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.