Clarkson R W, Chen C M, Harrison S, Wells C, Muscat G E, Waters M J
Centre for Molecular Biology and Biotechnology, University of Queensland St. Lucia, Australia.
Mol Endocrinol. 1995 Jan;9(1):108-20. doi: 10.1210/mend.9.1.7760844.
Using the 3T3-F442A preadipocyte line as a model of GH-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to GH addition were investigated. Addition of 50 ng/ml human GH to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant GH trans-activation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots, it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/EBP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit GH-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a GH-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus GH exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that GH-dependent phosphorylation results in the efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.
以3T3-F442A前脂肪细胞系作为生长激素（GH）依赖性分化的模型，研究了转录因子的DNA结合亲和力对添加GH的早期反应变化。在化学成分确定的培养基中向细胞添加50 ng/ml人GH，导致激活蛋白1（AP-1）和CCAAT增强子结合蛋白（C/EBP）的结合活性迅速增加，30分钟时显著增加，2小时时达到最大诱导水平（AP-1为3倍，C/EBP为2.5倍）。AP-1 DNA结合的诱导与先前描述的c-jun和c-fos基因的GH反式激活相关。在电泳迁移率变动分析和蛋白质免疫印迹中使用特异性抗体表明，C/EBP活性的增加是C/EBPβ两种可变翻译形式（40-C/EBPβ和23-C/EBPβ）合成增加的结果。这种蛋白质的增加并未伴随着mRNA水平的改变，并且可以被Janus激酶2酪氨酸激酶抑制剂和C激酶抑制剂在显示可抑制GH依赖性微管相关蛋白（MAP）激酶激活的浓度下阻断。与C/EBPβ的翻译激活增加同时，观察到C/EBPδ转录的GH依赖性增加。这伴随着C/EBPδ mRNA的增加，该增加被放线菌酮超诱导，并且与C/EBPβ蛋白的增加不同，胰岛素处理未观察到这种增加。因此，GH在两个水平上对C/EBP同工型发挥作用：C/EBPδ的转录激活和C/EBPβ的翻译激活。有人提出，GH依赖性磷酸化导致40-C/EBPβ和23-C/EBPβ（肝富集抑制蛋白的小鼠同源物）的有效翻译，并且与C/EBPδ的诱导一起，这些可能参与启动脂肪细胞分化程序。
