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-β in Hs578T Human Breast Cancer Cells

Abstract

Connective Tissue Growth Factor (CTGF) is a cysteine-rich peptide involved in human atherosclerosis and fibrotic disorders such as scleroderma. CTGF has considerable N-terminal sequence similarity with the insulin-like growth factor binding proteins (IGFBPs), including preservation of cysteines, and has been postulated to be a member of the IGFBP superfamily. Indeed, recent studies have shown that baculovirus generated CTGF, a secreted 38-kDa protein, binds IGFs in a specific manner, leading to the provisional renaming of CTGF as IGFBP-8 (or IGFBP-rP2). With immunoprecipitation and immunoblotting, using polyclonal anti-IGFBP-rP2 antibody generated against recombinant human IGFBP-rP2bac, IGFBP-rP2 can be identified in the serum-free conditioned media of Hs578T human breast cancer cells, as well as in various human biological fluids, such as normal sera, pregnancy sera, and cerebrospinal, amniotic, follicular and peritoneal fluids. Glycosylation studies with endoglycosidase F reveal that endogenous human IGFBP-rP2 is a secreted, glycosylated, approximately 32-38-kDa protein with 2-8-kDa of N-linked sugars 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 cells, transforming growth factor (TGF)-beta 2, a potent growth inhibitor for these cells, upregulates IGFBP-rP2 mRNA and protein levels. Expression of Hs578T IGFBP-rP2 is significantly increased by TGF-beta 2 treatment in a dose-dependent manner, with 2.5- and 6-fold increases in mRNA and protein levels, respectively, at a TGF-beta 2 concentration of 10 ng/ml. Our studies indicate that IGFBP-rP2 appears to be an important endocrine factor, and one of the critical downstream effectors of the critical downstream effectors of TGF-beta, similar to the role of IGFBP-3 in TGF-beta-induced growth inhibition in human breast cancer cells.

Matrix Metalloproteinases Cleave Connective Tissue Growth Factor and Reactivate Angiogenic Activity of Vascular Endothelial Growth Factor 165

“…This contrasts to the digestion patterns with leukocyte elastase and plasmin, since they degraded CTGF into small peptides by cleaving at multiple sites after a 24-h incubation. Previous immunoprecipitation studies (17) show that human biological fluids such as sera, cerebrospinal fluids, and ascites contain CTGF degradation fragments of 32, 24, and 18 kDa as well as intact 38-kDa CTGF. These fragments are also detected in the culture media from human breast cancer cells (17).…”

“…Previous immunoprecipitation studies (17) show that human biological fluids such as sera, cerebrospinal fluids, and ascites contain CTGF degradation fragments of 32, 24, and 18 kDa as well as intact 38-kDa CTGF. These fragments are also detected in the culture media from human breast cancer cells (17). Because the molecular sizes of the latter two fragments are similar to those found after digestion with MMPs and such biological fluids and culture media usually contain MMPs, it might be possible to speculate that the 24-and 18-kDa fragments are generated by digestion of CTGF with MMPs.…”

“…Plasmin is known to digest VEGF 165 and inactivate its angiogenic activity (16). Several fragments of CTGF have been detected in culture media of various cells (17), biological fluids such as sera (17) and uterine luminal flushing (18). However, no information is available for the proteinases that digest CTGF and modulate the angiogenic activity of the VEGF 165 ⅐CTGF complex.…”

Kallikrein-related Peptidase 12 Hydrolyzes Matricellular Proteins of the CCN Family and Modifies Interactions of CCN1 and CCN5 with Growth Factors

“…Proteolytic processing of CCN2 and CCN3 is also evidenced by observations showing that fragmented forms were present in the culture medium of cells overexpressing the recombinant full-length protein (43,45). In humans, CCN2 fragments were detected in several fluids such as normal sera, pregnancy sera, cerebrospinal, amniotic, follicular, and peritoneal fluids (46), and CCN3 fragments were observed in cerebrospinal fluid, amniotic fluid, and prepubertal and pubertal urine samples (47). Finally, N-terminal CCN2 fragments have been found in the plasma of patients with diabetic nephropathy (48) and multiple myeloma (49) and in the interstitial fluid and plasma of patients with scleroderma (50).…”

A Novel Member of the Insulin-Like Growth Factor Binding Protein Superfamily in Prostate Cancer

“…We have shown previously that IGFBP-rP2 is up-regulated by TGF-betal in the breast cancer cell line Hs578T (5), and IGFBP-rP2 induces apoptosis in the estrogen receptor-positive breast cancer cell line, . It is, thus, noteworthy that IGFBP-rP2 is up-regulated by TGF-beta and atRA, and down-regulated by IGF-I in prostatic epithelial cells.…”