Although there have been major advances in the treatment of rheumatoid arthritis with the advent of biological agents, the mechanisms that drive cytokine production and sustain disease chronicity remain unknown. Tenascin-C (encoded by Tnc) is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. Here we show that mice that do not express tenascin-C show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of tenascin-C promotes joint inflammation in vivo in mice, and addition of exogenous tenascin-C induces cytokine synthesis in explant cultures from inflamed synovia of individuals with rheumatoid arthritis. Moreover, in human macrophages and fibroblasts from synovia of individuals with rheumatoid arthritis, tenascin-C induces synthesis of proinflammatory cytokines via activation of Toll-like receptor 4 (TLR4). Thus, we have identified tenascin-C as a novel endogenous activator of TLR4-mediated immunity that mediates persistent synovial inflammation and tissue destruction in arthritic joint disease.

The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K i values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.Tissue inhibitors of matrix metalloproteinases (TIMPs) 1 are important regulators of matrix metalloproteinases (MMPs) that participate in the degradation of the extracellular matrix (1). To date, four isoforms of TIMP have been identified in humans that are designated TIMP-1, -2, -3, and -4 (2); these are homologous in sequence and have similar secondary and tertiary structures including six well conserved disulfide bonds. Structural and functional studies of TIMP-1 and TIMP-2 (3-6) have shown that the full inhibitory activity of TIMPs resides in the N-terminal domain that is stabilized by three disulfide bonds. Inhibition studies with recombinant TIMPs have shown that each TIMP binds to MMPs with varying degrees of affinity, implicating that they have distinct functions in vivo (2, 7).TIMP-3 was originally discovered as a transformation-induced protein in chicken fibroblasts (8), which was later shown to have inhibitory activity against MMPs (9). In addition to its function as an inhibitor of MMPs, TIMP-3 has been reported to inhibit the shedding of cell surface-anchored molecules such as tumor necrosis factor-␣ receptor (10), L-selectin (11), interleukin 6 receptor (12), and syndecans-1 and -4 (13). The release of these molecules is thought to be catalyzed by membrane-bound ADAMs (a disintegrin and a metalloproteinase domain), multidomain proteins containing an N-terminal propeptide, a metalloproteinase, a disintegrin-like, a transmembrane, and a cytoplasmic domain. The primary structures of the metalloproteinase domains of the MMPs and the ADAMs have little sequence similarity except near the catalytic Zn 2ϩ -binding motif, HEXXHXXGXXH (14). Direct evidence for the apparently unique ability of TIMP-3 to inhibit a broad spectrum of metalloproteinases is provided by the demonstration of its inhibitory actio...

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu 373 -Ala 374 bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu 1480 -Gly 1481 bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the Cterminal spacer domain deletion mutant more effectively hydrolyzed both the Glu 373 -Ala 374 and Glu 1480 -Gly 1481 bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein.Finding of ADAMTS-4 in the interleukin-1␣-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNFrelated apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-a, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.