Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
Rajesh Kumar Gandhirajan, … , Donald L. Gill, Muniswamy Madesh
Rajesh Kumar Gandhirajan, … , Donald L. Gill, Muniswamy Madesh
Published January 25, 2013
Citation Information: J Clin Invest. 2013;123(2):887-902. https://doi.org/10.1172/JCI65647.
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Research Article

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

Abstract

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1–dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1–/– mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3–dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release–activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

Authors

Rajesh Kumar Gandhirajan, Shu Meng, Harish C. Chandramoorthy, Karthik Mallilankaraman, Salvatore Mancarella, Hui Gao, Roshanak Razmpour, Xiao-Feng Yang, Steven R. Houser, Ju Chen, Walter J. Koch, Hong Wang, Jonathan Soboloff, Donald L. Gill, Muniswamy Madesh

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Figure 6

NOX2-derived ROS promotes STIM1-dependent Ca2+ entry and NFAT activation after LPS stimulation.

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NOX2-derived ROS promotes STIM1-dependent Ca2+ entry and NFAT activation...
Wild-type, gp91phox–/–, and Stim1 KD ECs were treated with LPS (1 μg/ml) for 16 hours. (A–C) Extracellular Ca2+ was removed for 5 minutes prior to assessment of SOCe activity in (A) wild-type, (B) gp91phox–/–, and (C) Stim1 KD ECs. Wild-type, gp91phox–/–, and Stim1 KD ECs were transduced with adenovirus encoding NFATc3-GFP for 36 hours and then treated with LPS (1 μg/ml) for an additional 16 hours. (D) Quantification of nuclear NFAT-positive cells. (E) gp91phox–/– cells loaded with Fluo-4 were treated with thapsigargin (2 μM) under nominally Ca2+-free conditions, followed by addition of 2 mM Ca2+ where indicated to assess SOCe. (F) Fluo-4–loaded gp91phox–/– ECs were treated with (right) or without (left) superoxide (xanthine + xanthine oxidase [O2.–]; 10 nmol/min). (G) gp91phox–/– ECs were transduced with AdNFATc3-GFP for 36 hours, and nuclear NFATc3-GFP was quantified after O2.– exposure. Scale bar: 20 μm. (H) gp91phox–/– ECs were transfected with either STIM1 wild-type or STIM1 C56A mutant constructs. Extracellular Ca2+ was removed for 5 minutes prior to the measurement of SOCe activity in Fluo-4–loaded ECs. (I) gp91phox–/– ECs transiently expressing either wild-type or STIM1 C56A plasmids were transduced with AdNFATc3-GFP for 36 hours, and nuclear NFATc3GFP was quantified. Scale bar: 20 μm. All data are the mean ± SEM of 3 independent experiments, each experiment consisting of triplicate analyses of 20–30 cells. **P < 0.01, ***P < 0.001.