Phorbol ester tumor promoters regulate insulin-like growth factor-binding protein-4 proteolysis.
Cultured human fibroblasts secrete a specific protease that alters extracellular insulin-like growth factor-binding protein-4 (IGFBP-4) structure and function. This enzyme appears to be secreted in a latent form and requires IGFs for activation. To study regulation of the IGFBP-4 protease, we treated normal adult human fibroblasts with various hormones and growth regulatory factors, and collected the human fibroblast-conditioned medium (HFCM) for analysis of IGFBP-4 protease activity. The IGFBP-4 protease assay involved incubation of 50 microliters HFCM with or without 5 nM IGF-II for 6 h at 37 C under cell-free conditions; IGF-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting. In HFCM from cells treated with vehicle, GH, insulin, epidermal growth factor, steroid hormones, or forskolin, IGF-II induced the select loss of detectable IGFBP-4 during the assay. In contrast, IGFBP-4 levels were maintained when HFCM from cells treated with phorbol ester tumor promoters was incubated with IGF-II under cell-free conditions. Hydrolysis of [125I]IGFBP-4 to 18,000 and 14,000 mol wt fragments also was prevented in HFCM from cells treated with phorbol esters. Phorbol esters had no effect on endogenous or exogenous IGFBP-4 proteolysis when added directly to HFCM during the assay, however. Treatment of cells with actinomycin-D or cycloheximide could prevent a phorbol ester-induced block of IGF-dependent IGFBP-4 proteolysis. These data suggest that phorbol ester tumor promoters stimulate human fibroblasts to produce and secrete an inhibitor of the IGFBP-4 proteolytic reaction. Alterations in IGFBP-4 protease activity could affect local IGF action through regulation of IGFBP-4 availability.
Conover
C A
CA
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Clarkson
J T
JT
Bale
L K
LK
D000328:Adult; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017077:Culture Media, Conditioned; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology*; D013006:Growth Hormone / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D018974:Insulin-Like Growth Factor Binding Protein 4; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology*; D011374:Progesterone / Q000494:pharmacology
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D007328:Insulin; D018974:Insulin-Like Growth Factor Binding Protein 4; D010703:Phorbol Esters; D003609:Dactinomycin; D005576:Colforsin; D011374:Progesterone; D004958:Estradiol; D004815:Epidermal Growth Factor; D007335:Insulin-Like Growth Factor II; D003907:Dexamethasone; D013006:Growth Hormone; D003513:Cycloheximide; D010450:Endopeptidases