Rogers J, Wiltrout L, Nanu L, Fant M E
Department of Pediatrics, UT-Southwestern Medical Center, Dallas 75235, USA.
Regul Pept. 1996 Mar 22;61(3):189-95. doi: 10.1016/0167-0115(95)00155-7.
Preterm, human, placental fibroblasts exhibit growth rates, in vitro, that vary with gestational age. The observed increase in proliferation rate is associated with enhanced mitogenic responsiveness to IGF-I. IGFBPs can either potentiate or inhibit IGF action at the cellular level. The production of IGFBPs by placental fibroblasts was studied as potential modulators of their responsiveness to IGFs. Human placental fibroblasts were obtained at various gestational ages and maintained in culture. IGFBP-3 protein and mRNA expression were assessed by Northern and ligand blot analyses. First, media conditioned by fibroblasts, in culture, were subjected to ligand blot analysis. Multiple species of IGFBPs were present in each cell line tested. IGFBP-3, migrating as a doublet at approx. 38/42 kDa, was the predominant IGFBP species present. Other IGFBPs of 22-35 kDa were also present. The secretion of IGFBP-3 exhibited a marked decrease at 10-15 weeks gestation relative to 8-9 week fibroblasts but began to increase again by 19 weeks. We next studied the expression of IGFBP-3 mRNA. Total cellular RNA was obtained from rapidly growing cells and subjected to Northern analysis. Placental fibroblasts exhibited decreased steady state levels of IGFBP-3 mRNA at 10-15 weeks gestation consistent with its decreased protein expression. The ability of IGFBP-3 to influence IGF-1 stimulated DNA synthesis was studied in 10 week placental fibroblasts as measured by [3H]thymidine incorporation. IGFBP-3 inhibited IGF-1 (3.3 nM) stimulated DNA synthesis in a dose-dependent manner when added simultaneously with IGF-1 or preincubated with the cells for 48 h prior to the addition of IGF-1. By contrast, maximum effective concentrations of IGFBP-3 (52 nM) potentiated the effect of IGF-1 50-200% when preincubated with bovine fibroblasts for 48 h prior to the addition of IGF-1. These data suggest that IGFBP-3 production is developmentally regulated in human placental fibroblasts and inhibits their mitogenic response to IGF-1. The regulated expression of IGFBP-3 may contribute to the altered growth rate and IGF responsiveness exhibited by placental fibroblasts, in vitro.
早产的人胎盘成纤维细胞在体外的生长速率随胎龄而变化。观察到的增殖速率增加与对胰岛素样生长因子-I（IGF-I）的促有丝分裂反应增强有关。胰岛素样生长因子结合蛋白（IGFBPs）在细胞水平上既可以增强也可以抑制IGF的作用。研究了胎盘成纤维细胞产生IGFBPs作为其对IGFs反应潜在调节因子的情况。在不同胎龄获取人胎盘成纤维细胞并进行培养。通过Northern印迹和配体印迹分析评估IGFBP-3蛋白和mRNA表达。首先，对培养的成纤维细胞条件培养基进行配体印迹分析。在每个测试的细胞系中都存在多种IGFBPs。IGFBP-3以约38/42 kDa的双峰形式迁移，是存在的主要IGFBP种类。还存在22 - 35 kDa的其他IGFBPs。与8 - 9周的成纤维细胞相比，IGFBP-3的分泌在妊娠10 - 15周时显著减少，但到19周时又开始增加。接下来我们研究了IGFBP-3 mRNA的表达。从快速生长的细胞中获取总细胞RNA并进行Northern分析。胎盘成纤维细胞在妊娠10 - 15周时IGFBP-3 mRNA的稳态水平降低，与其蛋白表达降低一致。通过[3H]胸苷掺入法测定，研究了IGFBP-3对10周胎盘成纤维细胞中IGF-1刺激的DNA合成的影响。当与IGF-1同时添加或在添加IGF-1之前与细胞预孵育48小时时，IGFBP-3以剂量依赖性方式抑制IGF-1（3.3 nM）刺激的DNA合成。相比之下，在添加IGF-1之前与牛成纤维细胞预孵育48小时时，IGFBP-3的最大有效浓度（52 nM）使IGF-1的作用增强了50 - 200%。这些数据表明，IGFBP-3的产生在人胎盘成纤维细胞中受到发育调控，并抑制它们对IGF-1的促有丝分裂反应。IGFBP-3的调控表达可能有助于胎盘成纤维细胞在体外表现出的生长速率改变和IGF反应性。
