Characterization of an insulin-like growth factor binding protein-5 protease produced by rat articular chondrocytes and a neuroblastoma cell line.
Both articular cartilage and the central nervous system are target organs for insulin-like growth factors (IGFs). We have previously described the hormonal regulation of IGF binding proteins (IGFBPs) in the conditioned media (CM) of rat articular chondrocytes and in a rat neuroblastoma cell line (B104). In the studies presented here, we have investigated the role of IGFBP-5 proteases in these complex systems. Proteolysis of [125I] IGFBP-5 was maximal after 2-3 h incubation with CM of either cell type and did not further increase, even with an incubation of 12 h. Assessment of the effect of pH on protease activity showed that proteolysis was active between pH 6 and pH 9, but not at more acidic pH. Among the various protease inhibitors, serine protease inhibitors [benzamidine (100 mM), aprotinin (1 mg/ml), PMSF (10 mM)] and metalloprotease inhibitors [EDTA (1 mM), 1,10-phenanthroline (10 mM)] were the most effective in inhibiting the proteolysis of IGFBP-5, whereas aspartic and cysteine protease inhibitors were ineffective. These results indicate that the IGFBP-5 protease in the conditioned medium of rat articular chondrocytes and B104 cells belongs to a family of serine-metallo proteases. Interestingly, divalent cations, such as Zn+2 (1 mM) and Ca+2 (10 mM) also inhibited the IGFBP-5 proteolysis. This effect was not observed with monovalent ions, such as Na+ and K+. We also examined the effect of IGFs on IGFBP-5 protease activity, and found that IGF-I and -II inhibited the proteolysis in cell-free conditioned medium, while des(1-3) IGF-I was less effective. The IGFs may act to protect [125I] IGFBP-5 from the proteases in the CM, although the precise mechanism remains unknown. Thus, IGFBP-5 protease activity produced by both rat articular cartilage and B104 cells is a serine-metallo protease, that is inhibited by divalent cations and in the presence of IGF.
Department of Pediatric, Oregon Health Sciences University, Portland, USA.
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Matsumoto
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Gargosky
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Kelley
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Rosenfeld
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002358:Cartilage, Articular / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism*; D009447:Neuroblastoma; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology
D001549:Benzamides; D002413:Cations, Divalent; D018975:Insulin-Like Growth Factor Binding Protein 5; D010618:Phenanthrolines; D011480:Protease Inhibitors; C037689:benzamide; D007611:Aprotinin; D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium; C025205:1,10-phenanthroline