CHARACTERIZATION OF CELL-ASSOCIATED AND SOLUBLE FORMS OF CONNECTIVE-TISSUE GROWTH-FACTOR (CTGF) PRODUCED BY FIBROBLAST CELLS IN-VITRO

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound heal ing, fibrotic disorders and uterine function. Although the primary tra nslational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pC TGF) genes an predicted to be secreted and of approximate M-r 38,000, 10 kDa biologically active forms of pCTGF have recently been described . In this report, we show that human foreskin fibroblasts (HFFs) and m ouse connective tissue fibroblasts contained 2.4kb CTGF transcripts, s tained positively with an anti-CTGF[81-94] peptide antiserum, and prod uced a 38 kDa protein that a as immunoprecipitated by an anti-CTGF[247 -260] peptide antiserum. While 38 kDa CTGF was readily detected in cel l lysates, it was non-or barely detectable in conditioned medium. 38 k Da CTGF remained cell-associated for at least 5 days after synthesis a nd was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibrobla st conditioned medium resulted in the isolation of 10-11 kDa CTGF prot eins that were heparin-binding, bioactive, and reactive with anti-CTGF [247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthes is in 3T3 cells to the same extent as platelet-derived growth factor ( PDGF)-AA, -AB, or -BB, it did not compete with I-125-PDGF-BB for bindi ng to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), di d not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, a nd was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally c ell-associated whereas low mass forms of CTGF are soluble and biologic ally active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF -R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mec hanism.