Activation of peroxisome proliferator-activated receptor-gamma by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells - PubMed (original) (raw)

Activation of peroxisome proliferator-activated receptor-gamma by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells

Jianguo Lin et al. Lab Invest. 2008 May.

Abstract

During hepatic fibrogenesis, reduction in the abundance of peroxisome proliferator-activated receptor-gamma (PPARgamma) is accompanied by activation of mitogenic signaling for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in hepatic stellate cells (HSCs), the major effector cells. We previously reported that curcumin, the yellow pigment in curry, interrupted PDGF and EGF signaling, stimulated PPARgamma gene expression, and enhanced its activity, leading to inhibition of cell proliferation of activated HSC in vitro and in vivo. The aim of this study was to elucidate the underlying mechanisms. We hypothesized that the enhancement of PPARgamma activity by curcumin might result in the interruption of PDGF and EGF signaling. Our experiments demonstrated that curcumin, with different treatment strategies, showed different efficiencies in the inhibition of PDGF- or EGF-stimulated HSC proliferation. Further experiments observed that curcumin dose dependently reduced gene expression of PDGF and EGF receptors (ie, PDGF-betaR and EGFR), which required PPARgamma activation. The activation of PPARgamma by its agonist suppressed pdgf-betar and egfr expression in HSC. In addition, curcumin reduced the phosphorylation levels of PDGF-betaR and EGFR, as well as their downstream signaling cascades, including ERK1/2 and JNK1/2. Moreover, activation of PPARgamma induced gene expression of glutamate-cysteine ligase, the rate-limiting enzyme in de novo synthesis of the major intracellular antioxidant, glutathione. De novo synthesis of glutathione was required for curcumin to suppress pdgf-betar and egfr expression in activated HSCs. Our results collectively demonstrated that enhancement of PPARgamma activity by curcumin interrupted PDGF and EGF signaling in activated HSCs by reducing the phosphorylation levels of PDGF-betaR and EGFR, and by suppressing the receptor gene expression. These results provide novel insights into the mechanisms of curcumin in the inhibition of HSC activation and the suppression of hepatic fibrogenesis.

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Figures

Figure 1

Curcumin, with different treatment strategies, showed different efficiencies in the inhibition of PDGF- or EGF-stimulated HSC proliferation. Cultured HSCs were divided into three groups. In the first two groups, HSCs were serum-starved in DMEM with 0.5% of FBS for 24 h prior to addition of PDGF (a) or EGF (b) at the indicated doses in the simultaneous presence (Cur + PDGF, or Cur + EGF) or absence (PDGF or EGF) of curcumin (20 μ_M) for an additional 24 h. In the third group, HSCs were pretreated with curcumin (20 μ_M) for 24 h in DMEM with 0.5% of FBS prior to the addition of PDGF (a) or EGF (b) at the indicated doses for an additional 24 h (Cur/PDGF, or Cur/EGF). Cell proliferation was determined by colorimetric MTS assays. Results were expressed as fold changes in the number of viable cells, compared with the untreated control (the first columns on the left). Values were expressed as means ± s.d. (n = 3). *P < 0.05 vs the untreated control (the first column on the left). §_P < 0.05 vs cells treated with PDGF or EGF only (the left first column in the same group). ‡_P < 0.05 vs cells simultaneously treated with curcumin plus PDGF or EGF (Cur + PDGF, or Cur + EGF) (the middle column in the same group).

Figure 2

Curcumin dose dependently suppressed gene expression of PDGF-β_R and EGFR in cultured HSCs, which might require activation of PPAR_γ. Cultured HSCs in DMEM with 10% of FBS were pretreated with or without the PD68235 (20 μ_M), a specific PPAR_γ antagonist, for 1 h prior to the addition of curcumin at the indicated concentrations for an additional 24 h. (a) Luciferase assays of cells transfected with pPDGF-_β_R-Luc, or pEGFR-Luc. Luciferase activities were expressed as relative units after _β_-galactosidase normalization (n = 6). *P < 0.05 vs the untreated control (the corresponding first column on the left). The floating schema denoted the pPDGF-_β_R-Luc, or pEGFR-Luc, luciferase reporter construct in use and the application of curcumin to the system; (b) real-time PCR analyses of the mRNA levels of PDGF-β_R or EGFR. GAPDH was used as an invariant control for calculating fold changes of target mRNA (n = 3). *P < 0.05 vs the untreated control (the corresponding first column on the left); ‡_P < 0.05 vs cells treated with curcumin at 20 _μ_M; (c) Western blotting analyses of the protein abundance of PDGF-_β_R or EGFR. _β_-Actin was used as an invariant control for equal loading. Representative result is shown from three independent experiments.

