Bale L K, Conover C A
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Endocrinology. 1992 Aug;131(2):608-14. doi: 10.1210/endo.131.2.1379162.
Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of the anabolic and mitogenic actions of the insulin-like growth factor (IGF) peptides. Previous studies have shown that the IGFs themselves can elevate levels of IGFBP-3 in vivo and in vitro. However, the regulatory mechanisms responsible for IGF-induced increases in IGFBP-3 are unclear. In this study we examined the expression of messenger RNA (mRNA) encoding IGFBP-3 in cultured bovine and human fibroblasts, two cell lines that secrete IGFBP-3 under IGF-I control. Northern analysis of bovine fibroblast RNA hybridized with a specific bovine IGFBP-3 complementary DNA probe indicated a single 2.8-kilobase (kb) transcript readily detectable within 2 h in IGF-I- or insulin-treated, but not in untreated, cells. IGFBP-3 mRNA abundance was maximal around 6 h, and remained elevated after 24 h of treatment. Secreted IGFBP-3 protein appeared more slowly. By Western ligand blotting, IGFBP-3 was not detected in medium from bovine fibroblasts incubated with IGF-I for 2, 4, or 6h, but was apparent after 24 h IGF-I treatment. Induction of IGFBP-3 mRNA was blocked when RNA synthesis was inhibited by actinomycin D. Furthermore, IGFBP-3 mRNA and protein was induced by different IGF-I analogs in direct relation to the ability of the peptides to bind to the type I IGF receptor, indicating a receptor-mediated process. GH had no effect on IGFBP-3 mRNA or protein levels in these cells. In contrast to its effect in bovine fibroblasts, IGF-I had no significant effect on steady state levels of IGFBP-3 mRNA in cultured human fibroblasts. A human IGFBP-3 complementary DNA probe hybridized to a single 2.8-kilobase mRNA species abundant in normal and SV40-transformed human fibroblasts under all culture conditions, and IGFBP-3 protein was secreted by these cells in the absence of exogenous stimuli. In human fibroblast cultures, IGF-I rapidly increased levels of IGFBP-3 in the medium without influencing transcript levels. Steady state levels of induced or constitutively expressed IGFBP-3 mRNA did not change significantly after 6h in the presence of actinomycin D, even though general RNA synthesis was inhibited more than 98%. These data demonstrate that expression of mRNA encoding IGFBP-3 is differentially controlled by IGF-I in bovine and human fibroblasts. Whereas cultured human fibroblasts may be suitable to study posttranscriptional regulation of IGFBP-3 availability, cultured bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3 gene expression and regulation by IGF-I.
胰岛素样生长因子结合蛋白-3（IGFBP-3）是胰岛素样生长因子（IGF）肽的合成代谢和促有丝分裂作用的重要调节因子。先前的研究表明，IGF本身可在体内和体外提高IGFBP-3的水平。然而，负责IGF诱导IGFBP-3增加的调节机制尚不清楚。在本研究中，我们检测了编码IGFBP-3的信使核糖核酸（mRNA）在培养的牛和人成纤维细胞中的表达，这两种细胞系在IGF-I的控制下分泌IGFBP-3。用特异性牛IGFBP-3互补DNA探针与牛成纤维细胞RNA进行Northern印迹分析表明，在IGF-I或胰岛素处理的细胞中，2小时内即可轻易检测到一条单一的2.8千碱基（kb）转录本，而未处理的细胞中则未检测到。IGFBP-3 mRNA丰度在6小时左右达到最高，并在处理24小时后仍保持升高。分泌的IGFBP-3蛋白出现得更慢。通过Western配体印迹法，在与IGF-I孵育2、4或6小时的牛成纤维细胞培养基中未检测到IGFBP-3，但在IGF-I处理24小时后则明显可见。当用放线菌素D抑制RNA合成时，IGFBP-3 mRNA的诱导被阻断。此外，不同的IGF-I类似物诱导IGFBP-3 mRNA和蛋白的能力与这些肽与I型IGF受体结合的能力直接相关，表明这是一个受体介导的过程。生长激素（GH）对这些细胞中的IGFBP-3 mRNA或蛋白水平没有影响。与对牛成纤维细胞的作用相反，IGF-I对培养的人成纤维细胞中IGFBP-3 mRNA的稳态水平没有显著影响。人IGFBP-3互补DNA探针与在所有培养条件下正常和SV40转化的人成纤维细胞中丰富的一条单一的2.8千碱基mRNA种类杂交，并且这些细胞在没有外源性刺激的情况下分泌IGFBP-3蛋白。在人成纤维细胞培养物中，IGF-I迅速增加培养基中IGFBP-3的水平，而不影响转录水平。在放线菌素D存在的情况下，诱导或组成性表达的IGFBP-3 mRNA的稳态水平在6小时后没有显著变化，尽管总体RNA合成被抑制了98%以上。这些数据表明，编码IGFBP-3的mRNA的表达在牛和人成纤维细胞中受到IGF-I的差异控制。培养的人成纤维细胞可能适合研究IGFBP-3可用性的转录后调节，而培养的牛成纤维细胞可能提供一个有用的模型系统来探究IGFBP-3基因表达和IGF-I调节的分子机制。
