PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells.
PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells.
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DOI：
10.1186/1465-9921-10-88
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发表时间：
2009-09-29
影响因子：
5.8
通讯作者：
Schmidt M
中科院分区：
文献类型：
--
作者：
Roscioni SS;Kistemaker LE;Menzen MH;Elzinga CR;Gosens R;Halayko AJ;Meurs H;Schmidt M
气道平滑肌通过分泌炎症介质如白细胞介素-8（IL-8）参与肺部疾病的发病机制。IL-8的产生部分地通过Gq-和Gs-偶联受体的活化来调节。在这里，我们研究的作用，环磷酸腺苷（cAMP）效应蛋白激酶A（PKA）和交换蛋白直接激活cAMP（Epac 1和Epac 2）在缓激肽诱导的IL-8释放的人气道平滑肌细胞系和这种反应的分子机制。在基础条件下和单独用缓激肽或与非诺特罗、Epac激活剂8-pCPT-2 ′-O-Me-cAMP和Sp-8-pCPT-2 ′-O-Me-cAMPS、PKA激活剂6-Bnz-cAMP和cGMP类似物8-pCPT-2 ′-O-Me-cGMP组合刺激后，通过ELISA评估IL-8释放。在指示的情况下，将细胞与药理学抑制剂艰难梭菌毒素B-1470（GTP酶）、U 0126（细胞外信号调节激酶ERK 1/2）和Rp-8-CPT-cAMPS（PKA）预孵育。环核苷酸类似物的特异性通过测量PKA底物血管舒张剂刺激的磷蛋白的磷酸化来证实。通过下拉技术评价Rap 1和Rap 2的GTP负载。通过蛋白质印迹评估Rap 1、Rap 2、Epac 1和Epac 2的表达。通过siRNA实现Epac蛋白表达的下调。使用非配对或配对双尾Student t检验。β2受体激动剂非诺特罗增加缓激肽释放IL-8。PKA激活剂6-Bnz-cAMP和Epac激活剂8-pCPT-2 '-O-Me-cAMP显著增加缓激肽诱导的IL-8释放。抗水解Epac激活剂Sp-8-pCPT-2 '-O-Me-cAMPS模拟8-pCPT-2'-O-Me-cAMP的作用，而阴性对照8-pCPT-2 '-O-Me-cGMP则没有。非诺特罗、毛喉素和6-Bnz-cAMP诱导VASP磷酸化，PKA抑制剂Rp-8-CPT-cAMPS可减弱VASP磷酸化。6-Bnz-cAMP和8-pCPT-2 '-O-Me-cAMP诱导Rap 1的GTP负载，但不诱导Rap 2的GTP负载。用毒素B-1470和U 0126单独或与PKA和Epac的激活剂组合处理细胞显著降低缓激肽诱导的IL-8释放。有趣的是，通过Rp-8-CPT-cAMPS抑制PKA和通过特异性siRNA沉默Epac 1和Epac 2表达大大降低了Rap 1的激活和PKA和Epac对缓激肽诱导的IL-8释放的增强。总的来说，我们的数据表明，PKA，Epac 1和Epac 2的一致行动，以调节气道平滑肌的炎症特性，通过信号传导到Ras样GTdR Rap 1和ERK 1/2。
Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. The β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac. Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.
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