Figure 3

Forced expression of PPAR_γ_ reduced the promoter activity of PDGF-_β_R and EGFR genes in passaged HSCs. HSCs in six-well culture plates in DMEM with 10% of FBS were co-transfected with a total of 4.5 _μ_g of a DNA mixture per well, including 2 _μ_g of pPDGF-_β_R-Luc or pEGFR-Luc, 0.5 _μ_g of pSV-_β_-gal and 2 _μ_g of pPPAR_γ_cDNA at various doses plus the empty vector pcDNA. The latter was used to ensure equal amount of total DNA in transfection assays. After recovery, cells were treated with or without curcumin (20 _μ_M) in media containing FBS (10%) for 24 h. Luciferase activities were expressed as relative units after _β_-galactosidase normalization (n = 6). *P < 0.05 vs cells with no treatment (the first columns on the left). The floating schema denoted the pPDGF-_β_R-Luc, or pEGFR-Luc, luciferase reporter construct in use and the application of the cDNA expressing plasmid pPPAR_γ_cDNA to the system.

Figure 4

The activation of PPAR_γ_ by PGJ2 suppressed pdgf-βr and egfr expression in activated HSCs in vitro. Cultured HSCs in DMEM with 10% of FBS were treated with curcumin (20 _μ_M) or PGJ2 at the indicated concentrations for 24 h. (a) Luciferase assays of cells transfected with pPDGF-_β_R-Luc, or pEGFR-Luc. Luciferase activities were expressed as relative units after _β_-galactosidase normalization (n = 6). *P < 0.05 vs the untreated control (the corresponding 1st column on the left); (b) real-time PCR analyses of the mRNA levels of PDGF-_β_R or EGFR. GAPDH was used as an invariant control for calculating fold changes of target mRNA (n = 3). *P < 0.05 vs the un-treated control (the corresponding 1st column on the left); (c) Western blotting analyses of the abundance of PDGF-_β_R or EGFR. _β_-Actin was used as an invariant control for equal loading. Representative result is shown from three independent experiments.

Figure 5

Curcumin reduced the phosphorylation levels of PDGF-_β_R, EGFR, and downstream inter-mediators EKR and JNK in activated HSC in vitro. Serum-starved HSCs in DMEM with 10% of FBS were pretreated with or without curcumin at 20 _μ_M for 30 min prior to stimulation with FBS (10%) for different lengths of time. Total protein extracts were prepared for detecting the levels of phosphorylated PDGF-_β_R, EGFR, ERK, and JNK by Western blotting analysis. Total PDGF-_β_R, EGFR, ERK, and JNK were used as the corresponding internal controls. Representative result is shown from three independent experiments.

Figure 6

The alteration in ERK or JNK activity changed the promoter activity of PDGF-_β_R and EGFR genes in activated HSCs in vitro. HSCs in six-well plates i n DMEM with 10% of FBS were co-tranfected with a fixed amount of a DNA mixture per well. It included 2 _μ_g of pPDGF-_β_R-Luc (a and b), or pEGFR-Luc (c and d), 0.5 _μ_g of pSV-_β_-gal and a plasmid expressing the active form of pa-ERK, or pa-JNK (a and c), or a plasmid expressing the dominant-negative form of pdn-ERK or pdn-JNK (b and d), plus the empty vector pcDNA. The latter was used to ensure equal amount of total DNA in transfection assays. The amount of DNA of pa-ERK, or pa-JNK, plus pcDNA was equalized to 0.7 _μ_g (a and c). The amount of DNA of pdn-ERK, or pdn-JNK, plus pcDNA was equalized to 2 _μ_g (b and d). After overnight recovery, cells were serum-starved in DMEM with 0.5% DMEM for 24 h prior to the stimulation with FBS (10%) in the presence or absence of curcumin (20 _μ_M) for an additional 24 h. Luciferase activities were expressed as relative units after β_-galactosidase normalization (n ≥ 6). *P < 0.05 vs cells with no treatment (the corresponding first column on the left). ‡_P < 0.05 vs cells with curcumin only, without co-transfected pa-cDNA (the corresponding third column on the left). The floating schema denotes the pPDGF-_β_R-Luc, or pEGFR-Luc, luciferase reporter construct in use and the application of a cDNA expressing plasmid to the system.

