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IKK? regulates endothelial thrombomodulin in a Klf2-dependent manner.

ABSTRACT: Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNF?) suppresses TM expression.TNF? has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-?B). However, because the TM promoter lacks an NF-?B binding site, the direct involvement of NF-?B has been controversial. We investigated the role of the upstream regulatory serine kinase, inhibitory kappa-B kinase-? (IKK?), in TM expression and function with or without TNF? treatment.Inhibition of IKK? was achieved by specific chemical inhibitors, siRNA or shRNA. TM expression was assessed by qRT-PCR, Western blot, flow cytometry, luciferase reporter assay and chromatin immune-precipitation (ChIP) assay. TM function was estimated by generation of activated protein C (APC). NF-?B activation was determined by immunocytochemistry.IKK? inhibition increased TM expression and function, and attenuated TNF?-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-?B protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKK? inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-?B pathway, respectively, resulted in TM over-expression. IKK? inhibition caused over-expression, increased promoter activity and enhanced binding of Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKK? inhibition-mediated TM up-regulation. We conclude that IKK? regulates TM in a Klf2-dependent manner.

SUBMITTER: Pathak R

PROVIDER: S-EPMC4163124 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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IKKβ regulates endothelial thrombomodulin in a Klf2-dependent manner.

Pathak R R Shao L L Chafekar S M SM Feng W W Ponnappan U U Fink L M LM Zhou D D Hauer-Jensen M M

Journal of thrombosis and haemostasis : JTH 20140811 9

<h4>Background</h4>Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression.<h4>Objective</h4>TNFα has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-κB). However, because the TM promoter lacks an NF-κB binding site, the direct involvement of NF-κB has been controversial. We investigated the role of the upstream regulato ...[more]

PMID: 25039491

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Project description: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.

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IKK? regulates endothelial thrombomodulin in a Klf2-dependent manner.

Publications

IKKβ regulates endothelial thrombomodulin in a Klf2-dependent manner.

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