TNFα Signals via p66Shc to Induce E-Selectin, Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Luigi Laviola,Rossella Labarbuta,Annalisa Natalicchio,Sabina Martemucci,Maura Roberta Orlando,Francesco Giorgino,Tiziana Cocco,Anna Leonardini,Consiglia Pacelli,Angelo Cignarelli,Maria Angela Incalza,Cristina Caccioppoli,Mariangela Melchiorre,Sebastio Perrini,Federica Tortosa,Masuko Ushio-Fukai

doi:10.1371/journal.pone.0081930

Luigi Laviola, Rossella Labarbuta + Show 14 more

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https://doi.org/10.1371/journal.pone.0081930

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Abstract

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66Shc on Ser36 and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66Shc in HUVEC resulted in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66Shc protein, in which Ser36 was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66Shc on Ser36.

Highlights

Endothelial dysfunction plays a major role in the pathogenesis of vascular damage [1]
TNFα action and signaling in human umbilical vein endothelial cells (HUVEC) Leukocyte transmigration through the endothelial monolayer, which represents a marker of endothelial activation, was significantly increased after exposure of HUVEC to TNFα for 1 h (P
These findings indicate that TNFα stimulates the increase of E-Selectin gene expression via JNK but not ERK signaling in HUVEC

Summary

Introduction

Endothelial dysfunction plays a major role in the pathogenesis of vascular damage [1]. The functional impairment of the endothelium induced by metabolic factors and/or cytokines precedes the development of morphological alterations [1,2], and is documented at a biochemical level by the abnormal expression of endothelial cell-specific genes [3]. E-Selectin is a Ca2+-dependent cell surface glycoprotein that recruits leukocytes under proinflammatory conditions [8], and is transcriptionally induced in response to inflammatory cytokines, such as IL-1β and TNFα [9]. PLOS ONE | www.plosone.org p66Shc in TNFα-Induced Endothelial Dysfunction associated with a pro-inflammatory condition, including diabetes, atherosclerosis, rheumatoid arthritis, and cancer [10,11,12]. Therapeutic strategies, which reduce vascular injury, result in significant down-regulation of ESelectin gene expression [13,14,15]

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