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Originally published In Press as doi:10.1074/jbc.M410078200 on November 9, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4959-4967, February 11, 2005
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Adipogenic Transcriptional Regulation of Hepatic Stellate Cells*

Hongyun She{ddagger}§, Shigang Xiong{ddagger}, Saswati Hazra{ddagger}, and Hidekazu Tsukamoto{ddagger}§

From the {ddagger}Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033 and the §Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073

Received for publication, September 1, 2004 , and in revised form, October 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {gamma} (PPAR{gamma}) 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) {alpha}, C/EBP{beta}, C/EBP{delta}, PPAR{gamma}, liver X receptor {alpha}, sterol regulatory element-binding protein 1c and of adipocyte-specific genes in the quiescent cells. Conversely, activated HSC have increased expression of PPAR{beta}, 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 PPAR{gamma} or SREBP-1c in these cells, induces a panel of adipogenic transcription factors, reduces PPAR{beta}, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transdifferentiation of vitamin A-storing hepatic stellate cells (HSC)1 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 (1). 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 (2). HSC are positive for a mesenchymal marker such as vimentin. Rodent HSC express desmin (3) and glial fibrillary acidic protein (4), suggesting smooth muscle cell and glial cell lineage, respectively. Upon activation, both rodent and human HSC lose vitamin A and begin to express {alpha}-smooth muscle actin (5, 6). Interestingly, undifferentiated HSC in fetal livers that do not yet exhibit vitamin A storage also express {alpha}-smooth muscle actin (7), 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 (8). Neurotrophins such as nerve growth factor, brain-derived neurotrophic factor (BDNF), neutrophin NT-3, and NT-4/5 are also expressed (9), and so are their receptors, Trk-A, B, and C (9, 10), further supporting the neural and glial lineage.

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) has been proposed as a potential molecular target for inhibition of HSC transdifferentiation (11–13). PPAR{gamma} level and activity are reduced in activated HSC, and the treatment of HSC with synthetic ligands for PPAR{gamma} such as thiazolidinediones effectively suppresses fibrogenic activity of HSC in vitro (11–13) and in vivo in experimental animals (13). However, these ligands are known to have PPAR{gamma}-independent effects (14), and it was yet to be tested whether PPAR{gamma} per se had a direct effect to suppress activation of HSC. To address this question, our laboratory has recently expressed PPAR{gamma}1 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 TGF{beta}1 or {alpha}1(I) procollagen and restored the ability to accumulate retinyl esters (15). More importantly, the fact that PPAR{gamma} is required for the maintenance of differentiated HSC highlights an analogy between differentiation of adipocytes and that of HSC (Fig. 1). PPAR{gamma} is considered as a master transcriptional regulator for adipogenesis, and along with other putative transcription factors such as C/EBP{alpha}, {beta}, and {delta} 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 (16, 17). If these cells are treated with mediators such as cytokines (tumor necrosis factor {alpha} and leptin) or growth factors (platelet-derived growth factor, epidermal growth factor/TGF{alpha}, and TGF{beta}) that suppress the activity of PPAR{gamma} and adipogenic transcriptional regulation, adipocyte differentiation is inhibited and preadipocyte differentiation ensues (18–20) (Fig. 1). Interestingly, these are the same mediators that are also implicated in activation/transdifferentiation of HSC (21) (Fig. 1). In fact, our recent work demonstrates that PPAR{gamma} activity is inhibited in HSC in a manner similar to that previously observed in adipocytes by the treatment with tumor necrosis factor {alpha} (22) that is known to cause early activation of HSC (21). Indeed, HSC was once called "fat-storing cells" because of their lipid content (23), and they do store neutral lipids besides retinyl esters (24). 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 (25). 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 PPAR{gamma}-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 PPAR{gamma} or SREBP-1c restores expression of the adipogenic transcription factors and reverses fully activated HSC to quiescent HSC.



