OUTSIDE-IN SIGNALING IN THE CHONDROCYTE - NITRIC-OXIDE DISRUPTS FIBRONECTIN-INDUCED ASSEMBLY OF A SUBPLASMALEMMAL ACTIN RHO-A FOCAL ADHESION KINASE SIGNALING COMPLEX

Elevated levels of fibronectin (Fn) in articular cartilage have been l inked to the progression of both rheumatoid and osteoarthritis. In thi s study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha 5 beta 1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation, Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clust ering of the alpha 5 beta 1 integrin receptor, which was accompanied b y the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines whi ch induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the tot al number of beads bound per cell nor the clustering of alpha 5 beta 1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (S p-cGMPS) either abolished or mimicked the NO effect, respectively, Adh erence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs, albumin), In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimula tory signal transduced by alpha 5 beta 1 and the FGF receptor, Both co nstitutive and FGF stimulated proteoglycan synthesis were completely i nhibited by nitric oxide, These data indicate that the ligation of alp ha 5 beta 1 in the chondrocyte induced the intracellular assembly of a n activation complex comprised of the cytoplasmic tail of alpha 5 beta 1 integrin, actin, and the signaling molecules rho A and FAK, We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracel lular aggregation of alpha 5 beta 1 integrin, These observations provi de a basis by which nitric oxide can interfere with chondrocyte functi ons by affecting chondrocyte-matrix interactions.