Shepard A R, Zhang W, Eberhardt N L
Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905.
J Biol Chem. 1994 Jan 21;269(3):1804-14.
We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.
我们在瞬时转染的大鼠垂体前叶肿瘤GC细胞中确定了介导人生长激素（hGH）基因cAMP反应性的顺式作用元件。对完整的hGH基因或与hGh cDNA、氯霉素乙酰转移酶或荧光素酶基因相连的hGH 5'-侧翼DNA（5'-FR）的分析表明，cAMP主要刺激hGH启动子活性。蛋白激酶A抑制蛋白cDNA的共转染证明cAMP反应是由蛋白激酶A介导的。hGH启动子的突变分析确定了位于核苷酸-187/-183（远端cAMP反应元件；dCRE）和-99/-95（近端cAMP反应元件；pCRE）的两个核心cAMP反应元件基序（CGTCA）以及位于核苷酸-123/-112（dGHF1）的垂体特异性转录因子（GHF1/Pit1）结合位点，这些是cAMP反应性所必需的。GHF1不是一个限制因素，因为共转染时GHF1的过表达增加了基础水平但没有增加福斯可林诱导水平。凝胶迁移分析表明，类似的、普遍存在的、耐热的蛋白质特异性结合pCRE和dCRE基序。CGTCA基序结合因子与cAMP反应元件结合蛋白（CREB）/激活转录因子-1（ATF-1）相关，因为DNA-蛋白质复合物被未标记的CREB共有寡核苷酸竞争，被抗CREB和ATF-1的抗血清特异性超迁移但不被抗ATF-2的抗血清超迁移，并且被纯化的CREB以与GC核提取物相同的相对结合亲和力（pCRE < dCRE < CREB）和迁移率结合。紫外线交联和蛋白质印迹分析揭示了多种DNA-蛋白质相互作用，其中约100 kDa和约45 kDa的蛋白质占主导；约45 kDa的蛋白质可能代表CREB。这些结果表明，CREB/ATF-1相关因子与细胞特异性因子GHF1协同作用，介导垂体前叶生长激素细胞中hGH-1基因转录的cAMP依赖性调节。
