Chen Bing-Kun, Overgaard Michael T, Bale Laurie K, Resch Zachary T, Christiansen Michael, Oxvig Claus, Conover Cheryl A
Endocrine Research Unit, Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Endocrinology. 2002 Apr;143(4):1199-205. doi: 10.1210/endo.143.4.8729.
The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A's proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, beta-phorbol 12,13-didecanoate (beta-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of beta-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with beta-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with beta-PDD induced pro-MBP mRNA and protein expression within 6 h. beta-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of beta-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.
胰岛素样生长因子结合蛋白4（IGFBP - 4）蛋白酶系统是局部IGF生物利用度和细胞生长的重要调节因子。最近，培养的人成纤维细胞分泌的IGF依赖性IGFBP - 4蛋白酶被鉴定为妊娠相关血浆蛋白A（PAPP - A）。在妊娠血清中，PAPP - A以与主要碱性蛋白前体形式（pro - MBP）的二硫键结合复合物形式循环，并且在该复合物中PAPP - A的蛋白水解活性不明显。在本研究中，我们分析了正常人成纤维细胞中的IGFBP - 4蛋白酶系统，以确定妊娠以外的调节机制。用佛波酯肿瘤启动子β - 佛波醇12,13 - 十二烷酸酯（β - PDD）处理导致细胞条件培养基中IGF依赖性IGFBP - 4蛋白酶活性呈时间依赖性抑制，在6小时时明显，24小时时完全抑制。PAPP - A mRNA在对照细胞中组成性表达，并且仅在β - PDD处理24小时后水平才降低。PAPP - A蛋白分泌到条件培养基中不受β - PDD处理的影响。另一方面，在对照人成纤维细胞中未检测到pro - MBP mRNA，用β - PDD处理在6小时内诱导pro - MBP mRNA和蛋白表达。RNA合成抑制剂放线菌素D可阻断β - PDD诱导的pro - MBP mRNA表达和蛋白酶抑制。在不存在或存在β - PDD的情况下，放线菌素D对PAPP - A mRNA水平均无影响。同样，用猿猴病毒40大T抗原转化人成纤维细胞导致pro - MBP mRNA和蛋白的合成以及IGFBP - 4蛋白酶活性的抑制。将成纤维细胞与用pro - MBP cDNA转染的细胞共培养导致IGFBP - 4蛋白水解活性受到抑制，而对PAPP - A合成没有任何影响。总之，佛波酯肿瘤启动子和猿猴病毒40转化通过诱导一种新型的PAPP - A抑制剂pro - MBP来调节人成纤维细胞中的IGFBP - 4蛋白水解。这些发现扩展了我们对IGFBP - 4蛋白酶系统的理解，并提示了局部细胞生长控制的额外水平。
