Department of Biology, American University of Beirut, Beirut, Lebanon.
Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
Vascul Pharmacol. 2020 Aug;131:106690. doi: 10.1016/j.vph.2020.106690. Epub 2020 May 11.
Cutaneous cold-induced vasoconstriction is a normal physiological reaction mediated by alpha 2C-adrenergic receptors (α-ARs) expressed in vascular smooth muscle cells (VSMCs). When this reaction is exaggerated, Raynaud's phenomenon (RP) ensues. RP is more prevalent in females compared to age-matched men. We previously established that 17-β estradiol (estrogen) upregulates α-ARs in human VSMCs via a cAMP/Epac/Rap pathway. We also showed that cAMP acts through JNK to increase α-AR expression. However, whether estrogen employs JNK to regulate α-AR is not investigated. Knowing that the α-AR promoter harbors an activator protein-1 (AP-1) binding site that can be potentially activated by JNK, we hypothesized that estrogen regulates αAR expression through an Epac/JNK/AP-1 pathway. Our results show that estrogen (10 M) activated JNK in human VSMCs extracted from cutaneous arterioles. Pretreatment with ESI09 (10 μM; an Epac inhibitor), abolished estrogen-induced JNK activation. In addition, pre-treatment with SP600125 (3 μM; a JNK specific inhibitor) abolished estrogen-induced expression of α-AR. Importantly, estrogen-induced activation of α-AR promoter was attenuated with SP600125. Moreover, transient transfection of VSMCs with an Epac dominant negative mutant (Epac-DN) abolished estrogen-induced activation of α-AR promoter. However, co-transfection of constitutively active JNK mutant overrode the inhibitory effect of Epac-DN on α-AR promoter. Moreover, estrogen caused a concentration-dependent increase in the activity of AP-1-driven reporter construct. Mutation of AP-1 site in the α-AR promoter abolished its activation by estrogen. This in vitro estrogen-increased α-AR expression was mirrored by an increase in the ex vivo functional responsiveness of arterioles. Indeed, estrogen potentiated α-AR-mediated cold-induced vasoconstriction, which was abolished by SP600125. Collectively, these results indicate that estrogen upregulates α-AR expression via an EPAC-mediated JNK/AP-1- dependent mechanism. These results provide an insight into the mechanism by which exaggerated cold-induced vasoconstriction occurs in estrogen-replete females and identify Epac and JNK as potential targets for the treatment of RP.
皮肤冷诱导血管收缩是一种由血管平滑肌细胞 (VSMCs) 中表达的α 2C-肾上腺素能受体 (α-AR) 介导的正常生理反应。当这种反应被夸大时，就会出现雷诺现象 (RP)。与年龄匹配的男性相比，女性中 RP 更为常见。我们之前已经证明，17-β 雌二醇 (雌激素) 通过 cAMP/Epac/Rap 途径在上皮细胞 VSMCs 中上调 α-AR。我们还表明，cAMP 通过 JNK 增加 α-AR 表达。然而，雌激素是否通过 JNK 来调节 α-AR 尚不清楚。由于 α-AR 启动子含有一个激活蛋白-1 (AP-1) 结合位点，该位点可以被 JNK 潜在激活，因此我们假设雌激素通过 Epac/JNK/AP-1 途径调节 αAR 表达。我们的结果表明，来自皮肤小动脉的人 VSMCs 中，雌激素 (10 M) 激活了 JNK。用 ESI09 (10 μM；Epac 抑制剂) 预处理，可消除雌激素诱导的 JNK 激活。此外，用 SP600125(3 μM；JNK 特异性抑制剂) 预处理可消除雌激素诱导的 α-AR 表达。重要的是，SP600125 可减弱雌激素诱导的 α-AR 启动子激活。此外，用 Epac 显性失活突变体 (Epac-DN) 瞬时转染 VSMCs 可消除雌激素诱导的 α-AR 启动子激活。然而，共转染组成型激活的 JNK 突变体可克服 Epac-DN 对 α-AR 启动子的抑制作用。此外，雌激素引起 AP-1 驱动的报告构建体活性的浓度依赖性增加。α-AR 启动子中的 AP-1 位点突变消除了其对雌激素的激活作用。这种体外雌激素诱导的 α-AR 表达增加与离体血管反应性增强相吻合。事实上，雌激素增强了α-AR 介导的冷诱导血管收缩，而 SP600125 则消除了这种作用。总的来说，这些结果表明，雌激素通过 EPAC 介导的 JNK/AP-1 依赖性机制上调 α-AR 表达。这些结果提供了一个深入了解雌激素丰富的女性中过度冷诱导血管收缩发生的机制，并确定 Epac 和 JNK 作为治疗 RP 的潜在靶点。
