Physiological and pathological degradation of cartilage extracellular matrix (ECM) is regulated by the balance between tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). We examined the potential of chondrocytes from normal bovine or human osteoarthritic (OA) cartilage to express RNA for the new inhibitor TIMP-3 and studied its regulation by an inducer of matrix synthesis, transforming growth factor-beta (TGF-beta). Freshly released chondrocytes constitutively expressed three transcripts of TIMP-3 that are induced by serum factors. In primary cultures of chondrocytes, one of these factors, TGF-beta, increased TIMP-3 mRNA in a dose-dependent fashion that required de novo protein synthesis and transcription. TGF-beta did not alter stability of the TIMP-3 transcripts in RNA decay time-courses, suggesting a transcriptional control. Nuclear run-on assays confirmed increased rate of TIMP-3 gene transcription by TGF-beta. An antiinflammatory glucocorticoid, dexamethasone, inhibited the basal, and suppressed partially the TGF-beta-inducible, TIMP-3 expression in primary bovine and human chondrocytes. DNA sequencing of bovine TIMP-3 cDNA revealed an open reading frame of a 211-amino-acid protein containing signal peptide and 12 conserved cysteines. The encoded protein differed from human TIMP-3 at four positions. The constitutive expression and evolutionary conservation of TIMP-3 imply its important function. TIMP-3 induction by TGF-beta suggests the role of this factor and TIMP-3 in cartilage remodeling with important implications for arthritis.

Tissue inhibitors of metalloproteinases (TIMPs) inhibit the extracellular matrix (ECM) metalloproteinases (MMPs). To determine the source of TIMPs in synovial fluids of patients with osteoarthritis (OA), the ability of chondrocytes to express TIMP-2 and its regulation by agents found in inflammed joints was investigated. The constitutive TIMP-2 mRNA expression was demonstrated in chondrocytes from normal bovine, human OA and normal cartilage. The cross-hybridization of human and bovine TIMP-2 suggested its evolutionary conservation. Serum, IL-1, IL-6 and TGC-beta were unable to augment considerably the basal expression of TIMP-2 mRNA. TIMP-1 RNA expression in chondrocytes from human OA cartilage was elevated compared to non-OA chondrocytes, while TIMP-2 mRNA levels were similar in both. IL-1 beta, IL-6 and TGF-beta did not affect TIMP-2 expression but TGF-beta induced TIMP-1 mRNA in human OA chondrocytes. TIMP-2 and TIMP-1 are therefore differentially regulated in chondrocytes and the basal TIMP-2 levels may be needed for the cartilage ECM integrity.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-beta), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-beta-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-beta. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-beta induction of TIMP-3. H7 and genistein also suppressed TGF-beta-induced TIMP-3 protein expression. These results suggest that TGF-beta signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-beta), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-beta-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-beta. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-beta induction of TIMP-3. H7 and genistein also suppressed TGF-beta-induced TIMP-3 protein expression. These results suggest that TGF-beta signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases.