The angiogenic factors Cyr61 and connective tissue growth factor induce adhesive signaling in primary human skin fibroblasts

The angiogenic inducers cysteine-rich angiogenic protein 61 (Cyr61) and con nective tissue growth factor (CTGF) are structurally related, extracellular matrix-associated heparin-binding proteins. Both can stimulate chemotaxis and promote proliferation in endothelial cells and fibroblasts in culture a nd induce neovascularization in vivo. Encoded by inducible immediate early genes, Cyr61 and CTGF are synthesized upon growth factor stimulation in cul tured fibroblasts and during cutaneous wound healing in dermal fibroblasts, Recently, we have shown that adhesion of primary human fibroblasts to immo bilized Cyr61 is mediated through integrin cr,P, and cell surface heparan s ulfate proteoglycans (HSPGs) (Chen, N,, Chen, C,-C,, and Lau, L,F, (2000) J , Biol, Chem, 275, 24953-24961), providing the first demonstration of an ab solute requirement for HSPGs in integrin-mediated cell attachment. We show in this study that CTGF also mediates fibroblast adhesion through the same mechanism and demonstrate that fibroblasts adhesion to immobilized Cyr61 or CTGF induces distinct adhesive signaling responses consistent with their b iological activities. Compared with fibroblast adhesion to fibronectin, lam inin, or type I collagen, cell adhesion to Cyr61 or CTGF induces 1) more ex tensive and prolonged formation of filopodia and lamellipodia, concomitant with formation of integrin alpha (6)beta (1)-containing focal complexes loc alized at leading edges of pseudopods; 2) activation of intracellular signa ling molecules including focal adhesion kinase, paxillin, and Rac with simi lar rapid kinetics; 3) sustained activation of p42/p44 MAPKs lasting for at least 9 h; and 4) prolonged gene expression changes including up-regulatio n of MMP-1 (collagenase-l) and MMP-3 (stromelysin-l) mRNAs and proteins sus tained for at least 24 h, Together, these results establish Cyr61 and CTGF as bona fide adhesive substrates with specific signaling capabilities, prov ide a molecular basis for their activities in fibroblasts through integrin alpha (6)beta (1) and HSPG-mediated signaling during attachment and indicat e that these proteins may function in matrix remodeling through the activat ion of metalloproteinases during angiogenesis and wound healing.