Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.
Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.
Durham
S K
SK
Division of Endocrinology and Metabolism, Mayo Clinic, Rochester, Minnesota 55905.
Riggs
B L
BL
Harris
S A
SA
Conover
C A
CA
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D010006:Osteoblasts / Q000201:enzymology*; D012516:Osteosarcoma / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D051381:Rats; D013539:Simian virus 40; D013002:Somatomedins / Q000494:pharmacology*; D013696:Temperature; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D010703:Phorbol Esters; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II; D010450:Endopeptidases