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 connective 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 and induce neovascularization in vivo. Encoded by inducible immediate early genes, Cyr61 and CTGF are synthesized upon growth factor stimulation in cultured fibroblasts and during cutaneous wound healing in dermal fibroblasts. Recently, we have shown that adhesion of primary human fibroblasts to immobilized Cyr61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans (HSPGs) (Chen, N., Chen, C.-C., and Lau, L.F. (2000) J. Biol. Chem. 275, 24953-24961), providing the first demonstration of an absolute 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 biological activities. Compared with fibroblast adhesion to fibronectin, laminin, or type I collagen, cell adhesion to Cyr61 or CTGF induces 1) more extensive and prolonged formation of filopodia and lamellipodia, concomitant with formation of integrin alpha(6)beta(1)-containing focal complexes localized at leading edges of pseudopods; 2) activation of intracellular signaling molecules including focal adhesion kinase, paxillin, and Rac with similar rapid kinetics; 3) sustained activation of p42/p44 MAPKs lasting for at least 9 h; and 4) prolonged gene expression changes including up-regulation of MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) mRNAs and proteins sustained for at least 24 h. Together, these results establish Cyr61 and CTGF as bona fide adhesive substrates with specific signaling capabilities, provide a molecular basis for their activities in fibroblasts through integrin alpha(6)beta(1) and HSPG-mediated signaling during attachment and indicate that these proteins may function in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing.
Chen
C C
CC
Department of Molecular Genetics, University of Illinois at Chicago College of Medicine, 60607-7170, USA.
Chen
N
N
Lau
L F
LF
D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D055515:Cysteine-Rich Protein 61; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006133:Growth Substances / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism* / Q000502:physiology*; D039121:Integrin alpha6beta1; D016023:Integrins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D007797:Laminin / Q000378:metabolism; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation; D014945:Wound Healing / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000378:metabolism
C501848:CCN1 protein, human; C526317:CCN2 protein, human; D055515:Cysteine-Rich Protein 61; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D006133:Growth Substances; D017874:Immediate-Early Proteins; D039121:Integrin alpha6beta1; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor; D012313:RNA; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; D020744:rac GTP-Binding Proteins