MPS 18-06 LRRC8A/C VOLTAGE-DEPENDENT ANION CHANNELS ARE REQUIRED FOR TNFALPHA-INDUCED SUPEROXIDE PRODUCTION BY NOX1 IN VASCULAR SMOOTH MUSCLE CELLS

Abstract

Objective: Tumor necrosis factor-α (TNFα), pro-inflammatory cytokine, is linked to cardiovascular risk, including inflammatory vascular lesions and hypertension. TNFα-induced superoxide (O2−•) production by NADPH oxidase 1 (Nox1) is required for inflammatory signaling in vascular smooth muscle cells (VSMC). TNFα also activates volume-regulated anion channels (VRACs). These multimeric Cl− channels are composed of Leucine Rich Repeat Containing 8A (LRRC8A) protein, combined with various closely-related isoforms (LRRC8B through E). This produces VRACs with differing biophysical properties that support VSMC proliferation and inflammation by an unknown mechanism. We hypothesized that VRAC current is required for Nox1 activation by TNFα. Design and Method: In VSMC, reducing expression (siRNA) of LRRC8A through E or inhibiting VRAC current (DCPIB 30 μM) were used to measure TNFα signaling. Extracellular O2−• production by Nox1 was detected by CAT1H spin probe using electron spin resonance. Biotinylated TNFα was used to visualize TNFα receptor (TNFR1) endocytosis with FITC-conjugated avidin. Immunoprecipitation-western blot analysis was performed with LRRC8A and p22phox subunit of Nox1. Results: siRNA of LRRC8A or C (but not the B, D, or E isoforms) or DCPIB inhibited NF-κB activation (72% decrease), induction of iNOS (52% decrease) and VCAM (36% decrease) expression, and proliferation (siControl 100%, siControl + TNFα 111 ± 1.5%, siLRRC8A + TNFα 101 ± 1.6%). siLRRC8A reduced extracellular O2−• production by Nox1 (siControl + TNFα 1259 ± 80, siLRRC8A + TNFα 792 ± 86 pmol/mg of protein). TNFR1, but not transferrin receptor endocytosis, was impaired by siNox1, siLRRC8A or DCPIB. Extracellular superoxide dismutase (500 U/mL) also potently inhibited TNFR1 endocytosis, while catalase (1000 U/mL) had no effect. Finally, LRRC8A co-immunoprecipitated with the p22phox subunit of Nox1 suggesting a direct functional link to activity of the Nox1 complex. Conclusions: LRRC8A/C VRACs are required for Nox1 activation at the plasma membrane and the extracellular O2−• that it produces is required for TNFR1 endocytosis. These data provide a novel mechanism for the anti-proliferative and anti-inflammatory effects of VRAC inhibition in VSMC.