title: LRRC8A channels support TNFα-induced superoxide production by Nox1 which is required for receptor endocytosis.
abstract: 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.
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, United States.
authors.forename: Hyehun
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ettinger
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, United States.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Rohrbough
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Dikalova
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, United States.
authors.forename: Hong N
authors.initials: HN
authors.lastname: Nguyen
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, United States. Electronic address: fred.s.lamb@vanderbilt.edu.
authors.forename: Fred S
authors.initials: FS
authors.lastname: Lamb
mesh_terms: D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007189:Indans / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000074624:NADPH Oxidase 1 / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism
chemical_list: C443139:4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-one-5-yl)oxybutyric acid; D003517:Cyclopentanes; D007189:Indans; C485976:LRRC8A protein, human; D008565:Membrane Proteins; D016328:NF-kappa B; D021122:Protein Subunits; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D013481:Superoxides; D013482:Superoxide Dismutase; D000074624:NADPH Oxidase 1; C402799:NOX1 protein, human; D048031:JNK Mitogen-Activated Protein Kinases
keywords: Leucine Rich Repeat Containing 8; NADPH oxidase 1; Superoxide; Tumor necrosis factor-α; c-Jun N-terminal kinase