypothesis that adipogenic transcriptional regulation is required for the maintenance of HSC quiescence. Comparative analysis of quiescent and activated HSC in culture reveals higher expression of putative adipogenic transcription factors such as CCAAT/enhancer-binding protein (C/EBP) α, C/EBPβ, C/EBPδ, PPARγ, liver X receptor α, sterol regulatory element-binding protein 1c and of adipocyte-specific genes in the quiescent cells. Conversely, activated HSC have increased expression of PPARβ, a transcription factor known to promote fatty acid oxidation. A treatment of activated HSC with the adipocyte differentiation mixture (isobutylmethylxanthine, dexamethasone, and insulin) or ectopic expression of PPARγ or SREBP-1c in these cells, induces a panel of adipogenic transcription factors, reduces PPARβ, and causes the phenotypic reversal to quiescent HSC. These results support the importance of adipogenic transcriptional regulation in HSC quiescence and provide a new framework for identifying novel molecular targets for the treatment of liver cirrhosis. Hepatic stellate cells (HSC) undergo transdifferentiation (activation) from lipid-storing pericytes to myofibroblastic cells to participate in liver fibrogenesis. Our recent work demonstrates that depletion of peroxisome proliferator-activated receptor γ (PPARγ) constitutes one of the key molecular events for HSC activation and that ectopic expression of this nuclear receptor achieves the phenotypic reversal of activated HSC to the quiescent cells. The present study extends these findings to test a novel hypothesis that adipogenic transcriptional regulation is required for the maintenance of HSC quiescence. Comparative analysis of quiescent and activated HSC in culture reveals higher expression of putative adipogenic transcription factors such as CCAAT/enhancer-binding protein (C/EBP) α, C/EBPβ, C/EBPδ, PPARγ, liver X receptor α, sterol regulatory element-binding protein 1c and of adipocyte-specific genes in the quiescent cells. Conversely, activated HSC have increased expression of PPARβ, a transcription factor known to promote fatty acid oxidation. A treatment of activated HSC with the adipocyte differentiation mixture (isobutylmethylxanthine, dexamethasone, and insulin) or ectopic expression of PPARγ or SREBP-1c in these cells, induces a panel of adipogenic transcription factors, reduces PPARβ, and causes the phenotypic reversal to quiescent HSC. These results support the importance of adipogenic transcriptional regulation in HSC quiescence and provide a new framework for identifying novel molecular targets for the treatment of liver cirrhosis. Transdifferentiation of vitamin A-storing hepatic stellate cells (HSC) 1The abbreviations used are: HSC, hepatic stellate cell(s); PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein; C/EBP, CCAAT/enhancer-binding protein; LXR, liver X receptor; MDI, isobutylmethylxanthine, dexamethazone, and insulin; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; TGF, transforming growth factor; MOI, multiplicity of infection; PBS, phosphate-buffered saline; SCAP, SREBP cleavage-activating protein; GFP, green fluorescent protein.1The abbreviations used are: HSC, hepatic stellate cell(s); PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein; C/EBP, CCAAT/enhancer-binding protein; LXR, liver X receptor; MDI, isobutylmethylxanthine, dexamethazone, and insulin; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; TGF, transforming growth factor; MOI, multiplicity of infection; PBS, phosphate-buffered saline; SCAP, SREBP cleavage-activating protein; GFP, green fluorescent protein. to vitamin A-depleted myofibroblastic cells represents a key cellular event in the genesis of cirrhosis, for which no effective medial treatments are currently available except for liver transplantation. Transdifferentiated (activated) HSC are proliferative, proinflammatory, and fibrogenic with induced ability to synthesize and deposit extracellular matrices (1Friedman S.L. J. Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, better understanding of the mechanism underlying HSC transdifferentiation is the pivotal step toward identification of molecular targets for new and effective treatments for the disease. The most fundamental prerequisite for the understanding of HSC transdifferentiation is defining the cell type of differentiated HSC. This question relates to the origin of HSC that continues to puzzle the field. HSC are believed to serve as pericytes for hepatic capillaries called sinusoids. They represent 5–8% of total liver cells and 15–23% of nonparenchymal cells in the normal liver (2Geerts A. Semin. Liver Dis. 2001; 21: 311-335Crossref PubMed Scopus (587) Google Scholar). HSC are positive for a mesenchymal marker such as vimentin. Rodent HSC express desmin (3Yokoi Y. Namihisa T. Kuroda H. Komatsu I. Miyazaki A. Watanabe S. Usui K. Hepatology. 1984; 4: 709-714Crossref PubMed Scopus (342) Google Scholar) and glial fibrillary acidic protein (4Gard A.L. White F.P. Dutton G.R. J. Neuroimmunol. 1985; 8: 359-375Abstract Full Text PDF PubMed Scopus (153) Google Scholar), suggesting smooth muscle cell and glial cell lineage, respectively. Upon activation, both rodent and human HSC lose vitamin A and begin to express α-smooth muscle actin (5Ramadori G. Veit T. Schwogler S. Dienes H.P. Knittel T. Meyer zum Rieder H. Buschenfelde K.H. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1990; 59: 349-357Crossref PubMed Scopus (281) Google Scholar, 6Enzan H. Himeno H. Iwamura S. Saibara T. Onishi S. Yamamoto Y. Hara H. Virchows Arch. 1994; 424: 249-256Crossref PubMed Scopus (119) Google Scholar). Interestingly, undifferentiated HSC in fetal livers that do not yet exhibit vitamin A storage also express α-smooth muscle actin (7Suskind D.L. Muench M.O. J. Hepatol. 2004; 40: 261-268Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), supporting a smooth muscle cell lineage. Synaptophysin, which controls exocytosis and the release of neurotransmitters in neurons and neuroendocrine cells, is also expressed in both rodent and human HSC (8Cassiman D. van Pelt J. De Vos R. Lommel VanF. Desmet V. Yap S.H. Roskams T. Am. J. Pathol. 1999; 155: 1831-1839Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Neurotrophins such as nerve growth factor, brain-derived neurotrophic factor (BDNF), neutrophin NT-3, and NT-4/5 are also expressed (9Cassiman D. Denef C. Desmet V.J. Roskams T. Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (190) Google Scholar), and so are their receptors, Trk-A, B, and C (9Cassiman D. Denef C. Desmet V.J. Roskams T. Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (190) Google Scholar, 10Trim N. Morgan S. Evans M. Issa R. Fine D. Afford S. Wilkins B. Iredale J. Am. J. Pathol. 2000; 156: 1235-1243Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), further supporting the neural and glial lineage. Peroxisome proliferator-activated receptor γ (PPARγ) has been proposed as a potential molecular target for inhibition of HSC transdifferentiation (11Miyahara 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 (425) Google Scholar, 12Marra F. Efsen E. Romanelli R.G. Caligiuri A. Pastacaldi S. Batignani G. Bonacchi A. Caporale R. Laffi G. Pinzani M. Gentilini P. Gastroenterology. 2000; 119: 466-478Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 13Galli 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). PPARγ level and activity are reduced in activated HSC, and the treatment of HSC with synthetic ligands for PPARγ such as thiazolidinediones effectively suppresses fibrogenic activity of HSC in vitro (11Miyahara 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 (425) Google Scholar, 12Marra F. Efsen E. Romanelli R.G. Caligiuri A. Pastacaldi S. Batignani G. Bonacchi A. Caporale R. Laffi G. Pinzani M. Gentilini P. Gastroenterology. 