Lowe W L, Kummer M, Karpen C W, Wu X D
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City.
Endocrinology. 1990 Dec;127(6):2854-61. doi: 10.1210/endo-127-6-2854.
Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.
成纤维细胞是肝外胰岛素样生长因子I（IGF-I）在体内的产生部位之一。在本研究中，将从新生大鼠皮肤制备的培养成纤维细胞用作模型，以评估血清在调节IGF-I信使核糖核酸（mRNA）水平中的作用。通过Northern印迹分析表明，IGF-I mRNA存在于培养的成纤维细胞中，由成纤维细胞培养20小时的无血清培养基中含有108 pg/ml的免疫反应性IGF-I。胎牛血清（FCS）以时间和剂量依赖性方式降低成纤维细胞中稳态IGF-I mRNA水平。在0.3%、0.6%或1% FCS存在下将成纤维细胞孵育18小时，可使IGF-I mRNA水平降至维持在含0.25%牛血清白蛋白的无血清培养基中的对照细胞水平的76%、56%和46%。在4-10% FCS时可见最大抑制作用，约为对照水平的20%。相反，随着FCS浓度增加，碱性成纤维细胞生长因子和β-肌动蛋白mRNA水平分别增加2倍和4倍。用10微克/毫升环己酰亚胺处理细胞导致FCS抑制作用部分消除，而细胞中的蛋白质合成降至对照水平的6%。向成纤维细胞中添加2微克/毫升胰岛素或15-100纳克/毫升IGF-I不会重现FCS的抑制作用。最后，FCS中存在的抑制因子通过用活性炭处理血清或对血清进行热灭活而被部分去除/失活，但用氯仿萃取使FCS脱脂对FCS的抑制作用没有影响。总之，FCS含有一种因子，该因子以时间和剂量依赖性方式降低培养的成纤维细胞中IGF-I mRNA水平。用环己酰亚胺处理使FCS抑制作用部分消除表明，这种作用至少部分依赖于新的蛋白质合成。此外，使用脱脂、热灭活和活性炭处理血清的研究表明，抑制因子是一种肽。
