TGF-β-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor

Transforming growth factor (TGF-beta 1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metal loproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metal loproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 transcription factor in TGF-beta-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-beta-time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U01 26, downregulated TGF-beta-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Spl -binding sites, we investigated whether Spl is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Spl to its consensus site, downregulated TGF-beta-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-beta-stimulated Spl binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (-940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-beta-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Spl transcription factor play a pivotal role in the induction of TIMP-3 by TGF-beta in chondrocytes. J. Cell. Physiol. 203: 345-352, 2005. (c) 2004 Wiley-Liss, Inc.