Decrease of Intracellular Chloride Concentration Promotes Endothelial Cell Inflammation by Activating Nuclear Factor-κB Pathway

Abstract

Recent evidence suggested that ClC-3 channel/antiporter is involved in regulation of nuclear factor (NF)-κB activation. However, the mechanism explaining how ClC-3 modulates NF-κB signaling is not well understood. We hypothesized that ClC-3-dependent alteration of intracellular chloride concentration ([Cl(-)](i)) underlies the effect of ClC-3 on NF-κB activity in endothelial cells. Here, we found that reduction of [Cl(-)](i) increased tumor necrosis factor-α (TNFα)-induced expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and adhesion of monocytes to endothelial cells (P<0.05; n=6). In Cl(-) reduced solutions, TNFα-evoked IκB kinase complex β and inhibitors of κBα phosphorylation, inhibitors of κBα degradation, and NF-κB nuclear translocation were enhanced. In addition, TNFα and interleukin 1β could activate an outward rectifying Cl(-) current in human umbilical vein endothelial cells and mouse aortic endothelial cells. Knockdown or genetic deletion of ClC-3 inhibited or abolished this Cl(-) conductance. Moreover, Cl(-) channel blockers, ClC-3 knockdown or knockout remarkably reduced TNFα-induced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocytes to endothelial cell adhesion, and NF-κB activation (P<0.01; n=6). Furthermore, TNFα-induced vascular inflammation and neutrophil infiltration into the lung and liver were obviously attenuated in ClC-3 knockout mice (P<0.01; n=7). Our results demonstrated that decrease of [Cl(-)](i) induced by ClC-3-dependent Cl(-) efflux promotes NF-κB activation and thus potentiates TNFα-induced vascular inflammation, suggesting that inhibition of ClC-3-dependent Cl(-) current or modification of intracellular Cl(-) content may be a novel therapeutic approach for inflammatory diseases.

Reduction of Intracellular Chloride Concentration Promotes Foam Cell Formation

“…6 Here, we found that lowering [Cl − ]i increases the expression of inflammatory genes such as IL-1β, IL-6 and TNF-α in peritoneal macrophages from high-fat-diet-fed apoE −/− mice and in oxLDL-treated human macrophages. These data suggest that lowering [Cl − ]i-induced foam cells are functionally inflammatory form of macrophages.…”

“…6 Briefly, RPMI 1640 medium lacking NaCl and KCl was initially obtained from Invitrogen (Grand Island, NE, USA). The normal chloride medium was prepared by adding 105 mmol/L NaCl and 5 mmol/L KCl.…”

“…6 Briefly, cells were washed 3 times with ice-cold PBS and then lysed with RIPA lysis buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Merck, Darmstadt, Germany). Equal proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA).…”

Endophilin A2 Influences Volume-Regulated Chloride Current by Mediating ClC-3 Trafficking in Vascular Smooth Muscle Cells

“…The isosmotic bath solution was in the pathology of hypertensive cerebrovascular remodeling, 10-12 atherogenesis, 13 and vascular inflammatory responses. 14, 15 16 We demonstrated that ClC-3 protein is distributed broadly in the cytoplasm and cell membrane of A10 VSMCs and primary culture of BASMCs. Even though ClC-3 is primarily localized in the cytoplasm of some cell types, ClC-3 protein could be transiently trafficked to the cell membrane during clathrin-mediated endocytosis, 17 suggesting the existence of a dynamic mechanism regulating subcellular distribution of ClC-3 protein.…”

“…15 Plasmids were transfected into the cells at 80% confluence with Lipofectamine 2000, according to the manufacturer's instruction (Invitrogen).…”