title: Novel mechanism of attenuation of LPS-induced NF-κB activation by the heat shock protein 90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, in human lung microvascular endothelial cells.
abstract: Heat shock protein (hsp) 90 inhibition attenuates NF-κB activation and blocks inflammation. However, the precise mechanism of NF-κB regulation by hsp90 in the endothelium is not clear. We investigated the mechanisms of hsp90 inhibition by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) on NF-κB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-κB was measured by luciferase reporter assay, gene expression by real-time RT-PCR, DNA binding of transcription factors by chromatin immunoprecipitation assay, protein-protein interaction by coimmunoprecipitation/immunoblotting, histone deacetylase (HDAC)/histone acetyltransferase enzyme activity by fluorometry, and nucleosome eviction by partial microccocal DNase digestion. In human lung microvascular endothelial cells, 17-AAG-induced degradation of IKBα was accomplished regardless of the phosphorylation/ubiquitination state of the protein. Hence, 17-AAG did not block LPS-induced NF-κB nuclear translocation and DNA binding activity. Instead, 17-AAG blocked the recruitment of the coactivator, cAMP response element binding protein binding protein, and prevented the assembly of a transcriptionally competent RNA polymerase II complex at the κB elements of the IKBα (an NF-κB-responsive gene) promoter. The effect of LPS on IKBα mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3 binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of 17-AAG was independent of HDAC. We conclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.
authors.affiliation: 1 Vascular Biology Center.
authors.forename: Gagan S
authors.initials: GS
authors.lastname: Thangjam
authors.forename: Chistiana
authors.initials: C
authors.lastname: Dimitropoulou
authors.forename: Atul D
authors.initials: AD
authors.lastname: Joshi
authors.forename: Nektarios
authors.initials: N
authors.lastname: Barabutis
authors.forename: Mary C
authors.initials: MC
authors.lastname: Shaw
authors.forename: Yevgeniy
authors.initials: Y
authors.lastname: Kovalenkov
authors.forename: Chistopher M
authors.initials: CM
authors.lastname: Wallace
authors.forename: David J
authors.initials: DJ
authors.lastname: Fulton
authors.forename: Vijay
authors.initials: V
authors.lastname: Patel
authors.forename: John D
authors.initials: JD
authors.lastname: Catravas
mesh_terms: D021581:Active Transport, Cell Nucleus; D016227:Benzoquinones / Q000494:pharmacology*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D054875:Ubiquitination
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D006657:Histones; D020672:I-kappa B Proteins; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides; D016328:NF-kappa B; C112765:tanespimycin; D006655:Histone Deacetylases