Durham S K, Riggs B L, Harris S A, Conover C A
Division of Endocrinology and Metabolism, Mayo Clinic, Rochester, Minnesota 55905.
Endocrinology. 1995 Apr;136(4):1374-80. doi: 10.1210/endo.136.4.7534697.
Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.
胰岛素样生长因子（IGF）结合蛋白-4（IGFBP-4）由多种成骨细胞分泌，似乎是骨细胞生理学的一个重要组成部分。我们之前报道过，正常人类成骨样（hOB）细胞分泌IGFBP-4以及一种新型IGFBP-4蛋白酶，其功能活性需要IGF。在本研究中，我们通过Western配体印迹法和无细胞IGFBP-4蛋白酶检测，评估了转化成骨细胞中的IGFBP-4/IGFBP-4蛋白酶系统。猿猴病毒40永生化hOB细胞（HOBIT）、人骨肉瘤细胞（TE-85）和大鼠骨肉瘤细胞（UMR 106-01、ROS 17/2.8）分泌IGFBP-4。与hOB培养基中快速且显著的蛋白水解不同，用外源性IGF-II在培养中或无细胞条件下检测时，这些细胞条件培养基没有明显的IGFBP-4蛋白酶活性。在存在外源性蛋白酶的情况下检测发现，HOBIT细胞而非骨肉瘤细胞系似乎产生了一种对放线菌酮敏感的IGFBP-4蛋白水解反应抑制剂。用猿猴病毒40 T抗原的温度敏感突变体转染的人成骨细胞在允许温度下孵育，或用佛波酯肿瘤启动子处理hOB细胞诱导的瞬时细胞转化，也导致了IGF依赖性IGFBP-4蛋白水解的抑制。如果在更换培养基时或蛋白酶表达并分泌后向培养物中添加佛波酯，就会观察到抑制作用。IGFBP-4蛋白水解的差异不能用IGFBP-4信使RNA表达或底物水平的变化来解释。这些数据表明，转化与成骨细胞中IGFBP-4/IGFBP-4蛋白酶系统的改变有关。正常人类成骨细胞分泌一种IGF依赖性IGFBP-4蛋白酶。IGF依赖性IGFBP-4蛋白水解反应抑制剂的诱导可能与早期转化过程有关。完全致瘤的骨细胞既不表达IGFBP-4蛋白酶也不表达蛋白酶抑制剂活性。