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  		FIG. 1.
The schematic diagram depicting a proposed analogy between preadipocyte-adipocyte differentiation and transdifferentiation of HSC. TNF, tumor necrosis factor.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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/EBP{beta} (C-19), C/EBP{delta} (M-17), SREBP-1 (2A4), PPAR{gamma} (H-100), PPAR{beta} (H-74), Kruppel-like factor 6/Zf9 (R-173), and {beta}-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 (26). 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 PPAR{gamma}1 was constructed as described before (15, 27). Adenoviral vector for SREBP-1c (28) was provided by Dr. Bruce M. Spiegelman of Harvard University. These vectors facilitated efficient transduction of PPAR{gamma}1 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 (PPAR{gamma}) 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 PPAR{gamma}, liver X receptor {alpha} (LXR{alpha}), {alpha}1(I) procollagen, TGF{beta}1, and glyceraldehyde-3-phosphate dehydrogenase (15), 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/EBP{alpha}, 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).


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  		TABLE I
Primers and probes used for real time PCR

 
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 x g for 30 min), the isolated organic phase was dried under N2 (45), 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (29). 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/EBP{beta} (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/EBP{beta} is known to be involved in a clonal expansion of preadipocytes following adipogenic stimulation (16, 17), whereas SREBP-1c causes transcriptional induction of genes involved in fatty acid and triglyceride synthesis (30). C/EBP{beta} also has a splice variant (liver-enriched transcriptional inhibitor protein) that acts as a negative regulator (31). 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). PPAR{gamma}, LXR{alpha}, and C/EBP{alpha} 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 PPAR{gamma} 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 (32). Further, the level of PPAR{beta} was also increased in day 3 and 7 HSC (Fig. 2A) as previously reported (33). The reciprocal induction of PPAR{beta} to the declined expression of adipogenic transcription factors is intriguing in light of its known stimulatory effects on fatty acid oxidation and energy dissipation (34), and these results suggest the anti-adipogenic or lipolytic nature of HSC activation. In fact, quiescent HSC are loaded with lipids besides vitamin A (24) 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 PPAR{gamma} 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).



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  		FIG. 2.
Adipocytic characteristics of quiescent HSC. A and B, depletion of adipogenic transcription factors in HSC activation. Proteins extracted from quiescent (day 1), activating (day 3), and activated (day 7) HSC were analyzed for the expression of C/EBP{beta} and nuclear form of SREBP-1c (nSREBP-1c) by immunoblot analysis along with the known HSC activation markers such as Kruppel-like factor 6 (KLF6) and PPAR{beta}. Real time PCR was performed for mRNA levels of PPAR{gamma}, LXR{alpha} and C/EBP{alpha}. *, p < 0.05. Note that the levels of all examined adipogenic transcription factors are high in quiescent day 1 HSC and reduced in activated HSC. C, activated HSC are devoid of lipid contents. Day 1 or day 7cultured HSC were fixed with 3.7% formaldehyde and stained with Oil Red O. The stained wells (lower panel) or HSC under a microscopic view (upper panels) were photographed. Note the more intense staining in day 1 HSC. D, adipocyte-specific genes are expressed in quiescent but not activated HSC. Total RNA extracted from day 1 or day 7 cultured HSC was analyzed for mRNA levels for adipsin and resistin by reverse transcriptase-PCR. They are expressed only in day 1 HSC.

 


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  		TABLE II
Adipogenic gene expression during HSC culture activation

 
PPAR{gamma} Transduction Induces Other Adipogenic Factors— Our previous study demonstrated that ectopic expression of PPAR{gamma} by an adenoviral vector caused a phenotypic reversal from activated to quiescent HSC (15). 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/EBP{beta} and the mRNA levels of LXR{alpha} and C/EBP{alpha} were increased in PPAR{gamma}-transduced HSC as compared with GFP-transduced cells (control). These results demonstrate that adipogenic transcriptional regulation is restored by PPAR{gamma} transduction and confirm the known cross-regulatory induction among them (35–38).



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  		FIG. 3.
PPAR{gamma} transduction induces other adipogenic transcription factors. Culture-activated HSC (day 7) were infected with the adenovirus expressing PPAR{gamma} or GFP (50 MOI) for 5 days. Cellular proteins or RNA were extracted, and adipogenic transcription factor expression was analyzed by immunoblotting (PPAR{gamma}, C/EBP{beta}, and nuclear form of SREBP-1) or real time PCR (C/EBP{alpha} and LXR{alpha}). *, p < 0.05. Note that the levels of all the adipogenic transcriptional factors are higher in PPAR{gamma}-transduced HSC (Ad-PPAR{gamma}).

