The constitutive function of native TRPC3 channels modulates vascular cell adhesion molecule-1 expression in coronary endothelial cells through nuclear factor kappaB signaling.

Abstract

Upregulation of endothelial vascular cell adhesion molecule (VCAM)-1 and the subsequent increase in monocyte recruitment constitute critical events in atherogenesis. We have recently shown that in human coronary artery endothelial cells (HCAECs) regulated expression of VCAM-1 depends, to a significant extent, on expression and function of the Ca(2+)-permeable channel transient receptor potential canonical (TRPC)3, regardless of the ability of the stimulatory signal to induce regulated Ca(2+) influx, leading to the hypothesis that TRPC3 constitutive, rather than regulated function, contributes to the underlying signaling mechanism. The present studies addressed this important question and gathered mechanistic insight on the signaling coupling constitutive TRPC3 function to VCAM-1 expression. In HCAECs, maneuvers that prevent Ca(2+) influx or knockdown of TRPC3 markedly reduced tumor necrosis factor (TNF)alpha-induced VCAM-1 and monocyte adhesion. TNFalpha also induced TRPC3 expression and TRPC3-mediated constitutive cation influx and currents. Stable (HEK293 cells) or transient (HCAECs) overexpression of TRPC3 enhanced TNFalpha-induced VCAM-1 compared to wild-type cells. IkappaBalpha phosphorylation/degradation was reduced by TRPC3 knockdown and increased by channel overexpression. Inhibition of calmodulin completely prevented nuclear factor kappaB activation, whereas blocking calmodulin-dependent kinases or NADPH oxidases rendered partial inhibition. Our findings indicate that in HCAECs expression of VCAM-1 and monocyte adhesion depend, to a significant extent, on TRPC3 constitutive function through a signaling mechanism that requires constitutive TRPC3-mediated Ca(2+) influx for proper activation of nuclear factor kappaB, presumably through Ca(2+)-dependent activation of the calmodulin/calmodulin-dependent kinase axis.