Conover C A, Clarkson J T, Bale L K
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Endocrinology. 1993 Sep;133(3):1347-51. doi: 10.1210/endo.133.3.7689953.
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.
培养的人成纤维细胞分泌一种特定的蛋白酶，该蛋白酶可改变细胞外胰岛素样生长因子结合蛋白4（IGFBP-4）的结构和功能。这种酶似乎以无活性形式分泌，需要胰岛素样生长因子（IGFs）来激活。为了研究IGFBP-4蛋白酶的调节机制，我们用各种激素和生长调节因子处理正常成人成纤维细胞，并收集人成纤维细胞条件培养基（HFCM）以分析IGFBP-4蛋白酶活性。IGFBP-4蛋白酶测定包括在无细胞条件下，将50微升HFCM与或不与5 nM IGF-II在37℃孵育6小时；通过Western配体印迹法评估IGF激活的IGFBP-4水解情况。在用载体、生长激素（GH）、胰岛素、表皮生长因子、类固醇激素或福斯可林处理的细胞的HFCM中，IGF-II在测定过程中导致可检测到的IGFBP-4选择性丢失。相反，当用佛波酯肿瘤启动子处理的细胞的HFCM在无细胞条件下与IGF-II孵育时，IGFBP-4水平得以维持。在用佛波酯处理的细胞的HFCM中，[125I]IGFBP-4水解为18,000和14,000道尔顿分子量片段的过程也受到了抑制。然而，在测定过程中直接向HFCM中添加佛波酯对内源性或外源性IGFBP-4蛋白水解没有影响。用放线菌素-D或环己酰亚胺处理细胞可以阻止佛波酯诱导的IGF依赖性IGFBP-4蛋白水解的阻断。这些数据表明，佛波酯肿瘤启动子刺激人成纤维细胞产生并分泌一种IGFBP-4蛋白水解反应的抑制剂。IGFBP-4蛋白酶活性的改变可能通过调节IGFBP-4的可用性来影响局部IGF作用。
