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ClC‐3 is required for superoxide production in early endosomes and subsequent NF‐kappaB activation
Author(s) -
Filali Mohammed S.,
Gross Jordan R.,
Collins Malia M.,
Volk Kenneth A.,
Lamb Fred S.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a447-b
Subject(s) - endosome , chemistry , nadph oxidase , microbiology and biotechnology , superoxide , reactive oxygen species , antiporter , hek 293 cells , lucigenin , biochemistry , intracellular , biology , receptor , membrane , enzyme
ClC‐3 is an early endosomal Cl − ‐H + antiporter. We have previously shown that ClC‐3 is required for endosomal reactive oxygen species (ROS) generation in response to TNFα in vascular smooth muscle cells. To further explore the role of ClC‐3 we assayed NF‐κB activation by TNFα using an AdNF‐κB luciferase reporter in HEK293T cells. We also quantified NADPH‐induced ROS production by affinity purified (Nox1‐immunoprecipitated) early endosomes using lucigenin‐enhanced chemiluminescence. TNF‐α caused an increase in NF‐κB activity which was ROS‐dependent (inhibited by 10mM NAC or 10μM DPI). This effect was also inhibited by 300μM niflumic acid or DIDS, demonstrating that anion transport is required for the response to TNFα. RNAi directed at ClC‐3 reduced both basal and TNFα‐induced NF‐κB activity, as did overexpression of a mutant ClC‐3 plasmid lacking two glutamate residues that are critical for proton transport by ClC‐3 (E224A, E281A of short ClC‐3). Thus, ClC‐3 is specifically involved in NF‐κB activation. Nox‐1 immunopurified endosomes produced ROS in response to NADPH and this effect was inhibited by DPI, niflumic acid and DIDS as well as by an anti‐ClC‐3 antibody. Antibodies to ClC‐4 or ClC‐5 had no effect. In summary, ClC‐3 is required for superoxide production by early endosomes and these ROS participate in intracellular signaling through NF‐κB. Supported by HL06248 .