Estrogen increases expression of vascular alpha 2C adrenoceptor through the cAMP/Epac/JNK/AP-1 pathway and potentiates cold-induced vasoconstriction
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Elsevier Inc.
Abstract
Cutaneous cold-induced vasoconstriction is a normal physiological reaction mediated by alpha 2C-adrenergic receptors (α2C-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 α2C-ARs in human VSMCs via a cAMP/Epac/Rap pathway. We also showed that cAMP acts through JNK to increase α2C-AR expression. However, whether estrogen employs JNK to regulate α2C-AR is not investigated. Knowing that the α2C-AR promoter harbors an activator protein-1 (AP-1) binding site that can be potentially activated by JNK, we hypothesized that estrogen regulates α2C-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 α2C-AR. Importantly, estrogen-induced activation of α2C-AR promoter was attenuated with SP600125. Moreover, transient transfection of VSMCs with an Epac dominant negative mutant (Epac-DN) abolished estrogen-induced activation of α2C-AR promoter. However, co-transfection of constitutively active JNK mutant overrode the inhibitory effect of Epac-DN on α2C-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 α2C-AR promoter abolished its activation by estrogen. This in vitro estrogen-increased α2C-AR expression was mirrored by an increase in the ex vivo functional responsiveness of arterioles. Indeed, estrogen potentiated α2C-AR-mediated cold-induced vasoconstriction, which was abolished by SP600125. Collectively, these results indicate that estrogen upregulates α2C-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. © 2020
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Ap-1, Estrogen, Jnk, Raynaud's phenomenon, Vasoconstriction, Α2c-adrenergic receptor, Animals, Arterioles, Cells, cultured, Cold temperature, Cyclic amp, Estradiol, Guanine nucleotide exchange factors, Humans, Jnk mitogen-activated protein kinases, Male, Mice, inbred c57bl, Muscle, smooth, vascular, Myocytes, smooth muscle, Raynaud disease, Receptors, adrenergic, alpha-2, Signal transduction, Tail, Transcription factor ap-1, Up-regulation, Ab 120657, Alpha 2c adrenergic receptor, Anthra[1,9 cd]pyrazol 6(2h) one, Binding protein, Epac protein, Esi 09, Protein inhibitor, Stress activated protein kinase, Transcription factor ap 1, Unclassified drug, Adra2c protein, human, Alpha 2 adrenergic receptor, Guanine nucleotide exchange factor, Rapgef3 protein, human, Animal cell, Animal experiment, Animal tissue, Arteriole, Article, Binding site, Cold, Controlled study, Drug antagonism, Drug mechanism, Enzyme activation, Ex vivo study, Genetic transfection, Human, Human cell, In vitro study, Mouse, Nonhuman, Priority journal, Promoter region, Protein expression, Protein function, Protein transport, Raynaud phenomenon, Transient transfection, Upregulation, Vascular smooth muscle cell, Animal, C57bl mouse, Cell culture, Drug effect, Enzymology, Genetics, Metabolism, Pathophysiology, Smooth muscle cell, Vascular smooth muscle, Vascularization