Effects of curcumin on peroxisome proliferator-activated receptor γ expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells

Peroxisome proliferator-activated receptors (PPARs) are soluble transcription factors that are activated by a diverse class of lipophilic compounds.¹ With the activation of PPARs a concomitant induction of a number of genes that code for peroxisomal fatty acid metabolizing enzymes was observed in mouse liver. There are at least three PPAR subtypes, PPARα, PPARβ and PPARγ. And each subtype is capable of binding to DNA after heterodimerizing with the retinoid X receptor.^1-3 It was reported that PPARγ expression is ten-fold higher in hepatic tissues than in peripheral-blood mononuclear cells, but the functional activities of PPARγ in the liver remain largely unknown.^3,4

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurres in many patients with chronic liver disease and involves the abnormal accumulation of extracellular matrix (ECM).⁵ hepatic stellate cells (HSCs) are non-parenchymal cells located perisinusoidally in the Space of Disse. Following liver injury of various etiologies HSCs can undergo the process of activation and trans-differentiate to a myofibroblast-like phenotype characterized by an increase in cell proliferation, loss of vitamin A-storing capability, expression of α-SMA (α-smooth muscle actin) and overproduction of ECM components, especially type I collagen.^6,7

HSCs activation is a highly pleiotropic process and dramatic phenotypic changes require global reprogramming of HSCs gene expression that in turn must be orchestrated by long-term changes in the expression and/or activity of key transcription factors of the HSCs genome. Modulation of transcription factor activity via post-translational modification exerts great regulatory influence on HSCs activation.^4,6,7 Previously we found that curcumin and SAB (salvianolic acid B) effectively inhibited HSC activation markers and reduced the phosphorylation of extracellular signal regulated kinase.⁸ Here we show the effect of curcumin on the PPARγ signal transduction pathway in the culture- activated HSCs.

Animals
Male Sprague Dawley rats (Quality eligible certificate: SCXK (Hu) 2003－0002), with a body weight between 500－650 g, were obtained from the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (TCM) and were supplied with food and water ad libitum.

Major compounds and reagents
Curcumin was purchased from Sigma (USA). SAB was provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences. TGZ (troglitazone) and GW9662 were products of Cayman (USA). MTT was purchased from Serva (USA). DMEM, MEM and M199 culture media were products of Gibco (USA). Fetal bovine sera were purchased from Hyclone (USA). Pronase E and type IV Collagenase were products of Sigma. DNase I was purchased from Roche. Nycodenz was produced by Axis-Shield (Norway). The cell apoptosis-Hoechst staining kit was purchased from Jiangsu Biyuntian Biological Technique Institute (China). Desmin rabbit polycolonal antibody was the product of NeoMarkers (USA). Rabbit anti-rat polyclonal PPARγ antibody was the product of Wuhan Boshide Company (China). Mouse anti-rat monoclonal β-actin and GAPDH antibodies were purchased from Kangcheng Company (China). Mouse anti-rat α-SMA monoclonal antibody was the product of Sigma. Rabbit anti-mouse type I collagen antibody was the product of Calbiochem (USA). Rabbit anti-rat polyclonal TGF β type I receptor antibody, mouse anti-rat Bax and Bcl-2 monoclonal antibodies, mouse monoclonal NFκB p65 antibody were products of Santa Cruz (USA). HRP-goat-anti-rabbit IgG (H+L) CYTM5 antibody was the product of Zymed company (USA). Monoclonal secondary antibody (Anti-mouse IgG, peroxidase-linked species-specific whole antibody (from sheep)) and polyclonal secondary antibody (Anti-rabbit IgG, peroxidase-linked species-specific whole antibody (from goat)) were products of Santa Cruz (USA). ProLong Antifade kit (p-7481) was the product of Molecular Probes (USA). Proteinase inhibitor (complete mini) was the product of Roche (Germany). The nuclei EZ prep nuclei isolation kit was the product of Sigma. DC protein quantification kit was the product of Bio-Rad (USA). Enhanced chemiluminescence reagents were purchased from Pierce (USA). TRizol reagent was the product of Invitrogen (USA). The cDNA synthesis kit and PCR Master Mix kit were purchased from Fermentas Company (Canada). DNA Maker was the product of Beijing TIANGEN Company (China). The specific primers were synthesized at Shanghai Shenggong Company (China).

