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Am J Physiol Gastrointest Liver Physiol 290: G883-G893, 2006. First published November 23, 2005; doi:10.1152/ajpgi.00450.2005
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HORMONES AND SIGNALING

Curcumin suppresses the expression of extracellular matrix genes in activated hepatic stellate cells by inhibiting gene expression of connective tissue growth factor

Shizhong Zheng1,3 and Anping Chen1,2

1Department of Pathology and 2Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, Louisiana; and 3Department of Pharmacology, Nanjing Medical University, Nanjing, China

Submitted 25 September 2005 ; accepted in final form 20 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Upon liver injury, quiescent hepatic stellate cells (HSCs), the most relevant cell type for hepatic fibrogenesis, become active and overproduce extracellular matrix (ECM). Connective tissue growth factor (CTGF) promotes ECM production. Overexpression of CTGF during hepatic fibrogenesis is induced by transforming growth factor (TGF)-beta. We recently demonstrated that curcumin reduced cell growth and inhibited ECM gene expression in activated HSCs. Curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-{gamma} and stimulated its activity in activated HSCs, which was required for curcumin to suppress ECM gene expression, including {alpha}I(I)-collagen. The underlying mechanisms remain largely unknown. The aim of this study was to elucidate the mechanisms by which curcumin suppresses {alpha}I(I)-collagen gene expression in activated HSCs. We hypothesize that inhibition of {alpha}I(I)-collagen gene expression in HSCs by curcumin is mediated by suppressing CTGF gene expression through attenuating oxidative stress and interrupting TGF-beta signaling. The present report demonstrated that curcumin significantly reduced the abundance of CTGF in passaged HSCs and suppressed its gene expression. Exogenous CTGF dose dependently abrogated the inhibitory effect of curcumin. Activation of PPAR-{gamma} by curcumin resulted in the interruption of TGF-beta signaling by suppressing gene expression of TGF-beta receptors, leading to inhibition of CTGF gene expression. The phytochemical showed its potent antioxidant property by significantly increasing the level of total glutathione (GSH) and the ratio of GSH to GSSG in activated HSCs. De novo synthesis of cellular GSH was a prerequisite for curcumin to interrupt TGF-beta signaling and inhibited gene expression of CTGF and {alpha}I(I)-collagen in activated HSCs. Taken together, our results demonstrate that inhibition of {alpha}I(I)-collagen gene expression by curcumin in activated HSCs results from suppression of CTGF gene expression through increasing cellular GSH contents and interruption of TGF-beta signaling. These results provide novel insights into the mechanisms underlying inhibition of HSC activation by curcumin.

collagen; fibrogenesis; phytochemicals; signal transduction; oxidative stress

LIVER FIBROSIS is characterized by abnormal accumulation of extracellular matrix (ECM), namely, fibrillar collagens. Hepatic stellate cells (HSCs), a liver-specific type of pericytes (previously termed fat- or vitamin A-storing cells, or Ito cells), are the most relevant cell type for the development of liver fibrosis. During hepatic fibrogenesis, quiescent HSCs become active, a process that is characterized by increased cell proliferation and excessive production and deposition of connective tissue elements and ECM, including {alpha}I(I)-collagen. The activation of HSCs is coupled to a dramatic reduction in the level of peroxisome proliferator-activated receptor (PPAR)-{gamma} (23, 34, 37). Prior studies have implicated a potential therapeutic value of PPAR-{gamma} activation in the treatment of liver fibrosis (23, 34, 37). Curcumin, the active ingredient of the rhizome of the plant turmeric (Curcuma longa Linn), possesses antiproliferative, antioxidant, anti-inflammatory, antiangiogenic, and antitumor effects (16, 38, 39, 46). We (55) recently reported that curcumin significantly inhibited HSC activation, including reducing cell proliferation, inducing apoptosis, and suppressing ECM gene expression, including {alpha}1(I)-collagen and fibronectin as well as {alpha}-smooth muscle actin ({alpha}-SMA), the marker of activated HSCs. In addition, curcumin induced the expression of PPAR-{gamma} and stimulated its trans-activation activity in activated HSCs in vitro (55), which was a necessity for curcumin to reduce HSC growth and suppress ECM gene expression (55, 56). The precise mechanisms of these actions remain largely to be defined.

Connective tissue growth factor (CTGF/CCN2) is a heparin-binding 38-kDa cysteine-rich peptide that promotes a broad range of cellular responses, including proliferation, chemotaxis, adhesion, migration, and ECM production (8). Accumulating evidence has suggested that upregulation of CTGF might be a central pathway during HSC activation and hepatic fibrogenesis by mediating many of the profibrotic effects of TGF-beta (1, 41, 42, 53). CTGF expression in HSCs is significantly enhanced during the process of activation in vitro and in vivo (41, 53). HSCs are the major cellular source of CTGF in the liver during liver fibrogenesis (42). In response to exogenous CTGF, HSCs demonstrate increased migration, proliferation, adhesion, and expression of type I collagen (6, 41). Blockade of CTGF synthesis by antisense oligonucleotides of CTGF resulted in downexpression of type I collagen mRNA in an animal model with experimental liver fibrosis (52). Overproduction of CTGF in a variety of fibrotic disorders, including hepatic fibrosis, is presumably secondary to the activation and production of TGF-beta. A brief exposure of fibroblasts to TGF-beta1 is sufficient to induce a prolonged high level of CTGF expression (26). The regulation of CTGF gene expression appears to be controlled primarily at the level of transcription, depending on sequences present in the 5'-flanking region of the CTGF gene promoter (26). CTGF is transcriptionally regulated by TGF-beta and mediates some of the ECM-inducing properties previously attributed to TGF-beta (7, 22, 32). The strong correlation between CTGF and TGF-beta, as well as the degree of fibrosis, is observed in cirrhotic liver tissue samples (42).

Oxidative stress is a major player in inducing HSC activation and hepatic fibrogenesis, regardless of etiology (19, 21, 24, 33, 51). Oxidative stress is a deleterious imbalance between the production and removal of reactive radicals, including reactive oxygen species (ROS). ROS, are generated from aerobic cells by various mechanisms, including inflammation, aerobic metabolism, and exposure to ionizing radiation (20, 28, 57). Responses of mammalian cells to oxidative stress occur through several antioxidant systems, including enzymes and nonenzymatic molecules. Among them, glutathione (GSH) is the most abundant thiol antioxidant (54). It reacts directly with ROS or functions as a cofactor of antioxidant enzymes (54). GSH is converted to its oxidized form, GSSG, leading to conversion of H2O2 and lipid peroxides to water and lipid alcohols, respectively. The GSH-to-GSSG ratio is regarded as a sensitive indicator of oxidant stress in cells (27). Although the antioxidant vitamin E inhibits HSC activation (33) and hepatic fibrogenesis (43), currently well-known antioxidants in protection of the liver from hepatic fibrosis are generally unimpressive (28). The antioxidant capability of curcumin is far greater than that of vitamin E and/or C (48), due to both the innate molecular structure and its ability to affect enzyme systems involved in the defense against oxidative stress (3, 18). These unique features might allow curcumin to succeed where other antioxidants have failed in inhibition of hepatic fibrogenesis.

