GPER Acts Through the cAMP/Epac/JNK/AP-1 Pathway to Induce Transcription of Alpha 2C Adrenoceptor in Human Microvascular Smooth Muscle Cells

Abstract

Raynaud's phenomenon, which results from exaggerated cold-induced vasoconstriction, is more prevalent in females than males. We previously showed that estrogen increases the expression of alpha 2C-adrenoceptors (α2C-AR), the sole mediator of cold-induced vasoconstriction. This effect of estrogen is reproduced by the cell-impermeable form of the hormone (E2:bovine serum albumin [BSA]), suggesting a role of the membrane estrogen receptor, G-protein-coupled estrogen receptor [GPER], in E2-induced α2C-AR expression. We also previously reported that E2upregulates α2C-AR in microvascular smooth muscle cells (VSMCs) via the cAMP/Epac/Rap/JNK/AP-1 pathway, and that E2:BSA elevates cAMP levels. We, therefore, hypothesized that E2uses GPER to upregulate α2C-AR through the cAMP/Epac/JNK/AP-1 pathway. Our results show that G15, a selective GPER antagonist, attenuates the E2-induced increase in α2C-AR transcription. G-1, a selective GPER agonist, induced α2C-AR transcription, which was concomitant with elevated cAMP levels and JNK activation. Pretreatment with ESI09, an Epac inhibitor, abolished G-1-induced α2C-AR upregulation and JNK activation. Moreover, pretreatment with SP600125, a JNK-specific inhibitor, but not H89, a PKA-specific inhibitor, abolished G-1-induced α2C-AR upregulation. In addition, transient transfection of an Epac dominant negative mutant (Epac-DN) attenuated G-1-induced activation of the α2C-AR promoter. This inhibitory effect of Epac-DN on the α2C-AR promoter was overridden by the cotransfection of constitutively active JNK mutant. Furthermore, mutation of AP-1 site in the α2C-AR promoter abrogated G1-induced expression. Collectively, these results indicate that GPER upregulates α2C-AR through the cAMP/EPAC/JNK/AP-1 pathway. These findings unravel GPER as a new mediator of cold-induced vasoconstriction, and present it as a potential target for treating Raynaud's phenomenon in estrogen-replete females.