LRRC8A channels support TNFα-induced superoxide production by Nox1 which is required for receptor endocytosis

• Published in: Free radical biology & medicine 2016-12, Vol.101, p.413-423
• Main Authors: Choi, Hyehun, Ettinger, Nicholas, Rohrbough, Jeffrey, Dikalova, Anna, Nguyen, Hong N., Lamb, Fred S.
• Format: Article
• Language: English
• Subjects: c-Jun N-terminal kinase; Cell Line; Cyclopentanes - pharmacology; Endocytosis - drug effects; Gene Expression Regulation; HEK293 Cells; Humans; Indans - pharmacology; JNK Mitogen-Activated Protein Kinases - genetics; JNK Mitogen-Activated Protein Kinases - metabolism; Leucine Rich Repeat Containing 8; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - genetics; Membrane Proteins - metabolism; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - metabolism; NADPH oxidase 1; NADPH Oxidase 1 - genetics; NADPH Oxidase 1 - metabolism; NF-kappa B - genetics; NF-kappa B - metabolism; Phosphorylation - drug effects; Protein Subunits - genetics; Protein Subunits - metabolism; Receptors, Tumor Necrosis Factor, Type I - genetics; Receptors, Tumor Necrosis Factor, Type I - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Signal Transduction; Superoxide; Superoxide Dismutase - pharmacology; Superoxides - metabolism; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-α; Vascular Cell Adhesion Molecule-1 - genetics; Vascular Cell Adhesion Molecule-1 - metabolism
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2016.11.003

Leucine Rich Repeat Containing 8A (LRRC8A) is a required component of volume-regulated anion channels (VRACs). In vascular smooth muscle cells, tumor necrosis factor-α (TNFα) activates VRAC via type 1 TNFα receptors (TNFR1), and this requires superoxide (O2•-) production by NADPH oxidase 1 (Nox1). VRAC inhibitors suppress the inflammatory response to TNFα by an unknown mechanism. We hypothesized that LRRC8A directly supports Nox1 activity, providing a link between VRAC current and inflammatory signaling. VRAC inhibition by 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) impaired NF-κB activation by TNFα. LRRC8A siRNA reduced the magnitude of VRAC and inhibited TNFα-induced NF-κB activation, iNOS and VCAM expression, and proliferation of VSMCs. Signaling steps disrupted by both siLRRC8A and DCPIB included; extracellular O2•- production by Nox1, c-Jun N-terminal kinase (JNK) phosphorylation and endocytosis of TNFR1. Extracellular superoxide dismutase, but not catalase, selectively inhibited TNFR1 endocytosis and JNK phosphorylation. Thus, O2•- is the critical extracellular oxidant for TNFR signal transduction. Reducing JNK expression (siJNK) increased extracellular O2•- suggesting that JNK provides important negative feedback regulation to Nox1 at the plasma membrane. LRRC8A co-localized by immunostaining, and co-immunoprecipitated with, both Nox1 and its p22phox subunit. LRRC8A is a component of the Nox1 signaling complex. It is required for extracellular O2•- production, which is in turn essential for TNFR1 endocytosis. These data are the first to provide a molecular mechanism for the potent anti-proliferative and anti-inflammatory effects of VRAC inhibition. [Display omitted] •Nox1 activation by TNFα requires functional LRRC8A anion channels.•TNFα receptor endocytosis requires Nox1-derived extracellular O2•-.•JNK provides negative feedback regulation of Nox1 at the plasma membrane.•Nox1-LRRC8A interdependence may allow anion channel blockers to inhibit inflammation.