IKKβ regulates endothelial thrombomodulin in a Klf2-dependent manner

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

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Background: Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression. Objective: TNFa 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 regulatory serine kinase, inhibitory kappa-B kinase-b (IKKb), in TM expression and function with or without TNFa treatment. Methods: Inhibition of IKKb 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-κB activation was determined by immunocytochemistry. Results and conclusions: IKKβ inhibition increased TM expression and function, and attenuated TNFa-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-κB protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKKb inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-κB pathway, respectively, resulted in TM over-expression. IKKβ inhibition caused over-expression, increased promoter activity and enhanced binding of Kr€uppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKb inhibition-mediated TM upregulation. We conclude that IKKβ regulates TM in a Klf2-dependent manner.

Original languageEnglish (US)
Pages (from-to)1533-1544
Number of pages12
JournalJournal of Thrombosis and Haemostasis
Volume12
Issue number9
DOIs
StatePublished - 2014
Externally publishedYes

Keywords

  • Endothelial cells
  • Inflammation
  • NF-kappa β
  • Thrombomodulin
  • Thrombosis

ASJC Scopus subject areas

  • Hematology

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