The aim of this study was to elucidate the mechanisms by which curcumin suppresses ECM gene expression in activated HSCs. We hypothesized that inhibition of ECM gene expression in HSCs by curcumin might be mediated by suppressing CTGF gene expression through attenuating oxidative stress and interrupting TGF-beta signaling. Results from this report support our hypothesis and provide novel insights into the mechanisms underlying the curcumin inhibition of HSC activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animal protocols were approved by the Institutional Animal Care and Use Committee of the Louisiana State University Health Sciences center (no. P-05-047).

Isolation and culture of HSCs. HSCs were isolated from male Sprague-Dawley rats (~200–250 g) as previously described (11). Cells were cultured in DMEM supplemented with 10% FBS. HSCs aged at passages 4–8 were used for experiments. All of the experiments were performed in media with 10% FBS unless specific indication. Curcumin (purity >94%) was purchased from Sigma (St. Louis, MO). PD-68235 is a specific PPAR-{gamma} antagonist (10) and was kindly provided by Pfizer (Ann Arbor, MI). Active TGF-beta1 and CTGF were purchased from Cell Sciences (Canton, MA).

Western blot analyses. Whole cell extracts were prepared from preconfluent passaged HSCs. SDS-PAGE with 10% resolving gel was used to separate proteins (25 µg/well). Separated proteins were detected by using primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized by utilizing chemiluminescence reagent (Kirkegarrd & Perry Laboratories, Gaithersburg, MD). After normalization with beta-actin, compared with no treatment, the effect of curcumin on reducing the level of CTGF was determined and expressed as a mean value (n = 3) using Quantity One 4.4.1 (Bio-Rad).

Plasmids and transient transfection assays. CTGF promoter luciferase (Luc) reporter plasmid pCTGF-Luc, a gift from Dr. Yuqing E. Chen (Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, GA), contains a fragment of the CTGF gene promoter (~2,000 bp nucleotides) subcloned into the Luc reporter plasmid pGL3 (22). PPAR-{gamma} expression plasmid pPPAR{gamma}cDNA, containing PPAR-{gamma} cDNA, was a gift from Dr. Reed Graves (Department of Medicine, University of Chicago, Chicago, IL). The plasmid p3TP-Lux is a TGF-beta-inducible Luc reporter containing the plasminogen activator inhibitor-1 gene promoter and was kindly provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center). The Luc reporter plasmid Tbeta-RI-Luc (pES1.0) was a gift from Dr. Michael Centrella (Yale University) and contains 965 bp of the gene promoter of TGF-beta receptor type 1 (Tbeta-RI) (30). Luc reporter plasmid pTbeta-RII-Luc was generously provided by Dr. Seong-Jin Kim [Laboratory of Chemoprevention, National Cancer Institute (NCI), Bethesda, MD]. pTbeta-RII contains 1,670 bp of the gene promoter of TGF-beta receptor type 2 (Tbeta-RII) (4). cDNA expression plasmid pdn-Tbeta-RIIcDNA was a gift from Dr. Robert J. Lechleider (NCI) and contains cDNA encoding the dominant-negative form of Tbeta-RII (17). Semiconfluent HSCs in six-well cell culture plates were transiently transfected using LipofectAMINE reagent (Life Technologies, Grand Island, NY). Each sample (3 µg of DNA/well) was triplicate in every experiment. Luc assays were performed as previously described (12). Transfection efficiency was determined by cotransfection of a beta-galactosidase reporter, pSV-beta-gal (0.5 µg/well; Promega). beta-Galactosidase activity was measured by a chemiluminescence assay kit (Tropix, Bedford, MA) according to the manufacturer's instructions. Results were combined from three independent experiments.

RNA isolation and real-time PCR. Total RNA was isolated by TRI Reagent (Sigma) following the protocol provided by the manufacturer. Real-time PCR was carried out as previously described (14). mRNA fold changes of target genes relative to the endogenous GAPDH control were calculated as suggested by Schmittgen et al. (47). The CTGF primers used in real-time PCR were as follows: forward 5'-TGT GTG ATG AGC CCA AGG AC-3' and reverse 5'-AGT TGG CTC GCA TCA TAG TTG-3'. Other primers used were previously described (56).

GSH assays. The levels of GSH and GSSG were determined using enzyme immune assay kit GSH-400 (Cayman Chemical, Ann Arbor, MI) following the protocol provided by the manufacturer.

Statistical analysis. Differences between means were evaluated using an unpaired, two-sided Student's t-test (with P < 0.05 considered significant). Where appropriate, comparisons of multiple treatment conditions with controls were made, using ANOVA with the Dunnett's test for post hoc analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Curcumin dose dependently suppresses gene expression of CTGF in activated HSCs. All of the following experiments were performed in media with 10% FBS unless specifically indicated. To evaluate the effect of curcumin on gene expression of CTGF in activated HSCs, passaged HSCs were treated with curcumin at the indicated concentrations for 24 h. Total RNA or whole cell protein extracts were prepared from these cells for real-time PCR or Western blot analyses, respectively. It was found that curcumin caused a dose-dependent reduction in the steady-state level of CTGF mRNA (Fig. 1A) and in the abundance of CTGF protein (Fig. 1B). Curcumin at 20 µM significantly reduced the levels of CTGF mRNA (Fig. 1A) and protein (Fig. 1B) by ~45% and ~60%, respectively. Similarly, curcumin caused a dose-dependent reduction in the concentration of secreted CTGF in the conditioned media (data not shown). To further evaluate the effect of curcumin on the regulation of CTGF gene expression, HSCs were transiently transfected with the CTGF promoter Luc reporter plasmid pCTGF-Luc, which contains a fragment of the CTGF gene promoter (~2,000 bp nucleotides) subcloned into the Luc reporter plasmid pGL3 (22). Cells were then treated with curcumin at the indicated concentrations. As shown in Fig. 1C by Luc assays, curcumin reduced Luc activity in a dose-dependent manner, suggesting that curcumin reduced the promoter activity of the CTGF gene in activated HSCs.