 
MDI Treatment Restores Both Adipogenic Transcription Factors and Quiescent HSC Phenotype—Preadipocytes can be treated with an adipocyte differentiation mixture (MDI) to drive them to differentiate into adipocytes through up-regulation of adipogenic transcription factors. Therefore, we asked next whether the MDI treatment could achieve the same effects on activated HSC and cause a phenotypic switch to quiescent HSC. The MDI treatment for 3 days resulted in up-regulation of C/EBP{beta} and {delta} and nSREBP-1 protein expression and PPAR{gamma} and LXR{alpha} mRNA expression regardless of whether the treatment was initiated on day 3 or day 7 HSC (Fig. 4A; real time PCR data are only shown for day 3 HSC). Conversely, the PPAR{beta} protein level was reduced by the treatment (Fig. 4A). The treatment caused a dramatic change in their morphology to that mimicking quiescent HSC with markedly reduced stress fibers (Fig. 4B). The prolonged treatment with MDI (7 days) also resulted in increased lipid content in HSC as shown by Oil Red O staining (Fig. 4C, left panels). Further, triglyceride synthesis as assessed by incorporation of [14C]acetate was increased by the MDI treatment in HSC (Fig. 4C, right panel), demonstrating enhanced de novo synthesis of lipids in MDI-treated HSC. Finally, the MDI treatment reduced DNA synthesis by 50% and mRNA levels of {alpha}1(I)procollagen and TGF{beta}1 by 95 and 80%, respectively (Fig. 4D), demonstrating that these bona fide markers for myofibroblastic transdifferentiation are clearly suppressed by MDI.



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  		FIG. 4.
Adipocyte differentiation mixture restores both adipogenic transcription factors and quiescent HSC phenotype. A, activating (day 3) or fully activated (day 7) HSC were treated with adipocyte differentiation mixture (MDI) or dimethyl sulfoxide (DMSO, vehicle) for 72 h, and the expression of adipogenic transcription factors were analyzed by immunoblotting (C/EBP{beta}, C/EBP{delta}, and nSREBP-1) and real time PCR (PPAR{gamma} and LXR{alpha}). Note the increased levels of all examined adipogenic transcription factors in MDI-treated HSC. *, p < 0.05. B, activated HSC (day 7) treated with MDI or Me2SO were stained for stress fibers with rhodamine phalloidin. Note the MDI treatment causes a morphological reversal of activated HSC to quiescent cells (upper panels) with reduced stress fibers (F-actin). C, activated HSC (day 7) and 3T3L1 pre-adipocytes (a positive control) were treated with MDI or Me2SO followed by staining with Oil Red O. Note increased staining in the MDI-treated HSC (left panels). Triglyceride synthesis as assessed by incorporation of [14C]acetic acid was enhanced in the MDI-treated HSC (right panel). D, DNA synthesis was evaluated by [3H]thymidine incorporation in HSC treated with MDI or Me2SO. The mRNA expression of two other HSC activation markers, {alpha}(1)I procollagen and TGF{beta}, were also assessed by real time PCR. All three of these activation parameters are inhibited by the MDI treatment.

 
Insig-1 Expression Inversely Correlates with nSREBP—We are intrigued by the abundant level of an active form of SREBP-1 (nSREBP-1) in quiescent HSC and its reduction in activated HSC. SREBP-1 as an inactive precursor, is bound to the endoplasmic reticulum membrane. SREBP-1 is escorted to the Golgi apparatus by SREBP cleavage-activating protein (SCAP), where two-step proteolysis of SREBP releases the NH3-terminal basic helix-loop-helix Zip domain for its nuclear translocation (39). Insulin-induced gene 1 (Insig-1) binds to SCAP and prevents SREBP activation (39). Therefore, we wondered whether the Insig-1 level might be inversely related to the nSREBP-1 level. We performed real time PCR for Insig-1 and indeed found that Insig-1 mRNA level is 2.7 times higher in activated day 7 HSC as compared with quiescent day 1 cells, whereas nSREBP-1 is abundant in day 1 but not day 7 cells (Fig. 5A). Further, the MDI treatment decreased Insig-1 level by 60% while raising the nSREBP-1 level in HSC (Fig. 5B). Thus, these results suggest that increased expression of Insig-1 might have reduced the nSREBP-1 abundance in activated HSC.



