Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice
Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam
Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam
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Research Article Inflammation Vascular biology

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice

Abstract

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a nonlethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16–24 hours postinjection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I IFN–like program and high expression of prothrombotic and proadhesive genes. Consistently, we observed intraluminal leukocyte adhesion and neutrophil extracellular trap–osis (NETosis), as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In human umbilical vein endothelial cells, pulse-chase experiments showed that SOCS3 protein had a half-life less than 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation led to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrate that the regulation of SOCS3 protein levels is critical to inhibit IL-6–mediated endotheliopathy during shock and provide a promising therapeutic avenue to prevent multiorgan dysfunction through stabilization of endothelial SOCS3.

Authors

Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam

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Figure 6

Levels of SOCS3 protein are rapidly degraded by the ubiquitin/proteasome pathway.

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Levels of SOCS3 protein are rapidly degraded by the ubiquitin/proteasome...
(A) Western blot analysis showing SOCS3 expression and STAT3 phosphorylation levels 2 hours after treatment with IL-6+R in the presence or absence of the proteasome inhibitor MG-132. (B) Pulse-chase experiment of cells treated for 6 hours with IL-6+R and then for the specified time with either PBS or the protein synthesis inhibitor CHX prior to lysis. Representative Western blots (left) and quantification of 3 independent experiments (right). Two-way ANOVA with Dunnett’s post hoc test (CHX vs. PBS). (C) Diagram of the SOCS3 overexpression strategy. (D) Western blots showing the expression levels of exogenous SOCS3 and their effects on IL-6+R–induced STAT3 phosphorylation. (E) Quantification of pulse-chase experiments by measuring the levels of Flag-SOCS3 after CHX. Two-way ANOVA with Dunnett’s post hoc test (K6Q- vs. WT-SOCS3). (F) Transendothelial electric resistance measurements of monolayers expressing the different constructs and treated with either PBS or IL-6+R. Two-way ANOVA of repeated measurements with Dunnett’s post hoc test (vs. PBS-treated, noninfected cells). Asterisks denote P < 0.05. Data representative of at least 3 independent experiments.