ClC‐3 is required for normal NF‐kB activation by inflammatory mediators in vascular smooth muscle (VSM)

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Inflammatory mediators such as interleukin-1beta (IL-1β) and tumor necrosis factor alpha (TNFα) alter VSM cell proliferation and apoptosis in a reactive oxygen species (ROS)-dependent manner involving the regulation of gene expression by nuclear factor kappa B (NF-κB). VSM proliferation is inhibited by antisense oligo-mediated reduction in the expression of the intracellular anion channel ClC-3 (Circ. Res. 91:E28–32, 2002). We observed that cultured Clcn3−/− VSM cells did not proliferate as rapidly as Clcn3+/+ cells, were more sensitive to H2O2-induced apoptosis, and displayed enhanced differentiation as reflected by increased expression of smooth muscle α-actin and myosin heavy chain. We hypothesized that altered ROS-dependent signaling explained these observations. Activation of NF-κB in Clcn3+/+ cells was blocked by the NADPH oxidase inhibitor DPI and the antioxidant N-acetyl cysteine. Clcn3−/− cells had lower basal NF-κB activity and failed to properly activate NF-κB in response to IL-1β or TNFα. Treatment of Clcn3+/+ VSM cells with DIDS (0.1–1mM) reduced basal NF-κB activity, blocked activation by mitogens and decreased proliferation. Finally, an anti-ClC-3 Ab precipitates Nox1 from human aortic VSM lysates, as well as several proteins associated with intracellular vesicles. We conclude that ClC-3 participates in NAPDH oxidase-dependent signaling involving redox-mediated activation of NF-κB, and hypothesize that superoxide anion generated by Nox1 in intracellular vesicles moves through ClC-3 into the cytoplasm where ROS signaling occurs. HL062483 and an AHA EI to FSL and HL081750 to FJM.