Figure 1
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  		Fig. 1. Curcumin (Cur) dose dependently suppresses gene expression of connective tissue growth factor (CTGF) in activated hepatic stellate cells (HSCs). Passaged HSCs grown in DMEM with 10% FBS were pretreated with or without PD-68235 (PD) at the indicated doses for 30 min before the addition of Cur at the indicated concentrations for an additional 24 h. Total RNA or whole cell extracts were prepared for analyses of gene expression of CTGF by real-time PCR (A; n = 3) or by Western blot analyses (B; n = 3), respectively. For real-time PCR, GAPDH was used as an invariant control for calculating mRNA fold changes. For Western blot analyses, beta-actin was used as an internal control for equal loading. Representative images shown are from 3 independent experiments. Numbers beneath the Western blots of CTGF are means (n = 3) of the amount of CTGF after normalization with beta-actin and comparison with the control. C: HSCs were transfected with the CTGF promoter luciferase (Luc) reporter plasmid pCTGF-Luc. Cells were then treated as described above. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). Values are expressed as means ± SD. *P < 0.05 vs. cells without Cur; {ddagger}P < 0.05 vs. cells with Cur at 20 µM.

 
We (55, 56) recently demonstrated that curcumin induced gene expression and activation of PPAR-{gamma} in passaged HSCs, which was a necessity for curcumin to reduce HSC growth and suppress ECM gene expression. To determine the role of PPAR-{gamma} activation in the inhibitory effect of curcumin on gene expression of CTGF, cells were pretreated with the PPAR-{gamma}-specific antagonist PD-68235 (10) for 30 min before the addition of curcumin. Inhibition of PPAR-{gamma} activation by PD-68235 resulted in partial abrogation of the inhibitory effect of curcumin on the promoter activity of the CTGF gene (Fig. 1C) and on the levels of CTGF mRNA (Fig. 1A) and protein (Fig. 1B) in passaged HSCs. Taken together, these results demonstrated that curcumin dose dependently suppressed gene expression of CTGF in activated HSCs in vitro, which required, at least in part, activation of PPAR-{gamma}.

Activation of PPAR-{gamma} results in inhibition of CTGF gene expression in activated HSCs. Although the level of PPAR-{gamma} is dramatically reduced in activated HSCs, it still responds to its agonists, leading to inhibition of HSC activation and suppression of collagen gene expression in vitro and in vivo (23, 34, 37). We (55) have shown that curcumin induces gene expression of PPAR-{gamma} and stimulates its trans-activation activity in activated HSCs in vitro. The results shown in Fig. 1 suggested that PPAR-{gamma} activation might be required for curcumin to inhibit CTGF gene expression in activated HSCs. Additional experiments were conducted to verify the role of PPAR-{gamma} activation in inhibition of CTGF gene expression. Passaged HSCs were treated with 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2), a natural PPAR-{gamma} ligand, at various concentrations for 24 h. As shown in Fig. 2, A and B, activation of endogenous PPAR-{gamma} by PGJ2 dose dependently suppressed gene expression of CTGF at both levels of transcription and translation. To further confirm the role of PPAR-{gamma} activation in inhibition of CTGF gene expression, passaged HSCs were cotransfected with the CTGF promoter Luc reporter plasmid pCTGF-Luc and the cDNA expression plasmid pPPAR{gamma}cDNA, which contains PPAR-{gamma} cDNA in a cytomegalovirus-driven expression vector. Prior experiments have shown that forced expression of PPAR-{gamma} cDNA results in an increase in the trans-activation activity of PPAR-{gamma} in HSCs (56). PPAR-{gamma} agonists are presumed to exist in the media with 10% FBS (29, 36, 56). As shown in Fig. 2C by Luc assays, forced expression of exogenous PPAR-{gamma} led to a dose-dependent reduction in Luc activity in these cells, indicating that the activation of PPAR-{gamma} inhibited the promoter activity of the CTGF gene. In summary, these results collectively demonstrated that activation of PPAR-{gamma}, regardless of the endogenous or exogenous receptor, resulted in mimicking the role of curcumin inhibition of CTGF gene expression in activated HSCs.


Figure 2
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  		Fig. 2. Activation of endogenous or exogenous peroxisome proliferator-actived receptor (PPAR)-{gamma} inhibits gene expression of CTGF and {alpha}I(I)-procollagen in activated HSCs in vitro. Passaged HSCs were treated with PGJ2 at the indicated concentrations or with Cur at 20 µM for 24 h. Total RNA or whole cell extracts were prepared for analyses of gene expression of CTGF and {alpha}I(I)-procollagen by real-time PCR (A; n = 3) or by Western blot analyses (B; n = 3), respectively. For real-time PCR, GAPDH was used as an invariant control for calculating mRNA fold changes. For Western blot analyses, beta-actin was used as an internal control for equal loading. C: passaged HSCs in 6-well cell culture plates were cotransfected with pCTGF-Luc and pPPAR{gamma}cDNA, which contains PPAR-{gamma} cDNA in a cytomegalovirus-driven expression vector. A total of 4.5 µg of plasmid DNA were used in each well for transfection, including 2 µg of pCTGF-Luc, 0.5 µg of pSV-beta-gal, pPPAR{gamma}cDNA at the indicated doses, and the empty vector pcDNA. The latter was used to ensure equal amounts of total DNA in transfection assays. The amount of DNA of pPPAR{gamma}cDNA plus pcDNA was equalized to 2 µg. Cells were treated with or without Cur at 20 µM for 24 h. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). Values are expressed as means ± SD. *P < 0.05 vs. cells cotransfected with no pPPAR{gamma}cDNA.

 
Exogenous CTGF dose dependently abrogates the inhibitory effect of curcumin on gene expression of {alpha}I(I)-collagen in activated HSCs. We hypothesized that inhibition of the expression of ECM genes, including {alpha}I(I)-collagen, by curcumin was at least partially mediated by suppression of CTGF gene expression in activated HSCs. To test the hypothesis, HSC were treated with curcumin (20 µM) in the presence or absence of exogenous CTGF at the indicated concentrations for 24 h. Total RNA and whole cell extracts were prepared from these cells. Real-time PCR assays (Fig. 3A) and Western blot analyses (Fig. 3B) demonstrated that curcumin significantly reduced, as expected, the abundance of {alpha}I(I)-procollagen at levels of translation and transcription. It was of interest to observe that coupled with the increase in the dose of exogenous CTGF, the inhibitory effect of curcumin was gradually diminished. These results collectively demonstrated that exogenous CTGF dose dependently eliminated the inhibitory effect of curcumin, indicating the role of CTGF in mediating the phytochemical inhibition of gene expression of {alpha}I(I)-collagen in activated HSCs.


Figure 3
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  		Fig. 3. Addition of exogenous CTGF dose dependently eliminates the inhibitory effect of Cur on gene expression of {alpha}I(I)-collagen in activated HSCs in vitro. HSCs were treated with Cur (20 µM) in the presence or absence of exogenous CTGF at the indicated concentrations for 24 h. Total RNA or whole cell extracts were prepared from these cells for analyses of {alpha}I(I)-collagen gene expression by real-time PCR assays (A; n = 3) or Western blot analyses (B; n = 3), respectively. mRNA fold changes were calculated by using GAPDH as an invariant control in real-time PCR. *P < 0.05 vs. cells with no treatment; **P < 0.05 vs. cells treated with Cur but without CTGF. beta-Actin was an internal control for equal loading in Western blot analyses. Representative images from 3 independent experiments are shown. The numbers beneath {alpha}I(I)-procollagen are means (n = 3) of the amount of {alpha}I(I)-procollagen after normalization with beta-actin and comparison with the control.

