GPR40 promotes tight junction assembly in human airway epithelial cells via AMPK-dependent mechanisms

Abstract

Tight junctions (TJ) play key roles in the regulation of airway barrier function, which is implicated in the pathogenesis of airway diseases, and stabilization of TJ may improve lung health. G-protein coupled receptor 40 (GPR40) has been identified as a receptor of polyunsaturated fatty acids, which have been shown to have a protective effect against inflammation-induced destruction of intestinal barrier. However, the physiological role of GPR40 in barrier function remains unknown. This study was aimed to investigate the function of GPR40 in regulating TJ assembly in human airway epithelial Calu-3 cells. Disassembly and reassembly of TJ were induced using a calcium switch technique. Protein and functional expression of GPR40 was validated by immunocytochemistry, immunoblot and intracellular calcium measurements. We found that GW9508, an agonist of GPR40, augmented the recovery of transepithelial electrical resistance and localization of zonular occludens-1 to an intercellular area, and inhibitors of GPR40 and phospholipase C significantly suppressed the effect of GW9508. Interestingly, the GPR40 agonist enhanced TJ assembly in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, GW9508 promoted AMPK phosphorylation in a dose-dependent and time-dependent manner. The GW9508-induced AMPK activation was mediated via GPR40 but not GPR120. Importantly, our results indicate that Ca2+/calmodulin-dependent protein kinase kinase β mediates the GPR40-induced AMPK activation and TJ assembly. Collectively, this study is the first to demonstrate the novel role of GPR40 in improving barrier integrity in airway epithelial cells as well as its underlying mechanisms involving AMPK.