Leucine Rich Repeat Containing 8A Is Required For NADPH Oxidase 1‐Dependent Superoxide Production and Tumor Necrosis Factor Alpha Receptor Endocytosis

Abstract

Tumor necrosis factor‐α (TNFα) induces superoxide (O2−•) production by NADPH oxidase 1 (Nox1) and activates volume‐regulated anion channel (VRAC) in vascular smooth muscle cells (VSMC). The Leucine Rich Repeat Containing 8A (LRRC8A) protein is part of VRAC, which supports cell proliferation and inflammation. We hypothesized that LRRC8A supports TNFα signaling. Reduction of LRRC8A expression (siLRRC8A) or inhibition of VRAC (DCPIB 30μM) inhibited responses to TNFα including; VRAC and NF‐kB activation (72% decrease), iNOS (52% decrease) and VCAM (36% decrease) expression, and proliferation (siControl 100%, siControl+TNFα 111±1.5%, siLRRC8A+TNFα 101±1.6%). siLRRC8A also reduced extracellular O2−• production (siControl+TNFα 1259±80, siLRRC8A+TNFα 792±86 pmol/mg of protein). Biotinylated TNFα was used to visualize TNFα receptor (TNFR) endocytosis with FITC‐conjugated avidin. TNFR, but not transferrin receptor endocytosis was impaired by siLRRC8A and DCPIB. Extracellular superoxide dismutase (500 U/mL) also inhibited TNFR endocytosis, while catalase (1000 U/mL) did not. LRRC8A co‐immunoprecipitated with the p22phox subunit of Nox1 suggesting a direct functional link to activity of the Nox1 complex. Thus, LRRC8A is required for Nox1‐mediated O2−• production, and extracellular O2−• is required for TNFR endocytosis. These data provide a novel mechanism for the anti‐proliferative and anti‐inflammatory effects of VRAC inhibition.