2000; 119: 466-478Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 13Galli 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) and in vivo in experimental animals (13Galli 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). However, these ligands are known to have PPARγ-independent effects (14Chawla A. Barak Y. Nagy L. Liao D. Tontonoz P. Evans R.M. Nat. Med. 2001; 7: 48-52Crossref PubMed Scopus (953) Google Scholar), and it was yet to be tested whether PPARγ per se had a direct effect to suppress activation of HSC. To address this question, our laboratory has recently expressed PPARγ1 by an adenoviral vector in culture-activated HSC. This manipulation reversed their phenotype to that of quiescent HSC with reduced expression of activation markers such as TGFβ1 or α1(I) procollagen and restored the ability to accumulate retinyl esters (15Hazra 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 (264) Google Scholar). More importantly, the fact that PPARγ is required for the maintenance of differentiated HSC highlights an analogy between differentiation of adipocytes and that of HSC (Fig. 1). PPARγ is considered as a master transcriptional regulator for adipogenesis, and along with other putative transcription factors such as C/EBPα, β, and δ and SREBP-1, it induces adipocyte-specific genes to promote adipocytic differentiation as demonstrated in preadipocytes such as 3T3L1 cells exposed to the adipocyte differentiation mixture (16MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (926) Google Scholar, 17Morrison R.F. Farmer S.R. J. Nutr. 2000; 130: 3116S-3121SCrossref PubMed Google Scholar). If these cells are treated with mediators such as cytokines (tumor necrosis factor α and leptin) or growth factors (platelet-derived growth factor, epidermal growth factor/TGFα, and TGFβ) that suppress the activity of PPARγ and adipogenic transcriptional regulation, adipocyte differentiation is inhibited and preadipocyte differentiation ensues (18Zhang B. Berger J. Hu E. Szalkowski D. White-Carrington S. Spiegelman B.M. Moller D.E. Mol. Endocrinol. 1996; 10: 1457-1466Crossref PubMed Scopus (306) Google Scholar, 19Camp H.S. Tafuri S.R. J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 20Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar) (Fig. 1). Interestingly, these are the same mediators that are also implicated in activation/transdifferentiation of HSC (21Pinzani M. Marra F. Semin. Liver Dis. 2001; 21: 397-416Crossref PubMed Scopus (402) Google Scholar) (Fig. 1). In fact, our recent work demonstrates that PPARγ activity is inhibited in HSC in a manner similar to that previously observed in adipocytes by the treatment with tumor necrosis factor α (22Sung C.K. She H. Xiong S. Tsukamoto H. Am. J. Physiol. 2004; 286: G722-G729Crossref PubMed Scopus (64) Google Scholar) that is known to cause early activation of HSC (21Pinzani M. Marra F. Semin. Liver Dis. 2001; 21: 397-416Crossref PubMed Scopus (402) Google Scholar). Indeed, HSC was once called “fat-storing cells” because of their lipid content (23Ito T. Nemoto M. Okajima. Folia Anat. Jpn. 1952; 24: 243-258Crossref PubMed Scopus (125) Google Scholar), and they do store neutral lipids besides retinyl esters (24Yamada M. Blaner W.S. Soprano D.R. Dixon J.L. Kjeldbye H.M. Goodman D.S. Hepatology. 1987; 7: 1224-1229Crossref PubMed Scopus (93) Google Scholar). Upon activation, HSC lose lipid content and become myofibroblastic with induced expression of type I and III collagen. Preadipocytes also have a fibroblastic phenotype with expression of these interstitial collagens. During adipocyte differentiation, this matrix expression shifts to that of basement membrane components including type IV collagen, laminin, entactin, and glycosaminoglycans (25Weiner F.R. Shah A. Smith P.J. Rubin C.S. Zern M.A. Biochemistry. 