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

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 alpha6beta1. As assessed by RT-PCR or Western blotting, CCN2(3) stimulated production of fibronectin and pro-collagen type IV(alpha5), 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 integrinbinding 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.