Abstract
IntroductionPeroxisome proliferator-activated receptor (PPAR)γ has been shown to exhibit anti-inflammatory and anti-catabolic properties and to be protective in animal models of osteoarthritis (OA). We have previously shown that interleukin-1β (IL-1) down-regulates PPARγ expression in human OA chondrocytes. However, the mechanisms underlying this effect have not been well characterized. The PPARγ promoter harbors an overlapping Egr-1/specificity protein 1 (Sp1) binding site. In this study, our objective was to define the roles of Egr-1 and Sp1 in IL-1-mediated down-regulation of PPARγ expression.MethodsChondrocytes were stimulated with IL-1 and the expression levels of Egr-1 and Sp1 mRNAs and proteins were evaluated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The role of de novo protein synthesis was evaluated using the protein synthesis inhibitor cycloheximide (CHX). The recruitment of Sp1 and Egr-1 to the PPARγ promoter was evaluated using chromatin immunoprecipitation (ChIP) assays. The PPARγ promoter activity was analyzed in transient transfection experiments. The roles of Egr-1 and Sp1 were further evaluated using small interfering RNA (siRNA) approaches. The level of Egr-1 in cartilage was determined using immunohistochemistry.ResultsDown-regulation of PPARγ expression by IL-1 requires de novo protein synthesis and was concomitant with the induction of the transcription factor Egr-1. Treatment with IL-1 induced Egr-1 recruitment and reduced Sp1 occupancy at the PPARγ promoter. Overexpression of Egr-1 potentiated, whereas overexpression of Sp1 alleviated, the suppressive effect of IL-1 on the PPARγ promoter, suggesting that Egr-1 may mediate the suppressive effect of IL-1. Consistently, Egr-1 silencing prevented IL-1-mediated down-regulation of PPARγ expression. We also showed that the level of Egr-1 expression was elevated in OA cartilage compared to normal cartilage.ConclusionsOur results indicate that induction and recruitment of Egr-1 contributed to the suppressive effect of IL-1 on PPARγ expression. They also suggest that modulation of Egr-1 levels in the joint may have therapeutic potential in OA.
Highlights
Peroxisome proliferator-activated receptor (PPAR)g has been shown to exhibit anti-inflammatory and anti-catabolic properties and to be protective in animal models of osteoarthritis (OA)
We showed that the level of early growth response gene 1 (Egr-1) expression was elevated in OA cartilage compared to normal cartilage
Our results indicate that induction and recruitment of Egr-1 contributed to the suppressive effect of IL-1 on PPARg expression
Summary
Peroxisome proliferator-activated receptor (PPAR)g has been shown to exhibit anti-inflammatory and anti-catabolic properties and to be protective in animal models of osteoarthritis (OA). Our objective was to define the roles of Egr-1 and Sp1 in IL-1mediated down-regulation of PPARg expression. OA is characterized by progressive degeneration of articular cartilage, synovial inflammation, and subchondral bone remodeling. It is characterized by increased levels of inflammatory mediators, among which interleukin 1 (IL-1) is considered a key player in the initiation and progression of the disease [1]. The mechanisms through which IL-1 exerts its effects include increased expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase 1 (mPGES-1), and the release of nitric oxide (NO) and prostaglandin E2 (PGE2) [1]. IL-1 promotes cartilage degradation by suppressing the synthesis of the major components of extracellular matrix proteoglycan and collagen and by enhancing the production of matrix metalloproteinases (MMPs) and aggrecanases [1]
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