HSCs isolation, culture, and treatment
HSCs were isolated from normal rats through an in situ perfusion of the liver with Pronase E followed by density gradient centrifugation with Nycondenz as described before.⁹ As the subcultured HSCs grew to confluence, cells were treated with curcumin (10－50 µmol/L), SAB (1 µmol/L), TGZ (20 µmol/L), GW9662 (20 µmol/L), curcumin+GW9662, SAB+GW9662 or TGZ+GW9662 respectively. Curcumin, TGZ and GW9662 were solubilized with dimethyl sulfoxide then dissolved in culture media. SAB was diluted with culture media. GW9662 was added 30 minutes in advance. The compound concentrations and additional protocols were based on our preliminary experiments.⁸

MTT analysis
The extent of HSCs proliferation was determined by MTT colorimetry as described before.⁸

Immunofluorescent staining
HSCs were cultured on glass coverslips in a 12-well plate, and then treated with curcumin for 24 hours. Then cells were fixed in methanol:acetone (1:1) at 4˚C overnight and washed with specific Buffers. After incubation with 3% goat sera, cells were incubated with PPARγ antibody at 4˚C overnight, then incubated with anti-rabbit IgG (H+L) CYTM5 at 37˚C for 1 hour. After wash, coverslips were covered with the mixture of ProLong mounting medium and ProLong Antifade reagent. Fluorescent images were observed under the confocal laser-scanning microscope TCS SP2 (Leica Company, Germany) and the 633 nm laser wavelength was used for analysis.

Semi-quantitative RT-PCR
The extraction of total RNA and semi-quantitative RT-PCR analysis were performed as described before.⁵ The specific primers are shown in Table and the Rotor-Gene RG-3000 PCR amplifier was used (Gene Company, Australia). The relative target mRNA expression level was corrected by β-actin in the same sample.

Western blotting
Total cellular protein was extracted as described before.^5,8 The nuclei protein was extracted by the nuclei EZ prep nuclei isolation kit according to the manufacture's protocol. Sample protein was resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis and then electrophoretically transferred to nitrocellulose. The primary antibodies were added and incubated at 4˚C overnight. Then membranes were incubated with corresponding secondary antibodies for 1 hour. Protein bands were revealed by the ECL kit according to the manufacturer's protocol. The amount of protein was corrected by β-actin or GAPDH in the same sample.

Hoechst 33258 staining
HSCs were cultured on glass coverslips and then treated with curcumin for 24 hours. Cells were fixed in fixation buffer at 4˚C overnight, then washed and exposed to 0.5 ml Hoechst 33258 in the dark at room temperature for 5 minutes. After washes, the ProLong Antifade reagent was dropped onto the coverslips. The fluorescent images were observed under a fluorescent microscope.

Gelatin zymograph assay
HSCs culture supernatants were harvested and centrifuged at 4˚C, 15 000 r/min for 15 minutes. Sixteen µl of supernatants and 1/4 volume of sample buffer were mixed. The 8% separating gel containing 0.1% gelatin and 5% condensation gel were prepared, and then deoxidizing non-denaturing electrophoresis was performed on ice at 80 V for 2.5 hours. The gel was washed and put into incubation buffer for 19 hours at 37˚C. Four hours after Coomassie brilliant blue R250 staining, decoloring buffers A, B and C were used for 15, 10 and 5 minutes sequentially. The target band was analyzed by FR-980 imaging system (Furi Company, China).

Statistical analysis
Statistical analysis was performed by using the SPSS11.0 statistical package. Each experiment was performed in triplicate. All results were expressed as mean ± standard deviation. Comparisons were analyzed by one-way analysis of variance (ANOVA). Differences were considered statistically significant if the P value was less than 0.05.

RESULTS

Curcumin inhibited HSCs proliferation dependent on PPARγ activation
From 10 to 50 µmol/L curcumin suppressed passaged HSCs proliferation in a dose-dependent manner (data not shown) and 30 µmol/L of curcumin was chosen in the following experiments. The working concentrations of SAB, TGZ and GW9662 were chosen as reported before.⁸ The specific agonist to PPARγ, TGZ significantly inhibited the proliferation of HSCs (P<0.01, Fig. 1); and the effect was abrogated by GW9662, the specific PPARγ antagonist (P<0.01).^1,3 Similarly, the inhibitory effect of curcumin on HSCs proliferation was also abrogated by GW9662 (P<0.05) and the results suggested that the inhibitory effect of curcumin on the proliferation of activated HSCs might be dependent on the activation of PPARγ. SAB also inhibited the proliferation of HSCs (P<0.01), however this effect could not be reversed by GW9662 (P>0.05), indicating that SAB's effect might not depend on the PPARγ pathway but on other pathways⁸ and it was not used in the following experiments.

