Effects of CTGF/Hcs24, a Product of a Hypertrophic Chondrocyte-Specific Gene, on the Proliferation and Differentiation of Chondrocytes in Culture1

Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wildtype mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis.

Section: Discussioncontrasting

confidence: 48%

Section: Discussionmentioning

confidence: 99%

Focal Adhesion Kinase/Src Suppresses Early Chondrogenesis

Pala¹,

Kapoor

Woods³

et al. 2008

“…Purified Cyr61 and CTGF mediate cell adhesion, stimulate cell migration, and augment growth factor-induced DNA synthesis (10 -12). Cyr61 and CTGF can promote chondrogenic differentiation, consistent with their expression in prechondrogenic mesenchyme during embryogenesis (13)(14)(15). Both Cyr61 and CTGF stimulate chemotaxis in endothelial cells through an integrin ␣ V ␤ 3 -dependent pathway and induce neovascularization in vivo (16,17).…”

mentioning

confidence: 72%

The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts

Chen

Lau

2001

289

279

The angiogenic inducers cysteine-rich angiogenic protein 61 (Cyr61) and connective tissue growth factor (CTGF) are structurally related, extracellular matrixassociated 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 ␣ 6 ␤ 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 ␣ 6 ␤ 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 ␣ 6 ␤ 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.

“…Full-length CTGF is ϳ36 kDa, but cleaved forms between 10 and 20 kDa have been observed in serum-stimulated mouse fibroblast cultures and physiological fluids (37). It promotes proliferation and differentiation of chondrocytes in culture (38) and has been detected in mouse fracture callus (39). Since CTGF is induced by TGF␤ (40), it may conceivably act downstream of activin.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Articular Cartilage Shows Increased Type II Collagen Synthesis in Osteoarthritis and Expression of Inhibin βA (Activin A), a Regulatory Molecule for Chondrocytes

Hermansson

Sawaji

Bolton

et al. 2004

156

141

We show that proteomic analysis can be applied to study cartilage pathophysiology. Proteins secreted by articular cartilage were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Cartilage explants were cultured in medium containing [ 35 S]methionine/cysteine to radiolabel newly synthesized proteins. To resolve the cartilage proteins by two-dimensional electrophoresis, it was necessary to remove the proteoglycan aggrecan by precipitation with cetylpyridinium chloride. 50 -100 radiolabeled protein spots were detected on two-dimensional gels of human cartilage cultures. Of 170 silverstained proteins identified, 19 were radiolabeled, representing newly synthesized gene products. Most of these were known cartilage constituents. Several nonradiolabeled cartilage proteins were also detected. The secreted protein pattern of explants from 12 osteoarthritic joints (knee, hip, and shoulder) and 14 nonosteoarthritic adult joints were compared. The synthesis of type II collagen was strongly up-regulated in osteoarthritic cartilage. Normal adult cartilage synthesized little or no type II collagen in contrast to infant and juvenile cartilage. Potential regulatory molecules novel to cartilage were identified; pro-inhibin ␤A and processed inhibin ␤A (which dimerizes to activin A) were produced by all the osteoarthritic samples and half of the normals. Connective tissue growth factor and cytokine-like protein C17 (previously only identified as an mRNA) were also found. Activin induced the tissue inhibitor for metalloproteinases-1 in human chondrocytes. Its expression was induced in isolated chondrocytes by growth factors or interleukin-1. We conclude that type II collagen synthesis in articular cartilage is down-regulated at skeletal maturity and reactivated in osteoarthritis in attempted repair and that activin A may be an anabolic factor in cartilage. Osteoarthritis (OA)1 is a common joint disease characterized by degeneration of articular cartilage. Since cartilage has very limited capacity for repair, the loss is effectively irreversible. Prevalence studies show that most people over the age of 65 have some evidence of the disease (1, 2). Little is known about the molecular mechanism of cartilage destruction in OA, particularly the early events. It is thought that there is an imbalance between anabolism and catabolism of the extracellular matrix, there being an increase in catabolism. It has been suggested that this increased breakdown of matrix is due to the production of degradative enzymes such as the matrix metalloproteinases (MMPs) and members of the disintegrin and metalloproteinase (ADAM) family (3, 4). The increase in proteinase expression may be due to inflammatory cytokines such as interleukin-1 (Il-1) and tumor necrosis factor (4, 5). However, it is unclear whether these degradative processes are a primary event or a secondary reaction.Articular cartilage consists mainly of extracellular matrix, the principal organic components of which are type II collagen fibers and aggregates of the large proteoglycan...