Integration of TRPC6 and NADPH oxidase activation in lysophosphatidylcholine-induced TRPC5 externalization

Abstract

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels, and the subsequent increase in intracellular Ca leads to TRPC5 activation. The goal of this study is to elucidate the steps in the pathway between TRPC6 activation and TRPC5 externalization. Following TRPC6 activation by lysoPC, extracellular regulated kinase (ERK) is phosphorylated. This leads to phosphorylation of p47 and subsequent NADPH oxidase activation with increased production of reactive oxygen species. ERK activation requires TRPC6 opening and influx of Ca as evidenced by the failure of lysoPC to induce ERK phosphorylation in TRPC6 endothelial cells. ERK siRNA blocks the lysoPC-induced activation of NADPH oxidase, demonstrating that ERK activation is upstream of NADPH oxidase. The reactive oxygen species produced by NADPH oxidase promote myosin light chain kinase (MLCK) activation with phosphorylation of MLC and TRPC5 externalization. Downregulation of ERK, NADPH oxidase, or MLCK with the relevant siRNA prevents TRPC5 externalization. Blocking MLCK activation prevents the prolonged rise in intracellular calcium levels and preserves endothelial migration in the presence of lysoPC.

TRPC channels: Structure, function, regulation and recent advances in small molecular probes

“…In cardiovascular system, TRPC channels are directly or indirectly regulated by several endogenous factors involved in the pathogenesis of various cardiovascular disorders. Recent studies show that brain-derived neurotrophic factor (BDNF) protects against myocardial infarction through TRPC3/6 channels (Hang et al, 2015); prolonged activation of the exchange protein directly activated by cAMP (EPAC) causes upregulation in the expression of TRPC3 and TRPC4 proteins and enhanced store-operated Ca 2+ entry in adult rat ventricular cardiomyocytes, which amounts to a proarrhythmic effect (Dominguez-Rodriguez et al, 2015); Transforming growth factor beta 1 (TGFβ1) induces upregulation of TRPC6 in vascular smooth muscle cells and in turn stress fiber formation that may underlie pathogenesis of vascular fibrosis (Park et al, 2017); lysophosphatidylcholine (LPC) activates TRPC6 channels in bovine aortic endothelial cells to inhibit endothelial cell migration and thereby delaying the healing of arterial injuries (Chaudhuri et al, 2008;Chaudhuri, Rosenbaum, Birnbaumer, & Graham, 2017). Other factors, such as atrial natriuretic peptide (ANP), endoglin, MicroRNAs, nitric oxide (NO) and protein kinase G, have also been shown to participate in various pathophysiological mechanisms by modulating TRPC channel function and/or expression [Chen et al, 2013;Feng, Xu, & Wang, 2018;Morine et al, 2016;.…”

The Role of Endothelial Ca2+ Signaling in Neurovascular Coupling: A View from the Lumen

“…It is, therefore, unlikely that it contributes to SOCE in mouse brain microvascular endothelial cells, which lack Orai1 [ 76 ], while it may assemble with polycystic TRP2 (TRPP2) to form a stretch-sensitive Ca 2+ -permeable channel [ 116 ]. In addition to SOCE, PLCβ-dependent signaling may lead to the activation of diacylglycerol (DAG)-sensitive Ca 2+ channels, such as TRPC3 [ 117 , 118 ] and TRPC6 [ 119 , 120 ]. The TRP superfamily of non-selective cation channels consists of 28 members that are classified into six sub-families based on their amino acid sequence homology and structural homology [ 121 , 122 ].…”

Endothelial Transient Receptor Potential Channels and Vascular Remodeling: Extracellular Ca2 + Entry for Angiogenesis, Arteriogenesis and Vasculogenesis

“…Earlier work showed that TRPC5 may negatively affect cell migration in BAECs. According to these studies, TRPC6-mediated Ca 2+ influx recruits myosin light chain kinase (MLCK) in an ERK-and NADPH oxidase-dependent manner, thereby inducing TRPC5 to translocate toward the plasma membrane and inhibit BAEC motility (Chaudhuri et al, 2008(Chaudhuri et al, , 2017. Recently, however, TRPC5 was found to sustain intracellular Ca 2+ oscillations and tube formation in EA.hy926 cells (Antigny et al, 2012).…”