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  		FIG. 5.
Insig-1 expression inversely correlates with nSREBP-1. A, total RNA extracted from quiescent (day 1) or activated (day 7) HSC was analyzed for Insig-1 mRNA by real time PCR and compared with nSREBP-1 expression as detected by immunoblotting. Insig-1 is induced as nSREBP-1 is depleted in activated HSC. *, p < 0.05. B, MDI treatment of HSC suppressed Insig-1 while inducing nSREBP-1. *, p < 0.05. DMSO, dimethyl sulfoxide.

 
Expression of SREBP-1 Induces Other Adipogenic Factors and HSC Quiescence—Next, we directly tested the effects of SREBP-1 expression on HSC by using an adenoviral expression vector. Infection with the vector induced SREBP-1 as confirmed by immunoblot analysis (Fig. 6A). This also resulted in a 25.6-fold induction in the mRNA level for PPAR{gamma}, a 2.9-fold increase in C/EBP{alpha}, and a 2.4-fold increase in LXR{alpha} (Fig. 6B). The mRNA levels of ACC and FAS, target genes of SREBP-1, were expectedly increased by 2.9- and 10.3-fold, respectively (Fig. 6B). Effects on HSC morphology were more drastic and immediate than those seen after PPAR{gamma} expression despite the less MOI used (25 MOI versus 50 MOI) and equally potent to that seen after the MDI treatment. Shrinkage of the cells and reappearance of dendritic processes occurred, and stress fibers were clearly reduced within 48 h after SREBP-1c transduction (Fig. 6C, top and middle panels). Oil Red O staining increased after 2–3 days in SREBP-1 transduced HSC (Fig. 6C, bottom panel). Further, type I collagen and proliferating cell nuclear antigen expression examined by immunoblot analysis (Fig. 6A) were decreased in these cells. These results suggest that activated HSC are morphologically and biochemically reversed to the quiescent cells by the ectopic expression of SREBP-1c.



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  		FIG. 6.
Expression of SREBP-1c induces other adipogenic factors and HSC quiescence. A, expression of nSREBP-1 is increased in activated HSC infected with an adenovirus expressing SREBP-1c (Ad-SREBP-1c) as compared with those infected with the control vector (Ad-GFP). In contrast, proliferating cell nuclear antigen and {alpha}(1)I procollagen, the known activation markers of HSC, are reduced in Ad-SREBP-1c infected cells. B, activated HSC (day 7) were infected with an adenovirus expressing SREBP-1c (Ad-SREBP-1c) or GFP (Ad-GFP) (50 MOI) for 3 days. Cellular proteins and RNA were prepared, and nSREBP-1 expression was assessed by immunoblot analysis. Expressions of other adipogenic transcription factors (PPAR{gamma}, C/EBP{alpha}, and LXR{alpha}) and lipogenic enzymes (ACC and FAS) were determined by real time PCR. Note ectopic expression of SREBP-1c results in enhanced expression of other adipogenic factors and SREBP-1c target genes (ACC and FAS) in HSC. *, p < 0.05. C, activated HSC (day 7) infected with Ad-SREBP-1c or Ad-GFP were fixed with 3.7% formaldehyde followed by staining with either rhodamine phalloidin for F-actin or Oil Red O for lipids. Note that SREBP-1 transduction reduces stress fiber formation and increases intracellular lipids.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates higher expression of a panel of putative adipogenic transcription factors in quiescent HSC and their coordinated depletion in culture-activated HSC. Quiescent HSC also express adipocyte specific genes, many of which are under the control of adipogenic transcription factors such as PPAR{gamma} and SREBP-1c. These results are in line with our central hypothesis that adipogenic transcriptional regulation is required for the maintenance of the quiescent HSC phenotype. To further test the hypothesis, we utilized three different gain-of-function approaches for adipogenic transcriptional regulation. They are the treatment with the adipocyte differentiation mixture MDI and ectopic expression of PPAR{gamma} or SREBP-1c, all of which are known to induce adipocyte differentiation (16, 17, 27, 28). These manipulations on activated HSC similarly result in coordinated induction of adipogenic transcription factors and a cellular phenotypic reversal to quiescent HSC. Even when PPAR{gamma} or SREBP-1c is ectopically expressed alone, it induces the expression of other key adipogenic factors. These findings confirm their known cross-activation in adipocyte differentiation (35–38) in the context of HSC and indicate its potential importance in HSC quiescence.