1989; 28: 4094-4099Crossref PubMed Scopus (103) Google Scholar). This is also a profile of extracellular matrices expressed by quiescent HSC in the perisinusoidal space of the liver. Based on these analogies, we proposed that the maintenance of the quiescent HSC phenotype requires transcriptional regulation similar if not identical to that known for adipocyte differentiation. To test this hypothesis, the present study examined the expression of putative adipogenic transcription factors in quiescent and culture-activated HSC, as well as PPARγ-transduced HSC. We also tested the effects of the adipocyte differentiation mixture (isobutylmethylxanthine, dexamethazone, and insulin (MDI)) on activated HSC. Finally, we transduced SREBP-1c via an adenoviral vector in activated HSC to restore its expression and to determine its effects on HSC phenotype. Our results indeed reveal that the expression of adipogenic transcription factors are high in quiescent rat HSC, and their levels rapidly decline during culture-induced transdifferentiation to myofibroblastic cells. Further, the MDI treatment or ectopic expression of PPARγ or SREBP-1c restores expression of the adipogenic transcription factors and reverses fully activated HSC to quiescent HSC. Materials—3-Isobutyl-1-methylxanthine, dexamethasone, insulin, and Oil Red O were purchased from Sigma. Rhodamine phalloidin (R-415) was obtained from Molecular Probes (Eugene, OR). Antibodies against C/EBPβ (C-19), C/EBPδ (M-17), SREBP-1 (2A4), PPARγ (H-100), PPARβ (H-74), Kruppel-like factor 6/Zf9 (R-173), and β-tubulin (H-235) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Type I collagen antibody was purchased from Rockland Inc. (Gilbertsville, PA). Primary Hepatic Stellate Cell Isolation and Culture—HSC were isolated from normal Wistar rats by in situ digestion of the liver and arabinogalactan gradient ultracentrifugation as described previously (26Tsukamoto H. Cheng S. Blaner W.S. Am. J. Physiol. 1996; 270: G581-G586PubMed Google Scholar). The purity of the cells was determined by phase contrast microscopy and ultraviolet-excited fluorescence microscopy, and the viability was determined by trypan blue exclusion (purity > 96%, viability > 94%). In vitro activation of HSC was achieved by culturing HSC on in Dulbecco's modified Eagle's medium with 1.0 g/liter glucose, 10% fetal bovine serum on a plastic dish for 3 or 7 days. They were treated with the adipogenic differentiation mixture (MDI, 0.5 mm isobutylmethylxanthine, 1 μm dexamethasone, and 1 μm insulin) and incubated for 24 or 72 h. Infection with Adenoviral Expression Vectors—Adenoviral vector for PPARγ1 was constructed as described before (15Hazra 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 (264) Google Scholar, 27Gurnell M. Wentworth J.M. Agostini M. Adams M. Collingwood T.N. Provenzano C. Browne P.O. Rajanayagam O. Burris T.P. Schwabe J.W. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 2000; 275: 5754-5759Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Adenoviral vector for SREBP-1c (28Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar) was provided by Dr. Bruce M. Spiegelman of Harvard University. These vectors facilitated efficient transduction of PPARγ1 or SREBP-1c in activated HSC for examination of their effects on the HSC phenotype and expression of other adipogenic transcription factors. These vectors also expressed GFP for assessment of the transduction, and the vector expressing GFP only was used as a control. Typically, HSC cultured on plastic for 7 days were infected by the vector with a MOI of 25 (SREBP-1c) or 50 (PPARγ) for 2–4 days depending on the cellular parameter to be examined. Generally, more