 
Curcumin interrupts TGF-beta signaling, resulting in inhibition of CTGF gene expression in passaged HSCs. Overproduction of CTGF in a variety of fibrotic disorders is presumably secondary to the activation and production of TGF-beta (25). Gene expression of CTGF is primarily regulated by TGF-beta in HSCs (41, 44). We, therefore, hypothesized that inhibition of CTGF gene expression by curcumin resulted from interruption of the TGF-beta signal transduction pathway in activated HSCs. To study the hypothesis, HSCs were transfected with the TGF-beta-inducible Luc reporter plasmid p3TP-Lux. Cells were treated with curcumin at the indicated concentrations for 24 h. The Luc assays shown in Fig. 4A demonstrated that the curcumin treatment of these cells caused a dose-dependent reduction in Luc activity, indicating that curcumin interrupted the TGF-beta signaling pathway. To verify the role of the blockade of TGF-betasignaling in inhibition of CTGF gene expression, passaged HSCs were cotransfected with the CTGF promoter reporter plasmid pCTGF-Luc and the plasmid pdn-Tbeta-RII at the indicated concentrations. pdn-Tbeta-RII contains the fragment of cDNA encoding the dominant-negative form of Tbeta-RII (17). Pilot experiments have demonstrated that transfection of HSCs with the plasmid pdn-Tbeta-RII interferes with the TGF-beta signal transduction pathway (data not shown). As shown in Fig. 4B by Luc assays, forced expression of dn-Tbeta-RII cDNA, like curcumin, resulted in a reduction in Luc activity in a dose-dependent manner, indicating that interruption of TGF-beta signaling by dominant-negative Tbeta-RII reduced the gene promoter activity of CTGF in passaged HSCs. Taken together, these experiments demonstrated that curcumin interrupted the signal transduction pathway of TGF-beta in activated HSCs, which led to inhibition of CTGF gene expression.


Figure 4
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  		Fig. 4. Cur interrupts transforming growth factor (TGF)-beta signaling, leading to inhibition of CTGF gene expression. A: passaged HSCs were transfected with p3TP-Lux. Cells were treated with Cur at the indicated concentrations for 24 h. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). *P < 0.05 vs. cells with no treatment. B: HSCs in 6-well cell culture plates were cotransfected with pCTGF-Luc and pdn-Tbeta-RII at the indicated concentrations. pdn-Tbeta-RII contains 1,670 bp of the gene promoter of TGF-beta receptor type 2 (Tbeta-RII). A total of 4.5 µg of plasmid DNA were used in each well for transfection, including 2 µg of pCTGF-Luc, 0.5 µg of pSV-beta-gal, pdn-Tbeta-RII at the indicated doses, and the empty vector pcDNA. The amount of pdn-Tbeta-RII plus pcDNA was equalized to 2 µg. Cells were treated with or without Cur at 20 µM for 24 h. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). Values are expressed as means ± SD. *P < 0.05 vs. cells cotransfected with no pdn-Tbeta-RII.

 
Interruption of the TGF-beta signal pathway by curcumin likely occurs at steps downstream from agonists, such as suppressing gene expression of TGF-beta receptors. Additional experiments were performed to elucidate the underlying mechanisms of curcumin in the interruption of the TGF-beta signaling pathway in activated HSCs. TGF-beta signaling is initiated by binding of active TGF-beta1 to Tbeta-RII, which leads to the phosphorylation and activation of Tbeta-RI (5, 35). The latter, in turn, phosphorylates Smad2/3 proteins, which subsequently form a complex with Smad4 and migrate into the nucleus to regulate the expression of target genes, including CTGF (41, 42). We (56) have previously demonstrated that curcumin significantly reduced the gene expression of Tbeta-RI and Tbeta-RII in passaged HSCs. Curcumin had no apparent impact on the production and activation of latent TGF-beta1 (56). We, therefore, hypothesized that interruption of the TGF-beta signaling pathway by curcumin likely occurred at steps downstream of agonists, such as suppressing gene expression of TGF-beta receptors. If this is true, the addition of exogenous TGF-beta1 after pretreatment of cells with curcumin for 24 h is expected to show little impact on activation of TGF-beta signaling and stimulation of gene expression of CTGF, because curcumin suppresses gene expression of TGF-beta receptors and reduces its bioavailability to the ligand TGF-beta1 (56). In great contrast, the simultaneous addition of exogenous TGF-beta1 and curcumin is expected to have an apparent effect on activation of TGF-beta signaling and stimulation of gene expression of CTGF. To study our hypothesis, HSCs were transfected with the TGF-beta-inducible Luc reporter plasmid p3TP-Lux. Cells were then divided into two groups. One group was pretreated with curcumin at 20 µM for 24 h before the addition of exogenous TGF-beta1 at various concentrations for an additional 24 h. The other group was simultaneously treated with curcumin at 20 µM plus exogenous TGF-beta1 at various concentrations for 24 h. As shown in Fig. 5A, pretreatment of cells with curcumin for 24 h, as expected, interrupted the TGF-beta signaling pathway. The subsequent addition of exogenous active TGF-beta1 showed no significant role in eliminating the curcumin-inhibitory effect until a very high dose (Fig. 5A). In great contrast, the simultaneous addition of exogenous active TGF-beta1 dose dependently abrogated the inhibitory effect of curcumin on TGF-beta signaling (Fig. 5B).


Figure 5
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  		Fig. 5. Interruption of the TGF-beta signal transduction pathway by Cur likely occurs at steps downstream of agonists, such as suppressing gene expression of TGF-beta receptors. Passaged HSCs were transfected with p3TP-Lux (A and B) or pCTGF-Luc (C and D). Cells in A and C were pretreated with Cur at 20 µM for 24 h before the addition of exogenous active TGF-beta1 at the indicated concentrations for an additional 24 h. The total treatment lasted for 48 h. Cells in B and D were simultaneously treated with Cur at 20 µM plus exogenous TGF-beta1 at the indicated concentrations for 24 h. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). *P < 0.05 vs. cells with no treatment (0 µM TGF-beta1 + 0 µM Cur); **P < 0.05 vs. cells with Cur at 20 µM without exogenous TGF-beta1 (0 µM TGF-beta1 + 20 µM Cur).

