Intrinsic biological activity of the thrombospondin structural homology repeat in connective tissue growth factor

Abstract

Connective tissue growth factor (CCN2) is a 349-residue mosaic protein that contains four structural modules (modules 1-4), which are presumptive domains for interactions with regulatory binding proteins and receptors. Module 3, corresponding to residues 199-243, is a thrombospondin structural homology repeat (TSR) and is flanked by regions that are highly susceptible to proteolytic cleavage. To test whether CCN2 module 3 (CCN2 3 ) has intrinsic biological properties, it was produced recombinantly in Escherichia coli (E. coli) and examined for its effects on the function of hepatic stellate cells (HSC), the principal fibrogenic cell type in the liver. CCN2 3 stimulated dose-dependent HSC adhesion and activity of p42/p44 mitogen activated protein kinase, the latter of which was antagonized by blocking the activity of focal adhesion kinase. HSC adhesion to immobilized CCN2 3 was attributed to binding interactions with cell surface integrin 6 1. As assessed by RT-PCR or Western blotting, CCN2 3 stimulated production of fibronectin and pro-collagen type IV( 5), both of which are downstream components of HSC-mediated fibrogenesis and which are constituents of high density matrix in fibrotic lesions. These data show that while the full length CCN2 protein is strongly associated with fibrosis and stellate cell function, key integrin-binding properties, signaling, and fibrogenic pathways are exhibited by module 3 alone. These data indicate that module 3 of CCN2 is intrinsically active and suggest that liberation of module 3 following CCN2 proteolysis may contribute to HSC-mediated fibrogenesis, as well as other CCN2-dependent processes.

Connective Tissue Growth Factor and Susceptibility to Renal and Vascular Disease Risk in Type 1 Diabetes

“…It was recently shown that CTGF N fragment mediates myofibroblast differentiation and collagen synthesis, whereas the C-terminal half mediates fibroblast proliferation (18). In addition, module 3 of CTGF was shown to exhibit integrin binding, signaling, and fibrogenic properties (19).…”

The contribution of adhesion signaling to lactogenesis

“…CTGF/CCN2 enhanced the levels of β1 integrin on the surface of the cells (Weston et al 2003;Morrison et al 2010) and contributed to the adhesion via binding to α6β1 integrin, a common laminin receptor (Chen et al 2001;Leu et al 2003;Tong and Brigstock 2006). CTGF/CCN2 also enhanced binding of cells to the common fibronectin receptor, α 5 β1 integrin (Frazier et al 1996;Blom et al 2001;Weston et al 2003).…”

Molecular regulation of CCN2 in the intervertebral disc: Lessons learned from other connective tissues

“…It is also reported that CCN2 binding to LRP1, attributed to the TSP domain, is required for the endocytic uptake and transcytosis of CCN2 (Gao and Brigstock 2003; Kawata, et al 2012). Additionally, a binding site for α6β1 integrin in this domain is important for CCN2-mediated adhesion and collagen deposition (Heng, et al 2006; Tong and Brigstock 2006). Lastly, the CT domain, which is a critical for the biological activity, binds HSPGs, and intergrins, including αVβ3 and α5β1 to regulate adhesion, mitogenic effects and extracellular matrix production (Gao and Brigstock 2006; Gao and Brigstock 2004; Gao and Brigstock 2003; Hoshijima, et al 2006).…”