than 75% of HSC were GFP positive at 24 h following the addition of viruses. Stress Fiber and Lipid Staining—HSC cultured in 24-well plates or slide chambers were washed with phosphate-buffered saline (PBS) and fixed in 3.7% paraformaldehyde. The cells were then washed and stained with rhodamine-labeled phalloidin (R-415, 1:50 v/v, 1% bovine serum albumin in PBS) in the dark. After washing with PBS, stress fiber fluorescence images were viewed by a confocal microscope (Nikon PCM 2000) equipped with Compix Imaging systems. For lipid staining, HSC were fixed with 10% formalin in PBS. Oil Red O (0.5% (w/v) in isopropanol) was diluted with 67% volume of water, filtered, and added to the fixed HSC. HSC were then washed, and the stained lipid droplets were visualized and photographed. RNA Extraction and Real Time Quantitative PCR—Total RNA was extracted using TRIzol reagent by Invitrogen. For real time PCR analysis for PPARγ, liver X receptor α (LXRα), α1(I) procollagen, TGFβ1, and glyceraldehyde-3-phosphate dehydrogenase (15Hazra 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 (264) Google Scholar), 5 ng of total RNA were reverse transcribed and amplified by 40 cycles using the TaqMan Gold reverse transcriptase-PCR kit (Applied Biosystems, Foster City, CA). Each Ct value was first normalized to the glyceraldehyde-3-phosphate dehydrogenase Ct value of a sample and subsequently to a control sample. For real time PCR for C/EBPα, Insig-1, ACC, FAS, and 36B4, the SyBer Green technique was used. The sequences of the primers and the probes used for real time PCR are listed in Table I. For validation of adipocyte-specific genes, reverse transcriptase-PCR analysis was performed for adipsin and resistin. The PCR primers used for these genes are as follows: 5′-ATGAGCAGTGGGTGCTGAG and 5′-AGAACGTTTTCAATCCACGG (adipsin); 5′-GAACCTTTCATTTCTCCTC and 5′-GTGACACATTGTATCCTCAC (resistin).Table IPrimers and probes used for real time PCRGeneAccession numberForward/reverse primersProbesPPARγNM_0131245′-CCTGAAGCTCCAAGAATACCAAA5′-CGATCAAAGTAGAGCCTGCGTCCCC5′-AGAGTTGGGTTTTTTCAGAATAATAAGGLXRαQ626855′-GGCCCTGCATGCCTATGT5′-CATCAACCACCCCCACGACCGA5′-CATTAGCATCCGTGGGAACAα1(I) collagenXM_2134405′-TCGATTCACCTACAGCACGC5′-TGTGGATGGCTGCACGAGTCACAC5′-GACTGTCTTGCCCCAAGTTCCTGFβ1NM_0215785′-AGAAGTCACCCGCGTGCTA5′-TGGTGGACCGCAACAACGCAAT5′-TGTGTGATGTCTTTGGTTTTGTCAGAPDHaGAPDH, glyceraldehyde-3-phosphate dehydrogenaseM177015′-TGCACCACCAACTGCTTAG5′-CAGAAGACTGTGGATGGCCCCAC5′-GGATGCAGGGATGATGTTCC/EBPαbSYBR primersNM_0125245′-AAGAAGTCGGTGGATAAGAACAG5′-GTTGCGCTGTTTGGCTTTATCTCACCbSYBR primersNM_0221935′-CCCAACAGAATAAAGCTACTCTGG5′-TCCTTTTGTGCAACTAGGAACGTFASbSYBR primersNM_0173325′-CCTGGACAGCATTCCAAACCT5′-AGCACATCTCGAAGGCTACACAInsig-1bSYBR primersQ087555′-TGCAGATCCAGCGGAATGT5′-CCAGGCGGAGGAGAAGATG36B4bSYBR primersNM_0224025′-TTCCCACTGGCTGAAAAGGT5′-CGCAGCCGCAAATGCa GAPDH, glyceraldehyde-3-phosphate dehydrogenaseb SYBR primers Open table in a new tab Preparation of Cellular Protein and Immunoblot Analysis—Total cell lysates were prepared with radioimmune precipitation assay buffer (PBS, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixture). The protein extracts were resolved on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were visualized by the ECL detection system (Pierce). Gene Expression Array Analysis—The GEArray pathway-specific expression array kit (SuperArray Inc., Bethesda, MD) was used for gene profiling of adipogenesis-related genes in HSC cultured on plastic for 1 or 7 days. Total RNA was extracted as above and reverse-transcribed for the preparation of 32P-labeled cDNA. The probes were hybridized to a cDNA expression array membrane consisting of 96 genes related to adipogenesis. The relative expression level of a given mRNA was assessed by normalizing to a housekeeping gene (cyclophilin A) and comparing with a