cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression
cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression
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DOI：
10.1074/jbc.m110.116905
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发表时间：
2010-05-07
影响因子：
4.8
通讯作者：
Pinsky, David J.
中科院分区：
文献类型：
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作者：
Liao, Hui;Hyman, Matthew C.;Pinsky, David J.
CD 39是一种跨膜酶，通过将ATP和ADP磷酸化水解为AMP来抑制血小板反应性和炎症。环磷酸腺苷（cAMP）是一种重要的第二信使，在调节血管稳态基因中起着重要作用.这些实验验证了cAMP可能正调控CD 39的表达从而调节重要的血管稳态特性的假设。CD 39 mRNA诱导13.8倍，RAW细胞处理的膜渗透cAMP类似物（8-溴环AMP; 8-Br-cAMP），刺激腺苷酸环化酶，或前列腺素类已知驱动cAMP反应。荧光激活细胞分选、免疫荧光和TLC分析表明，在用8-Br-cAMP处理的细胞中，CD 39蛋白表达和酶活性均增加，但在用针对CD 39的短发夹RNA转染的细胞中没有增加。这种类似物驱动的CD 39启动子的转录活性显着增加，但不是当启动子的cAMP反应元件位点突变。cAMP依赖性蛋白激酶（PKA），磷酸肌醇3-激酶（PI 3 K），或ERK抑制剂的预处理几乎消除了cAMP驱动的Cd 39 mRNA，蛋白质表达和启动子活性的增加。8-Br-cAMP以PKA-、PI 3 K-和ERK-依赖的方式显著增加CREB 1（Ser（133））和ATF 2（Thr（71））的磷酸化。染色质免疫沉淀分析表明，磷酸化CREB 1和ATF 2的cAMP反应元件样位点的结合显着增加与8-Br-cAMP治疗和PKA，PI 3 K，ERK抑制，而减少结合Creb 1和Atf 2过表达的转染结构增强cAMP驱动的Cd 39 mRNA的表达。用突变的Creb 1（S133 A）转染RAW细胞降低cAMP驱动的Cd 39 mRNA表达。此外，cAMP介导的诱导Cd 39 mRNA，蛋白质和磷酸水解活性在原代腹腔巨噬细胞中复制。这些数据确定cAMP是巨噬细胞CD 39表达的关键调节剂，并证明cAMP通过PKA/CREB、PKA/PI 3 K/ATF 2和PKA/ERK/ATF 2途径起作用，以控制关键的血管稳态介体。
CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser(133)) and ATF2 (Thr(71)) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.