Objective. To determine if integrin-mediated signaling results in activation of chondrocyte mitogenactivated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation.Methods. Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the ␣5␤1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay.Results. Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesionblocking antibodies to the ␣2␤1 and ␣5␤1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to ␣1, ␣3, and ␣V␤3 and a nonblocking ␣5 antibody had no effect.After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor B inhibitor was noted, followed by increased pro-and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-finduced activation of MMP-13.Conclusion. Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro-and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.Articular cartilage consists of an abundant extracellular matrix (ECM) composed chiefly of proteoglycans and type II collagen (CII) with lesser, but significant, amounts of other types of collagen and ECM proteins, including fibronectin (FN) (1,2). The chondrocyte is the only cell type present in articular cartilage and is therefore responsible for both synthesis and degradation of the cartilage ECM. The normal balance of cartilage ECM synthesis and degradation is disrupted in diseases such as osteoarthritis (OA) (2,3). Increased production of matrix metalloproteinases (MMPs), including collagenase 3 (MMP-13), has been noted in arthritic cartilage (4-7), but the exact driving forces responsible for the increase in MMPs are not known and were the subject of this study. Cytokines, in particular interleukin-1 (IL-1), may be important since IL-1 has been shown to stimulate chondrocyte MMP production

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.

Objective. Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors.Methods. Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzymelinked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis.Results. Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-Llysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the ␣5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the ␣5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 g/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of ␣v and ␣1 integrins or treatment with echistatin did not cause cell death. Addition of insulinlike growth factor 1 (IGF-1; 100 ng/ml) was not able to improve survival of ␣5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to ␣5 inhibition was associated with apoptosis.Conclusion. These results demonstrate that chondrocyte survival signals are transmitted via the ␣5␤1 FN receptor. Inhibition of matrix survival signals mediated by ␣5␤1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from ␣5␤1.Integrins are a large family of glycoprotein receptors that mediate cell-cell and cell-extracellular matrix adhesion (1,2). Integrins are heterodimeric transmembrane proteins consisting of an ␣ and a ␤ subunit. Both subunits have large extracellular domains that interact with extracellular matrix proteins and relatively small cytoplasmic domains that interact with cytoskeletal proteins. The extracellular matrix is an active, informationrich environment. It plays a pivotal role in regulating several biologic functions, such as cell growth, differentiation, adhesion, and migration. Signals from the extracellular matrix via the ␤1-subunit-containing integrins have been shown to play a role in chondrogenesis in vitro, although the exact function of integrins during chondrogenesis is not clear (3,4).In many cell types, the...