control value. Lipid Synthesis—Day 3 HSC were treated with MDI or Me2SO (vehicle) for 5 days and then incubated with [14C]acetic acid (1 μCi/ml) for 3 h at 37 °C, 5% CO2. The cells were lysed with ethanol followed by the addition of chloroform and vortexing. After mixing gently with HCl (0.1 n) and centrifugation (500 × g for 30 min), the isolated organic phase was dried under N2 (45Lawler Jr., J.F. Yin M. Diehl A.M. Roberts E. Chatterjee S. J. Biol. Chem. 1998; 273: 5053-5059Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and the lipid extracts were separated by TLC with dichloroethane-acetic acid (100:1, v/v) on silica gel plates. Cellular 14C-labeled triacylglycerol spots were exposed in a phosphorus imager and quantitated with Kodak one-dimensional image analysis software (EDAS 290). Data Analysis—The numerical data were expressed as the means ± S.D. Student's t test was performed to assess the statistical significance between the two sets of data, and p values less than 0.05 were considered significant. Putative adipogenic transcription factors are expressed in quiescent HSC. Freshly isolated rat HSC retain their quiescent phenotype with distinct stellate morphology and intracellular storage of vitamin A when cultured on plastic for 1–2 days. They begin to be activated by day 3 in culture and become fully activated by day 7 as morphologically characterized by a large, spread out, polygonal cell shape with large nuclei and lost vitamin A storage (29Friedman S.L. Wei S. Blaner W.S. Am. J. Physiol. 1993; 264: G947-G952PubMed Google Scholar). We first analyzed the expression of transcription factors known to be involved in adipocyte differentiation in day 1 (quiescent), day 3 (activating), and day 7 (fully activated) HSC by immunoblot or real time PCR analysis. Among the transcription factors examined, C/EBPβ (liver-enriched transcriptional activator protein) and an active, nuclear form of SREBP-1c (nSREBP-1c) were abundant in quiescent HSC as easily detected by immunoblot analysis, and their expression declined in day 3 and day 7 cells (Fig. 2A). C/EBPβ is known to be involved in a clonal expansion of preadipocytes following adipogenic stimulation (16MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (926) Google Scholar, 17Morrison R.F. Farmer S.R. J. Nutr. 2000; 130: 3116S-3121SCrossref PubMed Google Scholar), whereas SREBP-1c causes transcriptional induction of genes involved in fatty acid and triglyceride synthesis (30Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3632) Google Scholar). C/EBPβ also has a splice variant (liver-enriched transcriptional inhibitor protein) that acts as a negative regulator (31Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (855) Google Scholar). Indeed, our immunoblotting detected liver-enriched transcriptional inhibitor protein in quiescent HSC but with less intensity, and its level also diminished in culture activation (data not shown). We detected mostly nSREBP-1 (molecular mass = 68 kDa) (Fig. 2A) but a minimal if not undetectable level of precursor form of SREBP-1 (molecular mass = 125 kDa) in quiescent HSC (data not shown). PPARγ, LXRα, and C/EBPα were difficult to be detected by immunoblotting because of either the lack of optimal antibodies or low levels of expression. Thus, their expression were analyzed by real time PCR, and the data were normalized to those on day 1 (Fig. 2B). The mRNA levels of all three genes were also high in day 1 HSC and declined coordinately in day 3 and 7 cells. In particular, the PPARγ level was severely depleted in day 7 HSC. Because the levels of all these adipogenic transcription factors decreased in activating or activated HSC, the expression of Kruppel-like factor 6 increased in day 3 and 7 cells (Fig. 2A), confirming the previously described differential expression of this zinc finger