title: IKKβ regulates endothelial thrombomodulin in a Klf2-dependent manner.
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
  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 regulatory serine kinase, inhibitory kappa-B kinase-β (IKKβ), in TM expression and function with or without TNFα treatment.
  
  
  METHODS
  Inhibition of IKKβ 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.
  
  
  CONCLUSIONS
  IKKβ inhibition increased TM expression and function, and attenuated TNFα-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 IKKβ 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 Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKβ inhibition-mediated TM up-regulation. We conclude that IKKβ regulates TM in a Klf2-dependent manner.
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: R
authors.initials: R
authors.lastname: Pathak
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: L
authors.initials: L
authors.lastname: Shao
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: S M
authors.initials: SM
authors.lastname: Chafekar
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: W
authors.initials: W
authors.lastname: Feng
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: U
authors.initials: U
authors.lastname: Ponnappan
authors.affiliation: Desert Research Institute, Las Vegas, NV.
authors.forename: L M
authors.initials: LM
authors.lastname: Fink
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.
authors.forename: D
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.|Surgical Service, Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.
authors.forename: M
authors.initials: M
authors.lastname: Hauer-Jensen
mesh_terms: D000893:Anti-Inflammatory Agents / Q000737:chemistry; D000925:Anticoagulants / Q000737:chemistry; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011486:Protein C / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D000925:Anticoagulants; C096161:KLF2 protein, human; D051741:Kruppel-Like Transcription Factors; D016328:NF-kappa B; D011486:Protein C; D034741:RNA, Small Interfering; D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha; D051550:I-kappa B Kinase
keywords: NF-kappa β; endothelial cells; inflammation; thrombomodulin; thrombosis