The role of connective-tissue growth factor in TGF-beta-induced anchorage-independent growth and regulation of cell cycle of fibroblasts

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Gary Grotendorst, Committee Chair


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.


Biology, Anatomy; Biology, Cell; Biology, Animal Physiology

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