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Fig. 1. Effect of different drug treatments on the passaged rat HSCs proliferation. ^*P<0.01 vs the control group; ^△P<0.05 vs the curcumin group; ^#P<0.01 vs the TGZ (troglitazone) group; ^▲P>0.05 vs SAB (salvianolic acid B) group.

PPARγ expression decreased gradually upon HSCs activation, curcumin restored the expression and suppressed HSCs activation
The isolated HSCs were cultured on an uncoated polystyrene surface for 1, 4 and 7 days, which represented quiescent, intermediate and activated phenotypes respectively; and the phenotype of passaged HSCs was activated phenotype (figures not shown). While the HSCs underwent activation gradually as the duration of the culture was prolonged, the PPARγ nuclear expression level decreased gradually too. The results showed that the highest PPARγ level was found associated with the quiescent phenotype while the lowest level was found with the culture-activated phenotype.

Both PPARγ mRNA and protein levels showed the same decreasing trend but the protein reduction level lagged behind the mRNA level (Fig. 2). Curcumin up-regulated PPARγ not only at the transcriptional (P<0.05) but also at translational level (P<0.05); and the effect was lessened by GW9662 pretreatment (P<0.01, Fig. 3). Curcumin significantly inhibited the expression of α-SMA mRNA (P<0.001) and protein (P<0.05) and

the production of collagen I mRNA (P<0.001) and protein (P<0.05, Fig. 4). Combined with the MTT analysis findings these results showed that curcumin effectively inhibited the activation of HSCs through PPARγ signaling.

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Fig. 2. The expression level of PPARγ in the indicated time in primary culture cells and passaged cells, the mRNA was corrected by β-actin and the protein was corrected by GAPDH.

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Fig. 3. Effect of curcumin on the expression level of PPARγ, the mRNA was corrected by β-actin and the protein was corrected by GAPDH. ^*P<0.05 vs control; ^#P<0.01 vs curcumin.

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Fig. 4. Effect of curcumin on α-SMA and collagen I expression level, the mRNA and protein level was corrected by β-actin. ^* P<0.001 vs control; ^#P<0.01 vs curcumin; ^△P<0.05 vs control; ^▲P<0.05 vs curcumin.

Curcumin promoted PPARγ nuclear translocation/redistribution in HSCs
To determine the effect of curcumin on the subcellular localization of PPARγ, immunofluorescent labeling of the PPARγ protein was performed and the cells were observed under a confocal laser-scanning microscope. The results indicated that in untreated cells PPARγ was expressed not only in the cytoplasm but also in the nucleus and it was distributed evenly (Fig. 5A and C), after curcumin treatment PPARγ accumulated in the nucleus of HSCs and disappeared from the cytoplasm (Fig. 5B and D). Considered with the above Western blot analysis results, it indicated that curcumin not only up-regulated the expression level but also promoted the PPARγ translocation/redistribution from cytoplasm to nucleus.

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Fig. 5. Effect of curcumin on the subcellular localization of PPARγgin rat HSCs. A: Control; B: Curcumin treatment (PPARγ immunofluorescent staining, Original magnification×630). C: Control; D: Curcumin treatment (PPARγ immunofluorescent staining, original magnification×2520).

Curcumin induced HSCs apoptosis through changing the ratio of Bcl-2/Bax
As shown in Fig. 6, almost no apoptotic nuclei were observed in the untreated HSCs; the staining was elliptical blue and diffused in the nuclei. On the contrary, the typical apoptotic morphological changes such as condensed chromatin and shrunken crimpled and condensed gray-blue nuclei were found in the curcumin treated HSCs. The results showed that curcumin induced the apoptosis of culture-activated HSCs in vitro. In order to elucidate the mechanism of curcumin induced apoptosis of activated HSCs, the expression of Bcl-2 and Bax were determined. As shown in Fig. 7, the results showed that curcumin treatment increased the expression of pro-apoptotic Bax (mRNA P<0.01; protein P<0.05) and reduced the expression of anti-apoptotic Bcl-2 (mRNA P<0.01; protein P<0.01). GW9662 pretreatment antagonized the effects of curcumin (mRNAs P<0.05; proteins P<0.01). In the control group the ratios of Bcl-2/Bax mRNA and protein were about 1.35 and 1.31 respectively. Curcumin treatment reduced the ratios to 0.20 and 0.36 while GW9662 pretreatment increased the ratios to 0.66 and 0.96. The results indicated that curcumin induced the apoptosis of culture-activated HSCs through changing the ratio of anti-apoptotic/ pro-apoptotic regulators by the activation of PPARγ.

