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

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels, and the subsequent increase in intracellular Ca²⁺ 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^phox and subsequent NADPH oxidase activation with increased production of reactive oxygen species. ERK activation requires TRPC6 opening and influx of Ca²⁺ 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.