The Small GTPase RhoB Regulates TNFα Signaling in Endothelial Cells

Jeffrey Kroon,Sven Van Amstel,Simon Tol,Judith A Elias,Mar Fernandez-Borja,Venuprasad K Poojary

doi:10.1371/journal.pone.0075031

Jeffrey Kroon, Sven Van Amstel + Show 4 more

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https://doi.org/10.1371/journal.pone.0075031

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Journal: PLoS ONE	Publication Date: Sep 26, 2013
Citations: 64	License type: CC BY 4.0

Affiliation: Sanquin, University of Amsterdam

Abstract

The inflammatory response of endothelial cells triggered by cytokines such as TNFα and IL1β plays a pivotal role in innate immunity. Upon pro-inflammatory cytokine stimulation, endothelial cells produce chemokines and cytokines that attract and activate leukocytes, and express high levels of leukocyte adhesion molecules. This process is mediated by intracellular signaling cascades triggered by activation of e.g. the TNFα receptor (TNFR) that lead to the activation of the NFκB transcription factor and of MAP kinases, which in turn activate inflammatory gene transcription. We found that the small GTPase RhoB was strongly and rapidly upregulated in primary human endothelial cells by TNFα, IL1β and LPS. We subsequently investigated the role of RhoB in the regulation of TNFR signaling in endothelial cells by silencing RhoB expression with siRNA. We provide evidence that the TNFα-induced activation of p38 MAP kinase is strongly dependent on RhoB, but not on RhoA, while JNK activation is regulated by both RhoB and RhoA. Consistent with the important role of p38 MAP kinase in inflammation, we demonstrate that loss of RhoB impairs TNFα-induced ICAM-1 expression and reduces cell production of IL6 and IL8. In addition, we show that RhoB silencing alters the intracellular traffic of TNFα after endocytosis. Since RhoB is a known regulator of the intracellular traffic of membrane receptors, our data suggest that RhoB controls TNFα signaling through the regulation of the TNFR traffic.

Highlights

Tumor necrosis factor a (TNFa) is a pleiotropic pro-inflammatory cytokine that plays a pivotal role in the innate immune response to infection and tissue injury
We found that the pro-inflammatory mediators TNFa, IL1b and bacterial LPS potently stimulated RhoB expression in primary human endothelial cells, while RhoA expression was unchanged (Figure 1A)
GTP-RhoB could be detected in lysates of cells stimulated with TNFa (Figure 1B), which indicates that TNFa increases the level of activated RhoB in endothelial cells

Summary

Introduction

Tumor necrosis factor a (TNFa) is a pleiotropic pro-inflammatory cytokine that plays a pivotal role in the innate immune response to infection and tissue injury. Vascular endothelial cells respond to TNFa by upregulating the expression of cytokines and chemokines, such as IL-6 and IL-8, and of endothelial leukocyte adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin [1] These molecules enable TNFa-activated endothelial cells to attract, activate and recruit circulating leukocytes, which subsequently extravasate to reach the site of infection or injury. TNFR trimerizes and recruits TRAF-2 (TNFRassociated factor 2) and RIP1 (receptor interacting protein 1) to its cytoplasmic death domain. The formation of this signaling complex leads to the activation of the transcription factor NFkB and of the MAP kinases JNK and p38. One study has demonstrated that internalization of the TNFR from the plasma membrane is a required step for the activation of p38 and JNK MAP kinases [10]

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