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Fig. 6. Curcumin treatment induced apoptosis of culture-activated HSCs. A: Control; B: Curcumin treatment (Hoechst 33258 staining, original magnification×100).

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Fig. 7. Effect of cucumin on the expression levels of Bcl-2 and Bax by activating PPARγ, the mRNA and protein was corrected by β-actin. ^*P<0.01 vs Control; ^△P<0.01 vs Control; ^#P<0.05 vs Curcumin; ^▲P<0.01 vs Curcumin.

Curcumin down-regulated Cyclin D1, NFκB p65 and TGFβR-I expression in HSCs
The cyclin D1 gene was significantly down-regulated by curcumin treatment (P<0.001). As a cell cycle-stimulating factor, the inhibition of Cyclin D1 may reduce the proliferation of HSCs. The effect of curcumin on Cyclin D1 was limited by GW9662 pretreatment (P<0.001, Fig. 8).

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Fig. 8. Effect of curcumin on Cyclin D1 expression level by activating PPARγ, the mRNA was corrected by β-actin. ^*P<0.001 vs Control; ^#P<0.001 vs Curcumin.

The results indicated that curcumin inhibited the cell cycle-stimulating factor at least in part by activating PPARγ, which in turn inhibited HSCs proliferation. NFκB p65 antibody used in the study could only recognize the activated form of NFκB p65. Western blot analysis found that activated NFκB p65 protein expression in nuclei was inhibited by curcumin treatment and was PPARγ activation dependent (P<0.01, Fig. 9). Western blot analysis also found that TGFβR-I protein expression was inhibited by curcumin treatment (P=0.02, Fig. 10). The results showed that curcumin treatment interfered with TGFβ signaling, which in turn reduced ECM synthesis in HSCs.

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Fig. 9. Effect of curcumin on TGFβbRI protein level by activating PPARγ, the protein was corrected by GAPDH. ^*P=0.02 vs Control; ^#P=0.03 vs Curcumin.

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Fig. 10. Effect of curcumin on nuclear NFκB p65 protein level by activating PPARγ, the protein was corrected by GAPDH. ^*P<0.01 vs Control group; ^#P<0.01 vs Curcumin.

Curcumin enhanced activities of MMP-2 and MMP-9 secreted by HSCs
Culture-activated HSCs secreted the active forms of MMP-2 and MMP-9; which appeared at their characteristic 72- and 92-kD positions respectively (Fig.11). The gelatin zymograph assay found that the activities of MMP-2 and MMP-9 were enhanced significantly after curcumin treatment by activating PPARγ (both P<0.01).

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Fig. 11. Effect of curcumin on the activities of MMP2 and MMP9 by activating PPARγ. ^*P<0.01 vs Control; ^#P<0.01 vs Curcumin.

DISCUSSION

Curcuma Longa Linn is a traditional Chinese herbal medicine and has been widely applied in clinical therapy for centuries. Its extractions include curcumin, demethoxy curcumin and bistemethoxy curcumin. Curcumin is the most important active component with potent pharmacological effects^8,10,11 but its role in hepatic fibrosis is as yet largely unknown.⁸ HSCs are the primary source of excessive production of ECM and activation of HSCs is the central event in hepatic fibrogenesis. Resolution of HSCs activation represents an essential step toward reversibility of fibrosis.^12,13 Currently no ideal curative medical treatments are available in clinical practice for hepatic fibrosis.^6,8,12 In this study, we found that curcumin effectively suppressed the proliferation of activated HSCs, and this effect was dependent on the activation of PPARγ. Cyclin D1 is a cell cycle-stimulating factor and belongs to the D-type cyclins that play important roles in cell cycle progression. Cyclin D1 is a key factor in the transition of HSCs from G1 to S phase and the inhibition of cyclin D1 expression prevents cells from entering S phase.^14,15 We found that cyclin D1 expression was inhibited by curcumin in this study. Curcumin might prevent HSCs from entering S phase by inhibiting cyclin D1 through activating PPARγ, which in turn inhibit HSCs proliferation. These results support the idea that curcumin, like other PPARγ ligands, inhibits HSCs activation and liver fibrosis.^16-18 The inhibition of HSCs activation by curcumin is perhaps mediated by interrupting TGFβ signaling.¹⁹ This notion was confirmed by our finding that curcumin interfered with the TGFβ signaling pathway by inhibiting TGFβR-I expression in HSCs. The results provided a novel insight into the mechanisms underlying inhibition of HSCs activation by curcumin.

