A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

Abstract

Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation.Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme.Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.

WITHDRAWN: Acute cerebral infarction patients' 3-month mortality predicted by peripheral tumor necrosis factor alpha-induced protein 3 mRNA level

“…Second, A20 could inhibit NF-κB activation through the TNF-α, TLR4, nucleotide binding oligomer domain, T cell B, and IL-17 pathways, thereby inhibiting NF-κB transcription in the cytoplasm and downstream in ammatory factors to reduce the in ammatory response 31 . Furthermore, A20 is antiin ammatory in Endothelial Cell (EC )) and SMC through its ability to concomitantly inhibit NF-κB activation in response to in ammatory (TNF), immune (CD40), and oxidative insults and interrupt atherogenic interferon gamma (IFNγ) signaling 32 , A20/ Tumor necrosis factor alpha induced protein (TNFAIP3) Increases ENOS Expression in an ERK5Dependent Manner to Support Health in the Face of In ammation 32 .…”

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“…Meanwhile, the MDA level refects the degree of oxidative stress. Endothelium-derived NO exerts antioxidant and antiapoptotic efects in ECs to elicit an anti-AS efect [33]. Indeed, endothelial dysfunction is closely related to the decreased bioavailability of NO due to reduced NO generation in EC or increased inactivation of NO by ROS [34,35].…”

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“…Simultaneously, the expression of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) is enhanced, which, in turn, upregulates expression of vascular and intercellular cell adhesion molecules and promotes adherence of monocytes. The rise in the expression of TNF-α reduces endothelial NOS expression and interferes with NO production [ 56 , 57 ].…”