IDENTIFICATION OF GLYCOSYLATED 38-KDA CONNECTIVE-TISSUE GROWTH-FACTOR(IGFBP-RELATED PROTEIN-2) AND PROTEOLYTIC FRAGMENTS IN HUMAN BIOLOGICAL-FLUIDS, AND UP-REGULATION OF IGFBP-RP2 EXPRESSION BY TGF-BETA IN HS578T HUMAN BREAST-CANCER CELLS

Connective Tissue Growth Factor (CTGF) is a cysteine-rich peptide invo lved in human atherosclerosis and fibrotic disorders such as scleroder ma. CTGF has considerable N-terminal sequence similarity with the insu lin-like growth factor binding proteins (IGFBPs), including preservati on of cysteines, and has been postulated to be a member of the IGFBP s uperfamily. Indeed, recent studies have shown that baculovirus generat ed CTGF, a secreted 38-kDa protein, binds IGFs in a specific manner, l eading to the provisional renaming of CTGF as IGFBP-8 (or IGFBP-rP2). With immunoprecipitation and immunoblotting, using polyclonal anti-IGF BP-rP2 antibody generated against recombinant human IGFBP-rP2(bac), IG Fl3P-rP2 can be identified in the serum-free conditioned media of Hs57 8T human breast cancer cells, as well as in various human biological f luids, such as normal sera, pregnancy sera, and cerebrospinal, amnioti c, follicular and peritoneal fluids. Glycosylation studies with endogl ycosidase F reveal that endogenous human IGFBP-rP2 is a secreted, glyc osylated, approximately 32-38kDa protein with 2-8-kDa of N-linked suga rs and a 30-kDa core. There are 18- and 24-kDa proteins that appear to be IGFBP-rP2 degradation products. In Hs578T human breast cancer cell s, transforming growth factor (TGF)-beta 2, a potent growth inhibitor for these cells, upregulates IGFBP-rP2 mRNA and protein levels. Expres sion of Hs578T IGFBP-rP2 is significantly increased by TGF-beta 2 trea tment in a dose-dependent manner, with 2.5- and 6-fold increases in mR NA and protein levels, respectively, at a TGF-beta 2 concentration of 10 ng/ml. Our studies indicate that IGFBP-rP2 appears to be an importa nt endocrine factor, and one of the critical downstream effecters of T GF-beta, similar to the role of IGFBP-3 in TGF-beta-induced growth inh ibition in human breast cancer cells.