To date, the studied transcription factors have the common feature of being associated with the activated phenotype of HSCs. However it is also important to study the function of transcription factors that are active in quiescent HSCs.⁴ The present study demonstrated that PPARγ expression decreased markedly with the activation of HSCs, the finding showed that PPARγ plays an important role in maintaining the quiescent phenotype of HSCs. This was confirmed by our findings that curcumin reduced the expression of α-SMA and type I collagen of HSCs while restoring PPARγ nuclear expression. Curcumin treatment restored PPARγ nuclear expression and translocation/redistribution from the cytoplasm to the nucleus in culture-activated HSCs. This result was in accordance with published reports that used other cells types and other PPARγ ligands. 15d-PGJ2 treatment resulted in PPARγ nuclear translocation in endothelial cells. In HT-29 cells, activation of PPARγ results in a cascade of reactions including translocation or redistribution of PPARγ in the cell nucleus, conformational changes within PPARγ, recruitment of coactivators and binding to specific DNA sequence elements termed peroxisome-proliferator response elements. Using fluorescent microscopy to visualize the cellular distribution of a GFP-tagged PPARγ, a predominantly cytoplasmic distribution of the fluorescent label was found in unstimulated cells. Exposure of epithelial cells to 5-aminosalicylic acid resulted in a redistribution of PPARγ to the nucleus. The translocation of GFP-tagged PPARγ from the cytoplasm to the nucleus was also observed with rosiglitazone. Nuclear factor translocations or inversions can give rise to the activation of a gene through its positioning near a strong promoter or its fusion with another gene.²⁰

It has been demonstrated that curcumin is capable of inducing apoptosis in numerous cellular systems.²¹ In this study we found that curcumin induced the apoptosis of activated HSCs in vitro. The underlying mechanism is through the increase of pro-apoptotic Bax and reduction of anti-apoptotic Bcl-2 by activating PPARγ. To modify activated HSCs to a quiescent state or to induce apoptosis is a critical strategy for anti-fibrotic therapy.^4-8 But the fully activated human HSCs do not undergo spontaneous apoptosis and survive prolonged serum deprivation, Fas activation or exposure to nerve growth factor, TNF-α, oxidative stress mediators, doxorubicin or etoposide.²² This was confirmed by our Hoechst 33258 staining analysis that found almost no apoptotic cells in the untreated HSCs. The process of HSCs activation was accompanied by changes in expression of a set of genes involved in apoptosis control. It has been found that activated human HSCs in culture overexpressed Bcl-2. Bcl-2 is highly expressed in HSCs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis.²² It has been confirmed that stimulated HSCs trigger NFκB activation and NFκB inhibition triggered HSCs apoptosis.²³ The activation of NFκB can be interrupted by curcumin, thereby suppressing proliferation and inducing apoptosis.²⁴ We found that activated NFκB p65 protein expression in nuclei was inhibited by curcumin treatment through PPARγ activation. All these results showed that the induction of HSCs apoptosis by curcumin might be multifactorial.^25,26

Collagen turnover and ECM remodeling that occur during various physiological and pathological processes including tissue repair, wound healing, fibrosis and tumor invasion are largely dependent on the regulation of activities of MMPs. MMP-2 readily degrades gelatins, but also has activity on collagen types IV, V and VII, elastin and proteoglycan. MMP-9 is believed to participate in remodeling of basement membrane and cell migration. The basement membrane components, like type IV collagen and laminin, are presumably digested by activated MMP-9. Although Kupffer cells are believed to be the major source for MMP-9, we found that culture-activated HSCs also secreted active MMP-9; Kupffer cells were excluded as the source of MMP-9 by Desmin staining. Our results are consistent with a recent report.²⁷ Breakdown of the fibrillar collagen may be mediated by a coordinated action of multiple MMPs. Once HSCs are fully trans-differentiated into myofibroblastic cells by prolonged culture on plastic they no longer produce inducible MMPs. In the present study, it was found that the activities of MMP-2 and MMP-9 were enhanced by curcumin treatment and perhaps may have an influence on liver fibrogenesis.

In conclusion, our results suggest the pivotal roles of curcumin in molecular regulation of HSCs activation in liver fibrogenesis and solidify the notion that PPARγ serves as an important therapeutic target in liver fibrosis.^4,16-18 As a plant-derived natural substance that is relatively safe,⁵ curcumin may serve as an effective anti-fibrotic agent since it normalizes PPARγ expression while suppressing the activation marker genes, induces apoptosis and enhances MMP activity. In vivo studies on the development and progression of hepatic fibrosis is required to further elucidate the multiple mechanisms of curcumin.

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