title: Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.
abstract: BACKGROUND
  Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown.
  
  
  OBJECTIVE
  Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene.
  
  
  RESULTS
  Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions.
  
  
  CONCLUSIONS
  This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.). myron.cybulsky@utoronto.ca milstone@rascal.med.harvard.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Milstone
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Motoi
authors.initials: M
authors.lastname: Ilyama
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Mian
authors.initials: M
authors.lastname: Chen
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Peter
authors.initials: P
authors.lastname: O'Donnell
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Vannessa M
authors.initials: VM
authors.lastname: Davis
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Jorge
authors.initials: J
authors.lastname: Plutzky
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Brown
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Saptarsi M
authors.initials: SM
authors.lastname: Haldar
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Allan
authors.initials: A
authors.lastname: Siu
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Lau
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Su-Ning
authors.initials: SN
authors.lastname: Zhu
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Mayada F
authors.initials: MF
authors.lastname: Basheer
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Tucker
authors.initials: T
authors.lastname: Collins
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).
authors.forename: Jenny
authors.initials: J
authors.lastname: Jongstra-Bilen
authors.affiliation: From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.). myron.cybulsky@utoronto.ca milstone@rascal.med.harvard.edu.
authors.forename: Myron I
authors.initials: MI
authors.lastname: Cybulsky
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D050197:Atherosclerosis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D020212:Carotid Artery Injuries / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000502:physiology; D002791:Cholesterol, Dietary / Q000009:adverse effects; D019040:E-Selectin / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D025941:Protein Interaction Mapping; D012319:RNA Polymerase II / Q000378:metabolism; D011973:Receptors, LDL / Q000172:deficiency; D020218:Response Elements / Q000235:genetics / Q000528:radiation effects*; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic; D017539:Tunica Intima / Q000378:metabolism* / Q000473:pathology; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D002791:Cholesterol, Dietary; D019040:E-Selectin; D011973:Receptors, LDL; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; D019010:Vascular Cell Adhesion Molecule-1; D012319:RNA Polymerase II
keywords: NF-κB; atherosclerosis; chromatin immunoprecipitation; endothelial cells; gene expression; tunica intima; vascular cell adhesion molecule-1