Inhibiting endothelial cell Mst1 attenuates acute lung injury in mice
Zhi-Fu Guo, Nopprarat Tongmuang, Chao Li, Chen Zhang, Louis Hu, Daniel Capreri, Mei-Xing Zuo, Ross Summer, Jianxin Sun
Zhi-Fu Guo, Nopprarat Tongmuang, Chao Li, Chen Zhang, Louis Hu, Daniel Capreri, Mei-Xing Zuo, Ross Summer, Jianxin Sun
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Research Article Inflammation Vascular biology

Inhibiting endothelial cell Mst1 attenuates acute lung injury in mice

Abstract

Lung endothelium plays a pivotal role in the orchestration of inflammatory responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1) is a serine/threonine kinase that has been shown to play an important role in the regulation of apoptosis, stress responses, and organ growth. This study investigated the role of Mst1 in lung endothelial activation and acute lung injury (ALI). We found that Mst1 was significantly activated in inflamed lung endothelial cells (ECs) and mouse lung tissues. Overexpression of Mst1 promoted nuclear factor κ-B (NF-κB) activation through promoting JNK and p38 activation in lung ECs. Inhibition of Mst1 by either its dominant negative form (DN-Mst1) or its pharmacological inhibitor markedly attenuated cytokine-induced expression of cytokines, chemokines, and adhesion molecules in lung ECs. Importantly, in a mouse model of lipopolysaccharide-induced (LPS-induced) ALI, both deletion of Mst1 in lung endothelium and treatment of WT mice with a pharmacological Mst1 inhibitor significantly protected mice from LPS-induced ALI. Together, our findings identified Mst1 kinase as a key regulator in controlling lung EC activation and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective in the prevention and treatment of inflammatory lung diseases.

Authors

Zhi-Fu Guo, Nopprarat Tongmuang, Chao Li, Chen Zhang, Louis Hu, Daniel Capreri, Mei-Xing Zuo, Ross Summer, Jianxin Sun

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Figure 2

Mst1 induces NF-κB activation in ECs.

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Mst1 induces NF-κB activation in ECs. (A) Schematic representation of Ms...
(A) Schematic representation of Mst1 mutants. (B) EA.hy926 cells were transfected with 100 ng of p(NF-κB)3-Luc, 10 ng of pRL-RSV, and 200 ng of either empty vector (EV) or Mst1 mutants. Thirty-six hours after transfection, luciferase assays were performed (n = 4). (C) Lung ECs were transfected with 100 ng of p(NF-κB)3-Luc, 10 ng of pRL-RSV, and increasing amounts of pFlag-DN-Mst1. Thirty-six hours after transfection, luciferase assays were performed 6 hours after treatment with or without 20 ng/mL TNF-α (n = 4). (D) Lung ECs were transduced with adenoviruses bearing LacZ (Ad-lacZ) or DN-Mst1 (Ad-DN-Mst1) (at a multiplicity of infection [MOI] of 50) for 48 hours and then treated in the presence or absence of 20 ng/mL TNF-α for 1 hour. Nuclear protein was extracted, and EMSA was performed. The NF-κB complex was partially supershifted by anti–NF-κB p65 antibody or blocked by cold competitive probe. (E) Human lung ECs were transduced with Ad-LacZ or Ad-Mst1 (MOI, 50). Forty-eight hours after transduction, cell lysates were collected for Western blot analysis. (F) EA.hy926 cells were pretreated with DMSO vehicle control, JNK inhibitor (SP600125, 20 μmol/L) or p38 MAPK inhibitor (SB203580, 20 μmol/L) for 1 hour. Subsequently, the cells were transfected with 500 ng of p(NF-kB)3-Luc, 50 ng of pRL-RSV, 200 ng of either empty vector (pFlag-EV) or pFlag-Mst1. Twenty-four hours after transfection, luciferase assays were performed using Dual-Luciferase Reporter Assay System. Significance was determined by a 2-way ANOVA with Bonferroni’s post hoc test (B, C, and F).