Autophagy protein ATG7 is a critical regulator of endothelial cell inflammation and permeability

Mohammad Shadab,Antony Leonard,Spencer A Slavin,Michelle Warren Millar,Fabeha Fazal,Arshad Rahman

doi:10.1038/s41598-020-70126-7

Mohammad Shadab, Antony Leonard + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-70126-7

Copy DOI

Journal: Scientific Reports	Publication Date: Aug 13, 2020
Citations: 16	License type: open-access

Affiliation: University of Rochester

Abstract

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury. In this study, we investigated the role of ATG7, an essential autophagy regulator with no autophagy-unrelated functions, in the mechanism of EC inflammation and permeability. Knockdown of ATG7 using si-RNA significantly attenuated thrombin-induced expression of proinflammatory molecules such as IL-6, MCP-1, ICAM-1 and VCAM-1. Mechanistic study implicated reduced NF-κB activity in the inhibition of EC inflammation in ATG7-silenced cells. Moreover, depletion of ATG7 markedly reduced the binding of RelA/p65 to DNA in the nucleus. Surprisingly, the thrombin-induced degradation of IκBα in the cytosol was not affected in ATG7-depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. This is likely due to suppression of thrombin-induced phosphorylation and thereby inactivation of Cofilin1, an actin-depolymerizing protein, in ATG7-depleted cells. Actin stress fiber dynamics are required for thrombin-induced translocation of RelA/p65 to the nucleus, and indeed our results showed that ATG7 silencing inhibited this response via inactivation of Cofilin1. ATG7 silencing also reduced thrombin-mediated EC permeability by inhibiting the disassembly of VE-cadherin at adherens junctions. Together, these data uncover a novel function of ATG7 in mediating EC inflammation and permeability, and provide a mechanistic basis for the linkage between autophagy and EC dysfunction.

Highlights

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury
To determine how ATG7 silencing in EC affects thrombin-induced autophagic activity, we monitored the level of LC3-II through Western blot and the formation of LC3 puncta by confocal microscopy
We found that thrombin challenge did not affect the levels of ATG7 in cells transfected with specific for human ATG7 (si-ATG7) or siRNA control (si-Con) (Fig. S1)

Summary

Introduction

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury. IκBα undergoes phosphorylation at Ser[32] and Ser[36] leading to its proteasome-mediated degradation and subsequent release of NF-κB (predominantly RelA/p65 homodimer in EC)[13,14] This allows NF-κB to translocate to the nucleus and transcribe target proinflammatory genes including adhesion molecules, cytokines, and c hemokines[12,15,16]. Autophagy is a cellular process that facilitates the recycling of intracellular components like organelles and proteins[28] It plays a vital role when nutrients are scarce by utilizing the damaged organs or proteins to provide an alternative source of energy[28,29,30,31]. As cleavage and conjugation of LC3-I to LC3-II occurs only after induction of autophagy, detection of LC3-II has become a reliable readout for activation of the autophagic process

Methods

Results

Conclusion