Barthel F, Loeffler J P
Laboratoire de Neurophysiologie et de Neurobiologie des Systèmes Endocrines, URA 1446 du CNRS, Université Louis Pasteur, Strasbourg, France.
J Neurochem. 1993 Feb;60(2):696-703. doi: 10.1111/j.1471-4159.1993.tb03203.x.
We demonstrate that granular cerebellar neurons express functional corticotropin-releasing hormone (CRH) receptors. Activation of these receptors with CRH receptor agonists leads to a dose-dependent increase in cyclic AMP (cAMP) levels with an apparent EC50 close to 10(-9) M. Using the c-fos protooncogene as a system to evaluate genomic effects of CRH, we show that activation of CRH receptors regulates gene expression at the transcriptional level. CRH rapidly induced c-fos mRNA accumulation. Genetic studies, using chimera genes containing human c-fos promoter sequences coupled to a chloramphenicol acetyltransferase (CAT) reporter gene, confirmed and extended this observation. When protein kinase A (PKA) was specifically inactivated by gene transfer of a mutated regulatory subunit of PKA lacking cAMP binding sites, CRH-stimulated c-fos transcription was suppressed but the increase in cAMP level was not affected, indicating a key role of PKA in mediating CRH-stimulated transcription. As CRH clearly modulates gene expression via the cAMP pathway, we analyzed the genomic effect of this neurohormone on a deleted c-fos-CAT construct containing only the cAMP-responsive element (CRE) and on a heterologous promoter construct bearing the minimal palindromic consensus CRE (core sequence TGACGTCA). These minimal cAMP-responsive genes are induced by CRH. These inductions are dependent on functional PKA. Taken together, our results demonstrate the presence of functional CRH receptors in primary cerebellar cultures. Activation of these receptors stimulates gene expression via the cAMP/PKA pathway and the transacting factor CREB (cAMP-responsive element binding protein).
我们证明颗粒状小脑神经元表达功能性促肾上腺皮质激素释放激素（CRH）受体。用CRH受体激动剂激活这些受体导致环磷酸腺苷（cAMP）水平呈剂量依赖性增加，其表观半数有效浓度（EC50）接近10^(-9) M。利用c-fos原癌基因作为评估CRH基因组效应的系统，我们表明CRH受体的激活在转录水平调节基因表达。CRH迅速诱导c-fos mRNA积累。使用含有与氯霉素乙酰转移酶（CAT）报告基因偶联的人c-fos启动子序列的嵌合基因进行的遗传学研究证实并扩展了这一观察结果。当通过缺乏cAMP结合位点的PKA突变调节亚基的基因转移特异性灭活蛋白激酶A（PKA）时，CRH刺激的c-fos转录受到抑制，但cAMP水平的增加不受影响，表明PKA在介导CRH刺激的转录中起关键作用。由于CRH显然通过cAMP途径调节基因表达，我们分析了这种神经激素对仅含有cAMP反应元件（CRE）的缺失c-fos-CAT构建体和带有最小回文共有CRE（核心序列TGACGTCA）的异源启动子构建体的基因组效应。这些最小的cAMP反应基因由CRH诱导。这些诱导依赖于功能性PKA。综上所述，我们的结果证明在原代小脑培养物中存在功能性CRH受体。这些受体的激活通过cAMP/PKA途径和反式作用因子CREB（cAMP反应元件结合蛋白）刺激基因表达。