 
To further verify the effect of exogenous TGF-beta1 on CTGF gene regulation, HSCs were transfected with the CTGF promoter reporter plasmid pCTGF-Luc. Cells were similarly divided into two groups. As shown in Fig. 5C, pretreatment of cells with curcumin for 24 h, as expected, significantly reduced the promoter activity of CTGF in these cells. The subsequent addition of exogenous TGF-beta1 had no significant impact on the inhibitory effect. However, the simultaneous addition of exogenous TGF-beta1 with curcumin caused a dose-dependent and significant increase in Luc activity (Fig. 5D), suggesting that exogenous TGF-beta1 in this situation stimulated the promoter activity of the CTGF gene. Taken together, our results support the hypothesis that interruption of the TGF-beta signaling pathway by curcumin likely occurred at steps downstream of the ligand, such as suppression of gene expression of TGF-beta receptors.

Activation of PPAR-{gamma} inhibits gene expression of TGF-beta receptors, leading to interruption of TGF-beta signaling. We have previously shown that curcumin induces PPAR-{gamma} gene expression and its activity in activated HSCs in vitro. To evaluate the role of PPAR-{gamma} activation in the gene expression of TGF-beta receptors, passaged HSCs were treated with the natural PPAR-{gamma} agonist PGJ2 at various concentrations for 24 h. As shown in Fig. 6, A and B, activation of endogenous PPAR-{gamma} by PGJ2 dose dependently suppressed gene expression of Tbeta-RI and Tbeta-RII at both levels of transcription and translation. Transfection assays of HSCs with the TGF-beta receptor promoter reporter plasmids pTbeta-RI-Luc or pTbeta-RII-Luc confirmed the inhibitory effect of activation of PPAR-{gamma} by PGJ2 on the gene promoter activity of the two receptors in passaged HSCs (Fig. 6C). The plasmids pTbeta-RI-Luc and pTbeta-RII-Luc contain, respectively, 965 and 1,670 bp of the 5'-flanking region of the Tbeta-RI and Tbeta-RII gene promoter subcloned in a Luc reporter plasmid (4). In addition, PGJ2 caused a dose-dependent reduction in Luc activity in HSCs transfected with p3TP-Lux, suggesting that the activation of PPAR-{gamma} blocked TGF-beta signaling in activated HSCs. Taken together, these results indicated that the activation of PPAR-{gamma} inhibited gene expression of TGF-beta receptors, leading to interruption of TGF-beta signaling in activated HSCs.


Figure 6
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  		Fig. 6. Activation of PPAR-{gamma} reduces gene expression of TGF-beta receptors in passaged HSCs, leading to interruption of the TGF-beta signaling pathway. Passaged HSCs were treated with PGJ2 at the indicated concentrations or with Cur at 20 µM for 24 h. Total RNA or whole cell extracts were prepared for analyses of the expression of TGF-beta receptor type 1 (Tbeta-RI) and Tbeta-RII by real-time PCR (A; n = 3) or by Western blot analyses (B; n = 3), respectively. For real-time PCR, GAPDH was used as an invariant control for calculating mRNA fold changes. For Western blot analyses, beta-actin was used as an internal control for equal loading. C: passaged HSCs were transfected with the plasmids pTbeta-RI-Luc (which contains 965 bp of the gene promoter of Tbeta-RI), pTbeta-RII-Luc, or p3TP-Lux. Cells were treated with PGJ2 at the indicated concentrations for 24 h. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). Values are expressed as means ± SD. *P < 0.05 vs. cells without treatment.

 
Curcumin significantly increases the level of total cellular GSH. To further elucidate the underlying mechanisms of curcumin in the inhibition of CTGF gene expression in activated HSCs, we hypothesized that curcumin interrupted TGF-beta signaling and inhibited CTGF gene expression by improving the cellular redox and attenuating oxidative stress in activated HSCs. As shown in Fig. 7A, curcumin dose dependently increased the level of total cellular GSH (GSH + GSSG) in passaged HSCs. Inhibition of PPAR-{gamma} activation by its antagonist PD-68235 dramatically reduced the ability of curcumin to increase the level of cellular GSH, suggesting that PPAR-{gamma} activation is a prerequisite for curcumin to increase the level of cellular GSH in activated HSCs. Further analyses indicated that curcumin significantly increased the ratio of GSH to GSSG, a sensitive indicator of oxidant stress, in activated HSCs (Fig. 7B).


Figure 7
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  		Fig. 7. Cur dose dependently increases the level of total cellular GSH in activated HSCs. Passaged HSC were pretreated with or without the PPAR-{gamma} antagonist PD for 30 min before the addition of Cur at the indicated concentrations for an additional 24 h. A: levels of total intracellular GSH were determined on the basis of the content of reduced GSH plus GSSG. B: ratios of GSH to GSSG were calculated. Results are combined from 3 independent experiments performed in triplicate with each concentration (n = 3). *P < 0.05 vs. cells treated with no Cur; **P < 0.05 vs. cells treated with Cur at 20 µM.

 
De novo synthesis of GSH plays a pivotal role in interrupting TGF-beta signaling and inhibiting CTGF gene expression. Further experiments were performed to evaluate the role of the increase in the level of GSH in the interruption of TGF-beta signaling and inhibition of CTGF gene expression. The level of cellular GSH was altered by the following well-known manipulators of GSH synthesis (2, 50). N-acetylcysteine (NAC) was used to enhance cellular GSH content by supplying cysteine. In contrast, L-buthionine sulfoximine (BSO) was used as a specific inhibitor of glutamate-cysteine ligase (GCL), a critical rate-limiting enzyme in the de novo synthesis of GSH (54). Inhibition of GCL activity by BSO leads to depletion of intracellular GSH (50). As shown in Fig. 8A, NAC, like curcumin, significantly reduced Luc activity in cells transfected with the TGF-beta-inducible Luc reporter plasmid p3TP-Lux. Pretreatment of cells with BSO abrogated the effect of curcumin on interrupting TGF-beta signaling. These results suggested that simulation of de novo synthesis of GSH played a critical role in the interruption of TGF-beta signaling by curcumin. Further experiments demonstrated that NAC, mimicking curcumin, reduced the gene expression of Tbeta-RI and Tbeta-RII at both levels of transcription and translation in passaged HSCs (Fig. 8, B and C). The inhibitory effect of curcumin on gene expression was eliminated by depletion of cellular GSH in cells treated with BSO (Fig. 8, B and C). BSO itself had little, if any, stimulatory effect on Luc activity and on the expression of genes in passaged HSCs (data not shown). Additional experiments showed that increasing cellular GSH by the GSH precursor NAC significantly reduced, like curcumin, CTGF gene expression at levels of transcription and translation (Fig. 9, A and B). Depletion of cellular GSH by pretreatment of cells with BSO eliminated the inhibitory effect of curcumin on CTGF, suggesting that de novo synthesis of cellular GSH by curcumin might be required for curcumin to inhibit CTGF gene expression in activated HSCs in vitro. Furthermore, the antioxidant NAC, mimicking curcumin, reduced the steady-state level of mRNA and the protein abundance of {alpha}I(I)-procollagen, a target gene regulated by CTGF, and {alpha}-SMA, a marker of activated HSCs. Inhibition of GCL activity by BSO abolished the inhibitory effects of curcumin (Fig. 9). Taken together, these results demonstrated that curcumin significantly increased cellular GSH contents in activated HSCs, which played a critical role in interrupting TGF-beta signaling and inhibiting gene expression of CTGF and {alpha}I(I)-procollagen in activated HSCs.


