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 inhibitor of IGF action. hOB cells also secrete a protease, which requires IGFs for activation and specifically cleaves IGFBP-4. To study the regulation of this IGFBP-4 protease, hOB cells from 26 different adult donors were cultured in serum-free medium for 24 h in the absence or presence of hormones and other factors known to regulate bone growth. hOB cell-conditioned medium (hOB-CM) was collected for measurement of IGFBP-4 protease activity in a cell-free assay. This assay involved incubation of hOB-CM (50 microL) without or with IGF-II at 37 C for 6 h. IGF-II-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting and quantitated using laser densitometry. Conditioned medium from all hOB cells examined exhibited IGFBP-4 protease activity. PTH, GH, insulin, calcitonin, glucocorticoids, sex steroids, 1,25-dihydroxyvitamin D3, and epidermal growth factor had no significant effect on IGF-dependent IGFBP-4 protease activity. In comparison, hOB-CM from cells treated with transforming growth factor-beta (TGF beta) exhibited significantly augmented IGF-II-dependent proteolysis of endogenous IGFBP-4. Enhanced proteolysis of exogenous IGFBP-4 was also demonstrated: 1) 92% of recombinant human IGFBP-4 added to conditioned medium from TGF beta-treated 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 control hOB-CM in the presence of IGF-II. In addition to its effect on IGFBP-4 proteolysis, TGF beta treatment decreased IGFBP-4 messenger ribonucleic acid expression, as measured by Northern analysis. Our results indicate that the IGFBP-4-IGFBP-4 protease system in hOB cells can be controlled by two of the most abundant local growth factors for bone (IGF-II and TGF beta), with each acting via different mechanisms. Regulation of IGFBP-4 availability may play an important role in the modulation of bone cell responsiveness to IGFs.
Durham
S K
SK
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Riggs
B L
BL
Conover
C A
CA
D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D018974:Insulin-Like Growth Factor Binding Protein 4; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000378:metabolism*; D010006:Osteoblasts / Q000201:enzymology*; D011266:Pregnancy-Associated Plasma Protein-A; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D006133:Growth Substances; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D007335:Insulin-Like Growth Factor II; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A