Peroxisome proliferator-activated receptor gamma1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1beta in articular chondrocytes

Abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARgamma activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARgamma expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARgamma in normal and OA cartilage and to evaluate the effect of IL-1beta, a prominent cytokine in OA, on PPARgamma expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARgamma protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARgamma1 mRNA levels were about 10-fold higher than PPARgamma2 mRNA levels, and that only PPARgamma1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARgamma1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARgamma1 mRNA expression and PPARgamma1 promoter activity. TNF-alpha, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARgamma1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARgamma1 expression. Similarly, inhibitors of NF-kappaB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARgamma1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-kappaB signaling pathways. The IL-1-induced downregulation of PPARgamma expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation.

Figure 1

Expression of PPARγ protein in normal and osteoarthritis cartilage. Representative immunostaining of human normal cartilage (a) and cartilage from mild to moderate osteoarthritis (OA) (b) for peroxisome proliferator-activated receptor γ (PPARγ). (c) Normal specimens treated with anti-PPARγ antibody that was preadsorbed with a 20-fold molar excess of the protein fragment corresponding to amino acids 8 to 106 of human PPARγ (control for staining specificity). (d) Percentage of chondrocytes expressing PPARγ in normal and OA cartilage. The results are means ± SEM for 10 normal and 11 OA specimens. *p < 0.05 compared with normal cartilage.

Figure 2

PPARγ1 and PPARγ2 mRNA levels in normal and osteoarthritis human cartilage. RNA was extracted from normal (n = 7) and osteoarthritis (n = 8) cartilage, reverse transcribed into cDNA, and processed for real-time PCR. The threshold cycle values were converted to the number of molecules, as described in the Materials and methods section. Data were expressed as copies of the gene's mRNA detected per 1,000 glyceraldehyde-3-phosphate dehydrogenase copies. *p < 0.05 compared with normal samples. PPAR, peroxisome proliferator-activated receptor.

Figure 3

Effect of IL-1 on PPARγ1 protein expression in osteoarthritis chondrocytes. (a) Osteoarthritis (OA) chondrocytes were treated with 100 pg/ml IL-1 for the indicated periods. (b) OA chondrocytes were treated with increasing concentrations of IL-1 for 24 hours. Cell lysates were prepared and analyzed for peroxisome proliferator-activated receptor γ1 (PPARγ1) protein by Western blotting (upper panels). The blots were stripped and reprobed with a specific anti-β-actin antibody (lower panels). The blots are representative of similar results obtained from four independent experiments.

Figure 4

Effect of TNF-α, IL-17 and prostaglandin E₂ on PPARγ1 protein expression in osteoarthritis chondrocytes. Cells were treated with IL-1 (100 pg/ml), TNF-α (1 and 10 ng/ml), IL-17 (10 and 100 ng/ml), and prostaglandin E₂ (0.1 and 1 μM). After 24 hours, cell lysates were prepared and analyzed for peroxisome proliferator-activated receptor γ1 (PPARγ1) protein expression by Western blotting. In the lower panel, the blots were stripped and reprobed with a specific anti-β-actin antibody. The blots are representative of similar results obtained from four independent experiments.

Figure 5

IL-1 downregulates PPARγ1 expression at the transcriptional level. (a) Osteoarthritis (OA) chondrocytes were treated with increasing concentrations of IL-1 for 12 hours. Total RNA was isolated and reverse transcribed into cDNA, and peroxisome proliferator-activated receptor γ1 (PPARγ1) and glyceraldehyde-3-phosphate dehydrogenase mRNAs were quantified by real-time PCR. All experiments were performed in triplicate, and negative controls without template RNA were included in each experiment. (b) OA chondrocytes were co-transfected with 1 μg per well of the PPARγ1 promoter (pGL3-PPARγ1p3000) and 0.5 μg per well of the internal control pSV40-β-galactosidase, using FuGene 6 transfection reagent. The next day, transfected cells were treated with increasing concentrations of IL-1 for 18 hours. Luciferase activity values were determined and normalized to β-galactosidase activity. Results are expressed as percentage changes, taking the value of untreated cells as 100%, and show means ± SEM for four independent experiments. *p < 0.05 compared with untreated cells.

Figure 6

Effect of mitogen-activated protein kinase and NF-κB inhibitors on IL-1-induced downregulation of PPARγ1 expression. (a) Osteoarthritis (OA) chondrocytes were exposed to increasing concentrations of SB203580 (p38 mitogen-activated protein kinase inhibitor), SP600125 (c-Jun N-terminal kinase inhibitor) and PD98059 (extracellular signal-regulated kinase inhibitor) for 30 minutes before treatment with or without IL-1 (100 pg/ml). (b) OA chondrocytes were exposed to increasing concentrations of various inhibitors of NF-κB (pyrrolidine dithiocarbamate, MG-132, and SN-50) for 30 minutes before stimulation with or without IL-1 (100 pg/ml). After 24 hours, cell lysates were prepared and analyzed for peroxisome proliferator-activated receptor γ1 (PPARγ1) protein expression by Western blotting. In the lower panels, the blots were stripped and reprobed with a specific anti-β-actin antibody. The blots are representative of similar results obtained from four independent experiments.