Acidosis Activated GPR68 Promotes Vascular Smooth Muscle Cell Growth

Abstract

Cardiovascular disease (CVD) is the primary cause of death in the United States and worldwide. Localized extracellular acidosis has been theorized as a possible contributor to CVD pathogenesis, but its precise impact remains unclear. G protein coupled receptors (GPCRs) are seven transmembrane receptors that have wide-ranging functions in cardiovascular physiology and pathophysiology. A sub-family of proton sensing GPCRs has been identified that senses changes in extracellular pH and are activated in acidic conditions. One proton sensing GPCR, GPR68, is primarily localized in vascular smooth muscle (VSM) and is thought to signal through stimulatory Gs signaling. The Gs pathway is known to activate cyclic AMP and its downstream effectors PKA and EPAC, yet the influence of GPR68 and its cyclic AMP signals on VSM cell (VSMC) growth in the context of CVD in not known. The hypothesis of this study was to determine that acidic activation of proton sensing GPR68 and its Gs signals regulates VSMC growth via decreased cell proliferation and increased cell death, in turn implicating GPR68 as a potential cytostatic target to control pathologic VSM growth. In primary VSMCs under growth stimulated, normal pH conditions, loss of GPR68 (using GPR68 knockout (KO) models) results in increased proliferation with evidence pointing toward abbreviated cell cycle progression. Activation of GPR68 under acidic conditions suggests stimulation of Gs signaling through cAMP and the downstream effectors EPAC1, Rap1A/1B, and ERK1/2. Activation of GPR68 under acidic conditions also shows possible influence on intracellular calcium and the cytokine IL-6. In comparison, our in vivo arterial injury studies that mimic acidic vascular conditions suggest a decrease in GPR68 activation and that lack of GPR68 minimalizes remodeling and neointimal growth of VSMCs. Importantly these novel findings highlight the importance of GPR68 and Gs signaling in context-specific regulation of VSMC growth under both normal and acidic conditions. Clinically, a more complete understanding of possible biased signaling for GPR68 could elucidate contextual Gs signaling for deterring the proliferative hallmarks of occlusive CVD.