Figure 8
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  		Fig. 8. Inhibition of de novo GSH synthesis eliminates the effect of Cur on interrupting TGF-beta signaling and suppressing gene expression of TGF-beta receptors in activated HSCs. Passaged HSCs were treated for 24 h with N-acetylcysteine (NAC; 5 mM) or Cur (20 µM) or pretreated with the glutamate-cysteine ligase (GCL) inhibitor L-buthionine sulfoximine (BSO; 0.25 mM) for 1 h before the addition of Cur (20 µM). A: Luc assays of HSCs transfected with the TGF-beta-inducible reporter plasmid p3TP-Lux. Luc activity is expressed as relative units after beta-galactosidase normalization (n = 6). Values are expressed as means ± SD. *P < 0.05 vs. cells with no treatment (Ctr). B: real-time PCR analyses of the levels of Tbeta-RI and Tbeta-RII mRNA. mRNA fold changes were calculated by using GAPDH as an invariant control. Values are expressed as means ± SD; n = 3. *P < 0.05 vs. cells with no treatment (Ctr); **P < 0.05 vs. cells treated with Cur. C: Western blot analyses of Tbeta-RI and Tbeta-RII. beta-Actin was used as an internal control for equal loading. Representatives images shown are from 3 independent experiments.

 

Figure 9
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  		Fig. 9. Stimulation of de novo synthesis of GSH by Cur plays a critical role in inhibiting gene expression of CTGF and {alpha}I(I)-collagen in activated HSCs. Semiconfluent HSCs were treated for 24 h with NAC (5 mM) or Cur (20 µM) or pretreated with the GCL inhibitor BSO (0.25 mM) for 1 h before the addition of Cur (20 µM). Total RNA or whole cell protein extracts were prepared. A: real-time PCR analyses of the mRNA levels of CTGF, {alpha}I(I)-procollagen, and {alpha}-smooth muscle actin ({alpha}-SMA; n = 3). mRNA fold changes were calculated by using GAPDH as an invariant control. Values are expressed as means ± SD; n = 3. *P < 0.05 vs. cells with no treatment (Ctr); **P < 0.05 vs. cells treated with Cur. B: Western blot analyses of CTGF, {alpha}I(I)-procollagen, and {alpha}-SMA. beta-Actin was used as an internal control for equal loading. B/Cur, BSO + Cur. Representative images shown are from 3 independent experiments.

 

   DISCUSSION
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During the past decade, advances in the understanding of genes in promoting HSC activation are impressive. However, there have been few breakthroughs in the therapeutic intervention of hepatic fibrogenesis. Therefore, research identifying innocuous antifibrotic agents is of high priority and urgently needed. Most of the evolving antifibrotic therapies are aimed at inhibiting the activation of HSCs, including reducing cell proliferation, inducing apoptosis, and suppressing overproduction of ECM. Our recent studies, including the present report, have addressed this strategy and provided candidates for treatment and prevention of hepatic fibrosis (13, 14, 55, 56). In the present report, we demonstrated that curcumin dose dependently suppressed the gene expression of CTGF in activated HSCs, which required activation of PPAR-{gamma}. Exogenous CTGF dose dependently abrogated the effect of curcumin on the inhibition of {alpha}I(I)-collagen gene expression in HSCs. Activation of PPAR-{gamma} by curcumin resulted in the interruption of TGF-beta signaling, presumably by suppressing the gene expression of TGF-beta receptors, leading to the inhibition of CTGF gene expression. The phytochemical curcumin showed its potent antioxidant property by significantly increasing the level of total GSH and the ratio of GSH to GSSG in activated HSCs. De novo synthesis of cellular GSH was a prerequisite for curcumin to interrupt TGF-beta signaling and inhibit gene expression of CTGF and {alpha}I(I)-collagen in passaged HSCs. Taken together, our results support our initial hypothesis: inhibition of ECM gene expression in activated HSCs by curcumin was mediated by suppressing CTGF gene expression through reducing oxidative stress and interrupting TGF-beta signaling.

In the present report, we demonstrated that curcumin at 20 µM significantly inhibited the gene expression of CTGF by ~45–60% and caused a dramatic reduction in the production of {alpha}I(I)-procollagen by >50% (Figs. 3 and 9). The latter was dose dependently eliminated by exogenous CTGF (Fig. 3), which provides strong support for the conclusion of this report: Curcumin suppresses the expression of ECM genes in activated HSCs by inhibiting gene expression of CTGF. These results suggest the possible mechanisms and potential roles of curcumin in the inhibition of the progress of hepatic fibrogenesis in vivo. Overproduction of CTGF in a variety of fibrotic disorders is presumably secondary to the production and activation of TGF-beta, a key inducer of ECM synthesis. The promoter of the CTGF gene has a TGF-beta response element that regulates its expression (25). We demonstrated that curcumin interrupted TGF-beta signaling in activated HSCs in vitro (Fig. 4A), which presumably led to the suppression of CTGF gene expression. This assumption was supported by additional experiments showing that interruption of the TGF-beta signaling pathway by forced expression of dominant-negative Tbeta-RII resulted in the inhibition of the promoter activity of the CTGF gene (Fig. 4B), mimicking the inhibitory effect of curcumin shown in Fig. 1C.

Curcumin has no apparent inhibitory effect on the production and activation of latent TGF-beta1 in passaged HSCs (56). We, therefore, hypothesized that interruption of TGF-beta signaling by curcumin might occur at steps downstream of its agonists, such as receptors, by suppressing gene expression of the receptors and reducing its bioavailability to the ligand TGF-beta. Our hypothesis was supported by the observations showing that exogenous TGF-beta1 showed differential effects on TGF-beta signaling and CTGF gene expression, depending on when cells were exposed to the ligand. The simultaneous addition of TGF-beta1 with curcumin dramatically eliminated the inhibitory effects of curcumin on the interruption of TGF-beta signaling and inhibition of the promoter activity of the CTGF gene in HSCs (Fig. 5, B and D). In great contrast, the subsequent addition of exogenous TGF-beta1 after exposure of cells to curcumin for 24 h could not eliminate the effects of curcumin (Fig. 5, A and C). Treatment of cells with curcumin for 24 h significantly inhibits gene expression of TGF-beta receptors in activated HSCs (56). It bears indicating that these results do not exclude possible impacts of curcumin on other downstream steps in the TGF-beta signaling pathway.

