Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts
Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts
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DOI：
10.1007/s00210-008-0334-3
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发表时间：
2008-12-01
影响因子：
3.6
通讯作者：
Racke, K.
中科院分区：
文献类型：
--
作者：
Haag, S.;Warnken, M.;Racke, K.
在肺成纤维细胞中，EP2前列腺素受体的激活抑制了细胞的增殖，已知的EP2前列腺素受体与腺苷环化酶偶联。除了cAMP的经典靶标蛋白激酶A(PKA)外，还发现了其他的cAMP效应因子，其中包括EPAC(由cAMP激活的交换蛋白)。本研究旨在阐明EP2受体抑制肺成纤维细胞增殖的信号转导途径。用[~3H]-胸腺嘧啶核苷掺入法测定人肺成纤维细胞的增殖活性。选择性EP2受体激动剂Butaprost对~3H-胸腺嘧啶核苷的掺入抑制75%，这种作用被磷酸二酯酶抑制剂IBMX、稳定的cAMP类似物二丁酰cAMP和溴cAMP以及EPAC选择性cAMP类似物8-PCPT-2‘-O-Me-cAMP和Sp-8-PCPT-2’-O-Me-cAMP所模拟，而PKA选择性激动剂6-BNZ-cAMP则不起作用。PKA抑制剂RP-8-BR-cAMPS可抑制布他前列素诱导的CREB(cAMP反应元件结合蛋白)的磷酸化，但不影响布他前列素对[~3H]-胸腺嘧啶核苷掺入的抑制作用。部分阻断Epac1基因后，Butaprost的抑制作用明显减弱，而Epac2 siRNA或非沉默siRNA不影响Butaprost抑制[~3H]-TdR掺入的作用。综上所述，Epac1而不是经典的cAMP效应器PKA是介导EP2受体激活的抗增殖作用的信号转导通路中的关键元件。
In lung fibroblasts, proliferation is inhibited by activation of EP2 prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP2 receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [3H]-thymidine incorporation. The selective EP2 receptor agonist butaprost inhibited 3H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [3H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [3H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP2 receptor activation.