TY - JOUR
T1 - 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
AU - Thangjam, Gagan S.
AU - Dimitropoulou, Chistiana
AU - Joshi, Atul D.
AU - Barabutis, Nektarios
AU - Shaw, Mary C.
AU - Kovalenkov, Yevgeniy
AU - Wallace, Chistopher M.
AU - Fulton, David J.
AU - Patel, Vijay
AU - Catravas, John D.
PY - 2014/5
Y1 - 2014/5
N2 - 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 IKBa 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 kB elements of the IKBa (an NFkB-responsive gene) promoter. The effect of LPS on IKBa mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of17-AAGwas independent ofHDAC.Weconclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.
AB - 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 IKBa 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 kB elements of the IKBa (an NFkB-responsive gene) promoter. The effect of LPS on IKBa mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of17-AAGwas independent ofHDAC.Weconclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.
KW - CAMP response element binding protein binding protein
KW - Heat shock protein 90 inhibitor
KW - Human lung microvascular endothelial cells
KW - LPS
KW - NF-κB
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U2 - 10.1165/rcmb.2013-0214OC
DO - 10.1165/rcmb.2013-0214OC
M3 - Article
C2 - 24303801
AN - SCOPUS:84899732608
SN - 1044-1549
VL - 50
SP - 942
EP - 952
JO - American journal of respiratory cell and molecular biology
JF - American journal of respiratory cell and molecular biology
IS - 5
ER -