We (55) have previously shown that curcumin induces the gene expression of PPAR-{gamma} and stimulates its trans-activation activity in activated HSCs in vitro. PPAR-{gamma} agonists are presumed to exist in the media with 10% FBS (29, 36, 56). Activation of PPAR-{gamma} is required for curcumin to inhibit HSC activation, including reduced cell growth and suppressed ECM gene expression (8, 9). In the present report, we observed that blockade of PPAR-{gamma} activation by its antagonist abrogated the inhibitory effect of curcumin on CTGF gene expression (Fig. 1), suggesting that the inhibitory effect might be mediated by the activation of PPAR-{gamma}. The suggestion was supported by additional experiments. As shown in Fig. 2, A and B, activation of endogenous PPAR-{gamma} by PGJ2 dose dependently suppressed gene expression of CTGF at both levels of transcription and translation. In addition, activation of PPAR-{gamma} inhibited gene expression of TGF-beta receptors, leading to the interruption of TGF-beta signaling in activated HSCs (Fig. 6). The roles of PPAR-{gamma} activation in the suppression of CTGF gene expression and interruption of TGF-beta signaling are consistent with another prior report (22). Activation of PPAR-{gamma} inhibits TGF-beta-induced CTGF expression by directly interfering with the Smad3 signaling pathway (22).

Curcumin is a potent antioxidant (48), due to both its innate antioxidant chemical structure and its ability to affect enzyme systems involved in the defense against oxidative stress (3, 18). Curcumin inhibits lipid peroxidation (45, 49), nitric oxide synthase activity (9), and production of ROS (31). These unique features might allow curcumin to succeed where other regular antioxidants have failed in the inhibition of hepatic fibrogenesis. In the present report, we demonstrated that curcumin increased the ratio of GSH to GSSG, an indicator of cellular oxidative stress, and the level of total cellular GSH in activated HSCs. The latter required PPAR-{gamma} activation. Depletion of cellular GSH by the GCL inhibitor BSO dramatically eliminated the effects of curcumin on interruption of TGF-beta signaling and inhibition of gene expression of CTGF. These results collectively indicated that the level of GSH enhanced by curcumin played a crucial role in inhibiting CTGF gene expression in activated HSC. It remains unknown whether curcumin directly stimulates the activity of preexisting GCL, a rate-limiting enzyme of de novo synthesis of GSH, and/or induces gene expression of GCL in activated HSCs. Other studies (15, 40) have shown that the regulation of many detoxifying enzymes by dietary compounds is mediated by the electrophile response element (EpRE) located in the promoter of the genes. Curcumin induces gene expression of the GCL catalytic subunit in the human branchial epithelial cell line HBE1 by altering EpRE and activator protein-1 binding complexes (18). Our preliminary results suggest that activation of PPAR-{gamma} by curcumin might participate in the induction of gene expression of the GCL catalytic subunit in activated HSCs in vitro (data not shown). Additional experiments are ongoing to elucidate the molecular mechanisms of curcumin in the increase in the level of total cellular GSH in activated HSCs and in the involvement of PPAR-{gamma} activation in the process.

On the basis of our observations, we propose a model to elucidate the underlying mechanisms of curcumin in the inhibition of the production of ECM, including {alpha}I(I)-collagen, in activated HSCs (Fig. 10). The phytochemical curcumin dramatically increases the level of cellular GSH, which requires activation of PPAR-{gamma}. De novo synthesis of GSH by curcumin brings about the interruption of TGF-beta signaling by suppressing the gene expression of TGF-beta receptors, resulting in inhibition of CTGF gene expression. These effects ultimately lead to a reduction in the production of ECM, including {alpha}I(I)-collagen, in activated HSCs in vitro. It bears emphasis that our results and this model do not exclude any other possible mechanisms of curcumin in the suppression of CTGF gene expression and reduction of ECM production in activated HSCs. Results generated from our studies provide novel insights into the roles and mechanisms of curcumin in the inhibition of HSC activation. The unique characteristics of curcumin, including the potent capability to attenuate oxidative stress, inducing gene expression of endogenous PPAR-{gamma}, inhibiting HSC activation, and no adverse health effects, might make it a good antifibrotic candidate for the treatment and prevention of hepatic fibrosis.


Figure 10
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  		Fig. 10. Schema of the underlying mechanisms of Cur in the reduction of the production of extracellular matrix (ECM), including {alpha}I(I)-collagen, in activated HSCs in vitro.

 

