THE INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4 (IGFBP-4)-IGFBP-4 PROTEASE SYSTEM IN NORMAL HUMAN OSTEOBLAST-LIKE CELLS - REGULATION BY TRANSFORMING GROWTH-FACTOR-BETA

Insulin-like growth factor-binding protein-4 (IGFBP-4) is secreted by normal human osteoblast-like (hOB) cells and acts as a potent inhibito r of IGF action. hOB cells also secrete a protease, which requires IGF s for activation and specifically cleaves IGFBP-4. To study the regula tion of this IGFBP-4 protease, hOB cells from 26 different adult donor s were cultured in serum-free medium for 24 h in the absence or presen ce of hormones and other factors known to regulate bone growth. hOB ce ll-conditioned medium (hOB-CM) was collected for measurement of IGFBP- 4 protease activity in a cell-free assay. This assay involved incubati on of hOB-CM (50 mu L) without or with IGF-II at 37 C for 6 h. IGF-II- activated IGFBP-4 hydrolysis was assessed by Western Ligand blotting a nd quantitated using laser densitometry. Conditioned medium from all h OB cells examined exhibited IGFBP-4 protease activity. PTH, GH, insuli n, calcitonin, glucocorticoids, sex steroids, 1,25-dihydroxyvitamin D- 3 and epidermal growth factor had no significant effect on IGF-depende nt IGFBP-4 protease activity. In comparison, hOB-CM from cells treated with transforming growth factor-beta (TGF beta) exhibited significant ly augmented IGF-II-dependent proteolysis of endogenous IGFBP-4. Enhan ced proteolysis of exogenous IGFBP-4 was also demonstrated: 1) 92% of recombinant human IGFBP-4 added to conditioned medium from TGF beta-tr eated hOB cells was hydrolyzed during the assay in the presence of IGF -II compared to 45% of recombinant human IGFBP-4 added to control hOB- CM; and 2) increased radiolabeled IGFBP-4 fragments were generated in conditioned medium from TGF beta-treated hOB cells compared with contr ol hOB-CM in the presence of IGF-II. In addition to its effect on IGFB P-4 proteolysis, TGF beta treatment decreased IGFBP-4 messenger ribonu cleic acid expression, as measured by Northern analysis. Our results i ndicate that the IGFBP-4-IGFBP-4 protease system in hOB cells can be c ontrolled by two of the most abundant local growth factors for bone (I GF-II and TGF beta), with each acting via different mechanisms. Regula tion of IGFBP-4 availability may play an important role in the modulat ion of bone cell responsiveness to IGFs.