Faramaze Dehnade

Induction of matrix metalloproteinase-13 gene expression by TNF-α is mediated by MAP kinases, AP-1, and NF-κB transcription factors in articular chondrocytes

Abstract Tumor necrosis factor alpha (TNF-α), a major proinflammatory cytokine, induces arthritic joint inflammation and resorption of cartilage by matrix metalloproteinase-13 (MMP-13). RNA for MMP-13 is increased in human arthritic femoral cartilage. Mechanisms of this induction were investigated by pretreating primary human osteoarthritic (OA) femoral head chondrocytes or chondrosarcoma cells with the potential inhibitors of TNF-α signal transduction and downstream target transcription factors followed by stimulation with TNF-α and analysis of MMP-13 RNA/protein. TNF-α rapidly activated phosphorylation of extracellular signal-regulated kinases (ERKs), p38, and c-jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in human chondrocytes. Inhibitors of ERK (U0126, PD98059, and ERK1/2 antisense phosphorothioate oligonucleotide), JNK (SB203580, SP600125, and curcumin), and p38 (SB203580 and SB202190) pathways down-regulated the TNF-stimulated expression of MMP-13. Inhibitors of the transcription factors AP-1 (nordihydroguaiaretic acid, NDGA) and NF-κB (curcumin, proteasome inhibitors, and Bay-11-7085) suppressed TNF-α-induced MMP-13 expression in primary chondrocytes and SW1353 cells. These results suggest that induction of the MMP-13 gene by TNF-α is mediated by ERK, p38, and JNK MAP kinases as well as AP-1 and NF-κB transcription factors. Blockade of TNF-α signaling and its target transcription factors by the approaches tested here may be beneficial for reducing cartilage breakdown by MMP-13 in arthritis.

Oncostatin M-Induced Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase-3 Genes Expression in Chondrocytes Requires Janus Kinase/STAT Signaling Pathway

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.

Tissue inhibitor of metalloproteinases-4 (TIMP-4) gene expression is increased in human osteoarthritic femoral head cartilage

Tissue inhibitor of metalloproteinases‐4 (TIMP‐4), the newest member of the TIMP family, blocks the activities of several matrix metalloproteinases (MMPs) implicated in the arthritic cartilage erosion. By utilizing semi‐quantitative RT‐PCR, immunoblotting, and immunohistochemistry, we investigated whether the TIMP‐4 gene is expressed in human non‐arthritic and osteoarthritic (OA) cartilage. Directly analyzed femoral head cartilage showed TIMP‐4 RNA expression in 2 of 9 non‐arthritic and 12 of 14 OA patients. Femoral head cartilage from 6 of 9 OA patients had elevated TIMP‐4 protein compared to the low‐level expression in 3 of 8 non‐arthritic controls. In most patients, there was correlation between TIMP‐4 RNA and protein expression. TIMP‐4 protein was also detected immunohistochemically in the upper zone of OA cartilage. The widespread TIMP‐4 RNA and protein expression and augmentation in femoral OA cartilage suggests its important role in joint tissue remodeling and pathogenesis of OA. Increased TIMP levels in arthritic cartilage may not be a sufficiently effective defense against cartilage resorption by excessive multiple MMPs and aggrecanases. J. Cell. Biochem. 85: 295–303, 2002.

Enhanced Expression of Tissue Inhibitor of Metalloproteinases-4 Gene in Human Osteoarthritic Synovial Membranes and Its Differential Regulation by Cytokines in Chondrocytes

Objective: Tissue inhibitors of metalloproteinases (TIMPs) are multi-functional proteins with matrix metalloproteinases-inhibiting activities. We studied expression of anti-inflammatory, TIMP-4 gene in human joint tissues and its regulation by arthritis-associated cytokines. Results: TIMP-4 RNA expression originating from synovial fibroblasts was significantly (2.4 fold; p<0.001) elevated in 8 osteoarthritic (OA) versus 7 non-arthritic synovial membranes. Non-arthritic and OA femoral head and knee chondrocytes displayed substantial but variably constitutive expression of the TIMP-4 mRNA. In articular chondrocytes, transforming growth factor beta (TGF-β1) and oncostatin M (OSM) upregulated TIMP-4 RNA and protein expression while interleukin-1 (IL-1β) and tumor necrosis factor alpha (TNF-α) did not, suggesting differential regulation by arthritis-associated cytokines. Interleukin 17 (IL-17) mildly induced TIMP-4 mRNA. TGF-β1 induction of TIMP-4 expression was partly inhibited by ERK pathway and Sp1 transcription factor inhibitors. Conclusion: Enhanced TIMP-4 gene expression in OA synovial membranes and cartilage may be due to induction by TGF-β1, OSM and IL-17, suggesting its pathophysiological role in tissue remodeling in human joints. TGF-β1 induction of TIMP-4 expression is mediated partly by ERK pathway and Sp1 transcription factor.

Testing the eukaryotic promoters for efficient expression of exogenous genes in chondrocytes and synoviocytes

To identify suitable promoters for expressing exogenous genes in arthritic joints, the constitutive, simian virus 40 (SV 40) and IL‐1 or metal inducible, human stromelysin and metallothionein (MT) gene promoters were tested for their activity in chondrocytes and synovial fibroblasts. Transient transfection with plasmids containing the reporter chloramphenicol acetyltransferase (CAT) gene attached to these promoters showed that SV40, stromelysin and MT promoters drove CAT expression with different strengths in primary bovine chondrocytes. The MTI‐F and MT‐IG gene promoters were also functional in human chondrocytes. The SV40, IL‐1 inducible stromelysin‐1 and MT‐IG driven CAT activity was also detectable in human synoviocytes. Therefore, chondrocytes and synoviocytes have the trans‐acting factors necessary for transcription from the respective promoters which may be conserved in bovine and human cells. These promoters could be useful for expressing potentially therapeutic anti‐inflammatory and anti‐erosive genes in arthritic joints.