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47995 (to A. Chen) and by a Louisiana State University Health Sciences Center Bridge Award (to A. Chen).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Chen, Dept. of Pathology, Louisiana State Univ. Health Sciences Center, in Shreveport, 1501 Kings Hwy., Shreveport, LA 71130 (e-mail: achen{at}lsuhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Abou-Shady M, Friess H, Zimmermann A, di Mola FF, Guo XZ, Baer HU, and Buchler MW. Connective tissue growth factor in human liver cirrhosis. Liver 20: 296–304, 2000.[CrossRef][ISI][Medline]
  2. Anderson ME and Luo JL. Glutathione therapy: from prodrugs to genes. Semin Liver Dis 18: 415–424, 1998.[ISI][Medline]
  3. Araujo CC and Leon LL. Biological activities of Curcuma longa L. Mem Inst Oswaldo Cruz 96: 723–728, 2001.[ISI][Medline]
  4. Bae HW, Geiser AG, Kim DH, Chung MT, Burmester JK, Sporn MB, Roberts AB, and Kim SJ. Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene. J Biol Chem 270: 29460–29468, 1995.[Abstract/Free Full Text]
  5. Baker JC and Harland RM. From receptor to nucleus: the Smad pathway. Curr Opin Genet Dev 7: 467–473, 1997.[CrossRef][ISI][Medline]
  6. Ball DK, Rachfal AW, Kemper SA, and Brigstock DR. The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion. J Endocrinol 176: R1–R7, 2003.[Abstract]
  7. Blom IE, Goldschmeding R, and Leask A. Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biol 21: 473–482, 2002.[CrossRef][ISI][Medline]
  8. Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20: 189–206, 1999.[Abstract/Free Full Text]
  9. Brouet I and Ohshima H. Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 206: 533–540, 1995.[CrossRef][ISI][Medline]
  10. Camp HS, Chaudhry A, and Leff T. A novel potent antagonist of peroxisome proliferator-activated receptor gamma blocks adipocyte differentiation but does not revert the phenotype of terminally differentiated adipocytes. Endocrinology 142: 3207–3213, 2001.[Abstract/Free Full Text]
  11. Chen A and Davis BH. UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells. J Biol Chem 274: 158–164, 1999.[Abstract/Free Full Text]
  12. Chen A, Davis BH, Bissonnette M, Scaglione-Sewell B, and Brasitus TA. 1,25-Dihydroxyvitamin D3 stimulates activator protein-1-dependent Caco-2 cell differentiation. J Biol Chem 274: 35505–35513, 1999.[Abstract/Free Full Text]
  13. Chen A and Zhang l. The antioxidant (–)-epigallocatechin-3-gallate inhibits rat hepatic stellate cell proliferation in vitro by blocking the tyrosine phosphorylation and reducing the gene expression of platelet-derived growth factor-beta receptor. J Biol Chem 278: 23381–23389, 2003.[Abstract/Free Full Text]
  14. Chen A, Zhang L, Xu J, and Tang Jun. The antioxidant (–)-epigallocatechin-3-gallate inhibits activated hepatic stellate cell growth and suppresses acetaldehyde-induced gene expression. Biochem J 368: 695–704, 2002.[CrossRef][ISI][Medline]
  15. Chen C and Kong AN. Dietary chemopreventive compounds and ARE/EpRE signaling. Free Radic Biol Med 36: 1505–1516, 2004.[CrossRef][ISI][Medline]
  16. Chen H, Zhang ZS, Zhang YL, and Zhou DY. Curcumin inhibits cell proliferation by interfering with the cell cycle and inducing apoptosis in colon carcinoma cells. Anticancer Res 19: 3675–3680, 1999.[ISI][Medline]
  17. De Caestecker MP, Parks WT, Frank CJ, Castagnino P, Bottaro DP, Roberts AB, and Lechleider RJ. Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases. Genes Dev 12: 1587–1592, 1998.[Abstract/Free Full Text]
  18. Dickinson DA, Iles KE, Zhang H, Blank V, and Forman HJ. Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression. FASEB J 17: 473–475, 2003.[Abstract/Free Full Text]
  19. Fernandez-Checa JC, Kaplowitz N, Garcia-Ruiz C, and Colell A. Mitochondrial glutathione: importance and transport. Semin Liver Dis 18: 389–401, 1998.[ISI][Medline]
  20. Fridovich I. The biology of oxygen radicals. Science 201: 875–880, 1978.[Abstract/Free Full Text]
  21. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 275: 2247–2250, 2000.[Free Full Text]
  22. Fu M, Zhang J, Zhu X, Myles DE, Willson TM, Liu X, and Chen YE. Peroxisome proliferator-activated receptor gamma inhibits transforming growth factor beta-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3. J Biol Chem 276: 45888–45894, 2001.[Abstract/Free Full Text]
  23. Galli A, Crabb D, Price D, Ceni E, Salzano R, Surrenti C, and Casini A. Peroxisome proliferator-activated receptor gamma transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells. Hepatology 31: 101–108, 2000.[CrossRef][ISI][Medline]
  24. Greenwel P, Dominguez-Rosales JA, Mavi G, Rivas-Estilla AM, and Rojkind M. Hydrogen peroxide: a link between acetaldehyde-elicited alpha1(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells. Hepatology 31: 109–116, 2000.[CrossRef][ISI][Medline]
  25. Grotendorst GR. Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 8: 171–179, 1997.[CrossRef][Medline]
  26. Grotendorst GR, Okochi H, and Hayashi N. A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ 7: 469–480, 1996.[Abstract]
  27. Guo N, McIntosh C, and Shaw C. Glutathione: new candidate neuropeptide in the central nervous system. Neuroscience 51: 835–842, 1992.[CrossRef][ISI][Medline]
  28. Halliwell B. Drug antioxidant effects. A basis for drug selection? Drugs 42: 569–605, 1991.[ISI][Medline]
  29. Hazra S, Xiong S, Wang J, Rippe R, Chatterjee VK, and Tsukamoto H. PPARgamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. J Biol Chem 279: 11392–11401, 2003.[Medline]
  30. Ji C, Casinghino S, McCarthy TL, and Centrella M. Cloning, characterization, and expression of the transforming growth factor-beta type I receptor promoter in fetal rat bone cells. J Cell Biochem 63: 478–490, 1996.[CrossRef][ISI][Medline]
  31. Joe B and Lokesh BR. Role of capsaicin, curcumin and dietary n-3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages. Biochim Biophys Acta 1224: 255–263, 1994.[Medline]
  32. Leask A, Holmes A, Black CM, and Abraham DJ. Connective tissue growth factor gene regulation. Requirements for its induction by transforming growth factor-beta 2 in fibroblasts. J Biol Chem 278: 13008–13015, 2003.[Abstract/Free Full Text]
  33. Lee KS, Buck M, Houglum K, and Chojkier M. Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression. J Clin Invest 96: 2461–2468, 1995.[ISI][Medline]
  34. Marra F, Efsen E, Romanelli RG, Caligiuri A, Pastacaldi S, Batignani G, Bonacchi A, Caporale R, Laffi G, Pinzani M, and Gentilini P. Ligands of peroxisome proliferator-activated receptor gamma modulate profibrogenic and proinflammatory actions in hepatic stellate cells. Gastroenterology 119: 466–478, 2000.[CrossRef][ISI][Medline]
  35. Massague J and Chen YG. Controlling TGF-beta signaling. Genes Dev 14: 627–644, 2000.[Free Full Text]
  36. McIntyre TM, Pontsler AV, Silva AR, St Hilaire A, Xu Y, Hinshaw JC, Zimmerman GA, Hama K, Aoki J, Arai H, and Prestwich GD. Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARgamma agonist. Proc Natl Acad Sci USA 100: 131–136, 2003.[Abstract/Free Full Text]
  37. Miyahara T, Schrum L, Rippe R, Xiong S, Yee HF Jr, Motomura K, Anania FA, Willson TM, and Tsukamoto H. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 275: 35715–35722, 2000.[Abstract/Free Full Text]
  38. Mohan R, Sivak J, Ashton P, Russo LA, Pham BQ, Kasahara N, Raizman MB, and Fini ME. Curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor-2, including expression of matrix metalloproteinase gelatinase B. J Biol Chem 275: 10405–10412, 2000.[Abstract/Free Full Text]
  39. Mohandas KM and Desai DC. Epidemiology of digestive tract cancers in India. V. Large and small bowel. Indian J Gastroenterol 18: 118–121, 1999.[Medline]
  40. Myhrstad MC, Carlsen H, Nordstrom O, Blomhoff R, and Moskaug JO. Flavonoids increase the intracellular glutathione level by transactivation of the gamma-glutamylcysteine synthetase catalytical subunit promoter. Free Radic Biol Med 32: 386–393, 2002.[CrossRef][ISI][Medline]
  41. Paradis V, Dargere D, Bonvoust F, Vidaud M, Segarini P, and Bedossa P. Effects and regulation of connective tissue growth factor on hepatic stellate cells. Lab Invest 82: 767–774, 2002.[ISI][Medline]
  42. Paradis V, Dargere D, Vidaud M, De Gouville AC, Huet S, Martinez V, Gauthier JM, Ba N, Sobesky R, Ratziu V, and Bedossa P. Expression of connective tissue growth factor in experimental rat and human liver fibrosis. Hepatology 30: 968–976, 1999.[CrossRef]