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      Peroxisome proliferator-activated receptor-gamma down-regulates chondrocyte matrix metalloproteinase-1 via a novel composite element.

      The Journal of Biological Chemistry
      Animals, Binding Sites, Blotting, Northern, Blotting, Western, Cartilage, metabolism, Cell Nucleus, Cells, Cultured, Chondrocytes, Cloning, Molecular, DNA, Complementary, Dose-Response Relationship, Drug, Down-Regulation, Fibrinolytic Agents, pharmacology, Genes, Dominant, Humans, Immunohistochemistry, Interleukin-1, Kinetics, Ligands, Luciferases, Matrix Metalloproteinase 1, Mutagenesis, Site-Directed, Mutation, NF-kappa B, Promoter Regions, Genetic, Protein Binding, Proteoglycans, RNA, RNA, Messenger, Rabbits, Receptors, Cytoplasmic and Nuclear, Reverse Transcriptase Polymerase Chain Reaction, Sulfates, Thiazolidinediones, Time Factors, Transcription Factor AP-1, Transcription Factors, Transcription, Genetic, Transfection

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          Abstract

          Interleukin-1beta (IL-1beta) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-gamma isotype on IL-1beta-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-gamma in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1beta effects in chondrocytes. Low Rtz concentrations (close to K(d) values for PPAR-gamma, 0.1 to 1 microm) inhibited the effects of IL-1beta on (35)S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1beta-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-gamma enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-gamma abolished it, supporting the role of PPAR-gamma in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-gamma and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-gamma-dependent inhibitory mechanism on IL-1beta-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter.

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