Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells
Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells
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DOI：
10.1071/rd17483
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发表时间：
2018-10-01
影响因子：
1.9
通讯作者：
Yoshie, Mikihiro
中科院分区：
文献类型：
--
作者：
Kusama, Kazuya;Tamura, Kazuhiro;Yoshie, Mikihiro
在子宫内膜间质细胞(ESC)蜕膜形成过程中，蛋白激酶A(PKA)信号伴随细胞内cAMP水平的升高。CAMP信号的另一种中介--cAMP直接激活的交换蛋白(EPAC)促进了PKA类似物诱导的蜕膜化；然而，EPAC和PKA协同刺激蜕膜化的确切机制尚未被描述。为了研究CCAAT/增强子结合蛋白(C/EBP)在EPAC和PKA介导的原代人ESCs蜕膜形成中的作用，构建了含有蜕膜催乳素(DPRL)基因转录起始点上游332bp区域的报告载体，并用荧光素酶分析了启动子的活性。用PKA选择性cAMP类似物N-6-苯基-cAMP(Phe)处理转染ESCs后，DPRL启动子活性增加，与EPAC选择性cAMP类似物8-(4-氯苯硫基)-2‘-O-甲基cAMP(CPT)共处理后，DPRL启动子活性进一步增强。用腺苷环化酶激活剂Forsklin处理对报告活性也有类似的影响。DPRL启动子中C/EBPβ和/或C/EBP增量结合位点的定点突变消除了Phe/CPT介导的报告活性的上调。Epac2基因敲除显著降低了Phe刺激的C/EBPβ和C/EBP Delta mRNA水平，以及叉头盒O1(FOXO1)蛋白水平。这些结果表明，EPAC信号通过作用于基因启动子区域的C/EBP反应元件来增强PKA介导的DPRL在ESCs中的表达。
Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332 bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N-6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2'-O-methyl cAMP (CPT). Treatment with forskolin, an adenylylcyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBP beta and/or C/EBP delta-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBP beta and C/EBP delta mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.