protein in activated HSC in vitro and in vivo (32Kim Y. Ratziu V. Choi S.G. Lalazar A. Theiss G. Dang Q. Kim S.J. Friedman S.L. J. Biol. Chem. 1998; 273: 33750-33758Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Further, the level of PPARβ was also increased in day 3 and 7 HSC (Fig. 2A) as previously reported (33Hellemans K. Rombouts K. Quartier E. Dittie A.S. Knorr A. Michalik L. Rogiers V. Schuit F. Wahli W. Geerts A. J. Lipid Res. 2003; 44: 280-295Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The reciprocal induction of PPARβ to the declined expression of adipogenic transcription factors is intriguing in light of its known stimulatory effects on fatty acid oxidation and energy dissipation (34Wang Y.X. Lee C.H. Tiep S. Yu R.T. Ham J. Kang H. Evans R.M. Cell. 2003; 113: 159-170Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar), and these results suggest the anti-adipogenic or lipolytic nature of HSC activation. In fact, quiescent HSC are loaded with lipids besides vitamin A (24Yamada M. Blaner W.S. Soprano D.R. Dixon J.L. Kjeldbye H.M. Goodman D.S. Hepatology. 1987; 7: 1224-1229Crossref PubMed Scopus (93) Google Scholar) as evident by Oil Red O staining, and activated day 7 cells are devoid of lipids (Fig. 2C). Further, a microarray analysis of RNA samples obtained from day 1 versus day 7 HSC consistently demonstrated higher levels of expression in day 1 HSC of adipocyte-specific genes that are under the positive control of PPARγ and SREBP-1 (Table II). Reverse transcriptase-PCR analysis also confirmed the expression of adipocyte-specific genes such as adipsin and resistin in day 1 HSC as compared with day 7 cells (Fig. 2D).Table IIAdipogenic gene expression during HSC culture activationGenesmRNA ratio (HSC day 1/day 7)aThe mRNA ratio of each gene was calculated by dividing the mRNA level of day 1 HSC by that of day 7 HSC following standardization by a housekeeping gene (cyclophlin A)Target genes for PPARγ Acyl CoA oxidase3.44 Adipsin8.48 C/EBPβ2.54 C/EBPδ1.87 Glycerol phosphate dehydrogenase 1 Cytoplasmic2.77 Mitochondrial2.37 Phosphoenolpyruvate carboxykinase (mitochondrial)2.75 Phosphoenolpyruvate carboxylase (Pck2-Rik, PEPCK-M) PPARγ1.96 Resistin4.24 Solute carrier family 27 (fatty acid transporter) member 43.39 Uncoupling protein 2 (mitochondrial)2.06Target genes for SREBP-1 Acetyl-CoA carboxylase Acac a2.48 Acac b3.03 Fatty acid synthase3.10 Fructose bisphosphatase 15.83 Glucose-6-phosphatase1.58 Glucokinase2.40 Phosphoenolpyruvate carboxykinase (mitochondrial)2.75 Phosphoenolpyruvate carboxylase (Pck2-Rik, PEPCK-M) Pyruvate kinase 34.47 Pyruvate kinase (liver and red blood cells)2.39a The mRNA ratio of each gene was calculated by dividing the mRNA level of day 1 HSC by that of day 7 HSC following standardization by a housekeeping gene (cyclophlin A) Open table in a new tab PPARγ Transduction Induces Other Adipogenic Factors— Our previous study demonstrated that ectopic expression of PPARγ by an adenoviral vector caused a phenotypic reversal from activated to quiescent HSC (15Hazra 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 (264) Google Scholar). We wanted to examine next whether this reversal is associated with restoration of other adipogenic transcription factors whose expression are all reduced in activation. As shown in Fig. 3, the protein levels of nSREBP-1c and C/EBPβ and the mRNA levels of LXRα and C/EBPα were increased in PPARγ-transduced HSC as compared with GFP-transduced cells (control). These results demonstrate that adipogenic transcriptional regulation is restored by PPARγ transduction and confirm the known cross-regulatory induction among them (35Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1394) Google Scholar, 36Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. 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