Regulation of tissue inhibitor of metalloproteinases-3 gene by growth factors and antioxidants in connective tissue cells

The matrix metalloproteinases (MMPs) play a key role in the physiological and pathological degradation of cartilage extracellular matrix (ECM). The tissue inhibitors of metalloproteinases (TIMPs) are specific endogenous inhibitors of the MMPs.; 1. Transforming growth factor (TGF-β), a major stimulant of cartilage ECM synthesis and osteophyte formation in arthritic joints, induces tissue inhibitor of metalloproteinases-3 (TIMP-3) mRNA and protein in articular chondrocytes. N-acetylcysteine (NAC) and related thiol antioxidants inhibited TGF-β-upregulation of TIMP-3. TGF-β rapidly stimulated Smad2 phosphorylation in bovine and human chondrocytes that was inhibited by NAC and other thiols. NAC also abolished intracellular reactive oxygen species (ROS) production triggered by TGF-β Our findings suggest that thiols inhibit TGF-β induction of TIMP-3 partly by upstream suppression of the key Smad2 phosphorylation step in the Smad signaling pathway.; 2. NAC and the related thiol antioxidants have also been reported to have the ability to promote chondrocyte survival in culture. Mechanisms of this action of NAC were studied in primary human and bovine chondrocytes. NAC dose-dependently activated phosphorylation of the extracellular signal regulated kinases (ERKs). This mitogen-activated protein kinase (MAPK) pathway is generally activated by growth factors. The activation was inhibited by the mitogen activated protein kinase kinase (MAPKK) inhibitor, PD98059. The induction was mimicked by other thiols, L-cysteine, reduced glutathione and pyrrolidine dithiocarbamate (PDTC) but not by non-thiol, N-acetylalanine.; 3. Oncostatin M (OSM) is a cytokine of the interleukin-6 (IL-6) family whose level is increased in the serum and synovial fluids of patients with rheumatoid arthritis. We found that OSM induced TIMP-3, collagenase-1 (MMP-1), collagenase-3 (MMP-13), and stromelysin-1 (MMP-3) in articular chondrocytes. Investigation of the induction mechanisms revealed that tyrosine kinases and MAPK cascades might be involved in OSM signaling leading to TIMP-3 and MMPs expression. We showed that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, 2, 3 and signal transducer and activator of transcription 1 (STAT1), as well as ERKs, protein 38 (p38) and c-Jun N-terminal kinase (JNK) in primary cultures of bovine and human chondrocytes. A specific JAK3 inhibitor blocked OSM-mediated STAT1 tyrosine phosphorylation, DNA binding activity of STAT1 as well as TIMP-3, MMP-1, MMP-3, and MMP-13 RNA expression. (Abstract shortened by UMI.)...