Figure 7

The inhibition of ERK or JNK activity reduced pdgf-βr and egfr expression in activated HSCs in vitro. Cultured HSCs in DMEM with 10% of FBS were treated with curcumin (20 _μ_M), or the ERK inhibitor PD98059 (a, c, and e), or the JNK inhibitor SP600125 (b, d, and f), at the indicated doses for 24 h. (a and b). Luciferase assays of cells transfected with pPDGF-_β_R-Luc, or pEGFR-Luc. Luciferase activities were expressed as relative units after _β_-galactosidase normalization (n = 6). *P < 0.05 vs the un-treated control (the corresponding first column on the left). The floating schema denotes the pPDGF-_β_R-Luc, or pEGFR-Luc, luciferase reporter construct in use and the application of a kinase inhibitor to the system; (c and d) real-time PCR analyses of the mRNA levels of PDGF-_β_R or EGFR. GAPDH was used as an invariant control for calculating fold changes of target mRNA (n = 3). *P < 0.05 vs the untreated control (the corresponding first column on the left); (e and f) Western blotting analyses of the abundance of PDGF-_β_R or EGFR. _β_-Actin was used as an invariant control for equal loading. Representative result is shown from three independent experiments.

Figure 8

The inhibition of de novo GSH synthesis eliminated the inhibitory effect of curcumin on pdgf-βr and egfr expression in activated HSCs in vitro. HSCs in DMEM with 10% of FBS were treated for 24 h with curcumin (20 μ_M) or NAC (5 mM) with or without the pre-exposure to BSO (0.25 mM) for 1 h. Values were means ± s.d. (n ≥ 3). *P < 0.05, vs cells with no treatment (the corresponding first column on the left); ‡_P < 0.05, vs cells treated with curcumin, or NAC, only (the corresponding second or the third column on the left). (a) Luciferase assays of cells transfected with pPDGF-_β_R-Luc, or pEGFR-Luc. Luciferase activities were expressed as relative units after _β_-galactosidase normalization (n = 6). The floating schema denotes the pPDGF-_β_R-Luc, or pEGFR-Luc, luciferase reporter construct in use and the application of NAC or curcumin to the system; (b) real-time PCR analyses of the mRNA levels of PDGF-_β_R, or EGFR. GAPDH was used as an invariant control for calculating fold changes of target mRNA (n = 3); (c) Western blotting analyses of the abundance of PDGF-_β_R, or EGFR. _β_-Actin was used as an invariant control for equal loading. Representative result is shown from three independent experiments.

Figure 9

The activation of PPAR_γ_ induced gclc and gclm expression in activated HSCs in vitro. (a) Cultured HSCs in DMEM with 10% of FBS were pretreated with or without the PD68235 (20 μ_M), a specific PPAR_γ antagonist, for 1 h prior to the addition of curcumin at the indicated concentrations for an additional 24 h. Western blotting analyses of GCLc and GCLm. _β_-Actin was used as an invariant control for equal loading. Representative was shown from three independent experiments. (b and c) Cultured HSCs in DMEM with 10% of FBS were treated with curcumin (20 _μ_M) or PGJ2 at the indicated concentrations for 24 h. (b) Real-time PCR analyses of the mRNA levels of GCLc and GCLm. GAPDH was used as an invariant control for calculating fold changes of target mRNA (n = 3). *P < 0.05 vs the untreated control (the corresponding first column on the left); (c) Western blotting analyses of the abundance of GCLc and GCLm. _β_-Actin was used as an invariant control for equal loading. Representative result is shown from three independent experiments.

Figure 10

Schema of the underlying mechanism of curcumin in the interruption of PDGF and EGF signaling in activated HSC in vitro. Curcumin rapidly interrupts PDGF-β_R and EGFR signaling in activated HSCs by reducing the phosphorylation levels of PDGF-β_R and EGFR, as well as of their downstream signaling inter-mediators ERK and JNK. This instant action causally stimulates gene expression and its activity of PPAR_γ, which critically leads to attenuation of oxidative stress by induction of gene expression of GCL and elevation of the level of cellular GSH. The actions of curcumin, including activation of PPAR_γ and elevation of GSH contents, collectively result in suppression of pdgf-βr and egfr expression, leading to long-term interruption of PDGF-_β_R and EGFR signaling and inhibition of cell proliferation.

References

1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–218. - PMC - PubMed
1. Friedman SL. Stellate cells: a moving target in hepatic fibrogenesis. Hepatology. 2004;40:1041–1043. - PubMed
1. Wong L, Yamasaki G, Johnson RJ, et al. Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture. J Clin Invest. 1994;94:1563–1569. - PMC - PubMed
1. Bachem MG, Riess U, Gressner AM. Liver fat storing cell proliferation is stimulated by epidermal growth factor/transforming growth factor alpha and inhibited by transforming growth factor beta. Biochem Biophys Res Commun. 1989;162:708–714. - PubMed
1. Komuves LG, Feren A, Jones AL, et al. Expression of epidermal growth factor and its receptor in cirrhotic liver disease. J Histochem Cytochem. 2000;48:821–830. - PubMed

Activation of peroxisome proliferator-activated receptor-gamma by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells - PubMed (original) (raw)