Estrogen increases expression of vascular alpha 2C adrenoceptor through the cAMP/Epac/JNK/AP-1 pathway and potentiates cold-induced vasoconstriction

Abstract

Cutaneous cold-induced vasoconstriction is a normal physiological reaction mediated by alpha 2C-adrenergic receptors (α_2C-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_2C-ARs in human VSMCs via a cAMP/Epac/Rap pathway. We also showed that cAMP acts through JNK to increase α_2C-AR expression. However, whether estrogen employs JNK to regulate α_2C-AR is not investigated. Knowing that the α_2C-AR promoter harbors an activator protein-1 (AP-1) binding site that can be potentially activated by JNK, we hypothesized that estrogen regulates α_2C-AR expression through an Epac/JNK/AP-1 pathway. Our results show that estrogen (10^-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_2C-AR. Importantly, estrogen-induced activation of α_2C-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_2C-AR promoter. However, co-transfection of constitutively active JNK mutant overrode the inhibitory effect of Epac-DN on α_2C-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_2C-AR promoter abolished its activation by estrogen. This in vitro estrogen-increased α_2C-AR expression was mirrored by an increase in the ex vivo functional responsiveness of arterioles. Indeed, estrogen potentiated α_2C-AR-mediated cold-induced vasoconstriction, which was abolished by SP600125. Collectively, these results indicate that estrogen upregulates α_2C-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.

Keywords: AP-1; Estrogen; JNK; Raynaud's phenomenon; Vasoconstriction; α(2C)-adrenergic receptor.