The findings from the present study also raise an intriguing notion that the regulation demonstrated for HSC transdifferentiation may be similar to that known for differentiation or transdifferentiation among mesenchymal cells. For instance, PPAR{gamma} insufficiency favors differentiation of mesenchymal stem cells into osteoblasts over adipocytes (40). Even after commitment, 3T3-F442A preadipocytes transdifferentiate into osteoblasts when treated with bone morphogenic protein and retinoic acid (41). They also differentiate into smooth muscle-like cells if the homeobox gene HOXB7 (42) or the smooth muscle cell-specific gene, aortic carboxypeptidase-like protein (43), is expressed. Thus, the plasticity seen in these committed or differentiated mesenchymal cells may also underlie HSC transdifferentiation.

The level of nSREBP-1c in particular is abundant in quiescent HSC, and the restoration of this factor in activated HSC induces a drastic reversal of the cell phenotype to quiescent HSC. The level of nSREBP-1 correlates with the expression of genes involved in fatty acid synthesis (ACC and FAS) as demonstrated in quiescent HSC (Table II) and SREBP-1c-transduced cells (Fig. 6B) and with de novo synthesis of triglycerides as shown in the MDI-treated HSC (Fig. 4C). Indeed, quiescent HSC contains ample triglycerides (24) that presumably serve as a source of fatty acids for esterification of retinol and storage of vitamin A. Activation of the precursor SREBP-1c by proteolytic processing takes place in response to sensing low sterol level mediated by SREBP. SCAP escorts SREBP to Golgi for its two-step proteolysis and consequent formation of the mature form of SREBP-1 (nSREBP-1) (30). SREBP-1 is also activated in a sterol-independent manner by caspase 3 in response to a proapoptotic stimulus (44) or by ceramide-mediated signaling after tumor necrosis factor {alpha} treatment (45). Insig was identified as a negative regulator of SREBP-1 activation. This gene encodes two isoforms designated Insig-1 (39) and Insig-2 (46), both of which bind to SCAP and, by doing so, retain SREBP in endoplasmic reticulum and prevent its activation. Insig-1 is considered as a key mediator to suppress lipogenesis in adipocyte and to inhibit differentiation of preadipocytes (47). Real time PCR analysis for Insig-1 performed in the present study, demonstrates increased expression of Insig-1 in culture-activated HSC with decreased nSREBP-1c levels. MDI treatment reduces Insig-1 expression while increasing nSREBP-1c (Fig. 5). This inverse relationship between the level of the mature nSREBP-1c and Insig-1 mRNA suggests regulation of Insig-1 expression as a potential determinant for the differential nSAREBP-1 levels in quiescent and activated HSC. Regulatory mechanisms for Insig-1 expression and parallel analysis of SCAP expression will need to be addressed to explore this possibility in future studies on HSC transdifferentiation.

Because SREBP-1 transduction also causes induction of PPAR{gamma} (Fig. 6B) and ectopic PPAR{gamma} expression attains HSC quiescence (15), it is possible that the effects rendered by SREBP-1c are mediated at least in part by PPAR{gamma}. This hypothesis is suggested by the known cooperative actions of these two adipogenic transcription factors in achieving optimal adipocyte differentiation. Furthermore, the contributions of other adipogenic transcription factors cannot be ruled out. These include LXR{alpha} and C/EBP{alpha}. Although their pivotal roles in adipocyte differentiation are evident, their biological significance in HSC is totally unknown at the present time. Detailed elucidation of respective roles of these transcription factors will not only improve the understanding of the molecular basis for HSC differentiation but also help to identify novel molecular targets for the treatment of cirrhosis.


   FOOTNOTES
 
* This work is supported by National Institutes of Health Grants R37 AA006603, P50 AA11999, R24 AA12885 (Non-Parenchymal Liver Cell Core), and P30 DK48522 (Molecular Biology and Confocal Microscopy Cores) and by the Medical Research Service of Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Keck School of Medicine of the University of Southern California, 1333 San Pablo St., MMR-402, Los Angeles, CA 90033-9141. Tel.: 323-442-5107; Fax: 323-442-3126; E-mail: htsukamo{at}usc.edu.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Bruce Spiegelman of Harvard University for the gift of the SREBP-1c adenoviral vector.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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