Connective tissue growth factor (CTGF) is a 38 kDa cysteine-rich peptide whose synthesis and secretion are uniquely induced by transforming growth factor type-beta (TGF-beta) in connective tissue cells. TGF-beta has the unique ability to stimulate the growth of normal fibroblasts in soft agar, a property of transformed cells.I have conducted experiments to investigate the role of CTGF in TGF-beta stimulated anchorage-independent growth (AIG). The results of these studies indicate that CTGF cannot substitute for TGF-beta as an inducer of AIG in NRK fibroblasts. However, CTGF is required during the induction of AIG by TGF-beta. This was confirmed by using CTGF specific antibodies and an anti-sense CTGF gene (NRK-ASCTGF), both of which inhibited TGF-beta induced AIG. It was also possible to block the TGF-beta-induction of CTGF expression and AIG by upregulating levels of intracellular cAMP through the addition of compounds such as cholera toxin (CTX), forskolin or 8-Br-cAMP to the fibroblasts. Under these conditions, AIG could be restored in a cell cycle dependent manner by addition of recombinant CTGF (rCTGF) to the cells. Neither fibroblast growth factor (FGF) nor platelet-derived growth factor (PDGF) could substitute for CTGF in this process. These studies demonstrate that the TGF-beta stimulation of NRK fibroblast AIG is dependent on events induced via the synergistic action of CTGF-dependent and CTGF-independent signaling pathways.Further detailed studies into the molecular mechanism of CTGF action indicate that CTGF controls cell cycle progression through late G1 and S-phase of NRK fibroblast suspension cultures. CTGF allows S-phase entry by upregulating cyclin A levels. The molecular mechanism for cyclin A induction appears to be via reduction of P27Kip1 levels which results in hyperphosphorylation of the retinoblastoma protein (pRb) and release of E2F, a known transcriptional regulator for the cyclin A gene. These data indicate that CTGF acts as a mediator of TGF-beta induced fibroblast proliferation in suspension cultures by modulating cyclin dependent kinase (cdk) activities
