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

Gagan S. Thangjam, Chistiana Dimitropoulou, Atul D. Joshi, Nektarios Barabutis, Mary C. Shaw, Yevgeniy Kovalenkov, Chistopher M. Wallace, David J Fulton, Vijaykumar Surendrakant Patel, John D. Catravas

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)942-952
Number of pages11
JournalAmerican journal of respiratory cell and molecular biology
Volume50
Issue number5
DOIs
StatePublished - Jan 1 2014

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tanespimycin
HSP90 Heat-Shock Proteins
Endothelial cells
Endothelial Cells
Chemical activation
Lung
Nucleosomes
Histones
Transcriptional Activation
Assays
Histone Acetyltransferases
Cyclic AMP Response Element-Binding Protein
Fluorometry
Phosphorylation
Proteins
Histone Deacetylase Inhibitors
Histone Deacetylases
Deoxyribonucleases
RNA Polymerase II
Chromatin Immunoprecipitation

Keywords

  • CAMP response element binding protein binding protein
  • Heat shock protein 90 inhibitor
  • Human lung microvascular endothelial cells
  • LPS
  • NF-κB

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

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. / Thangjam, Gagan S.; Dimitropoulou, Chistiana; Joshi, Atul D.; Barabutis, Nektarios; Shaw, Mary C.; Kovalenkov, Yevgeniy; Wallace, Chistopher M.; Fulton, David J; Patel, Vijaykumar Surendrakant; Catravas, John D.

In: American journal of respiratory cell and molecular biology, Vol. 50, No. 5, 01.01.2014, p. 942-952.

Research output: Contribution to journalArticle

Thangjam, Gagan S. ; Dimitropoulou, Chistiana ; Joshi, Atul D. ; Barabutis, Nektarios ; Shaw, Mary C. ; Kovalenkov, Yevgeniy ; Wallace, Chistopher M. ; Fulton, David J ; Patel, Vijaykumar Surendrakant ; Catravas, John D. / 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. In: American journal of respiratory cell and molecular biology. 2014 ; Vol. 50, No. 5. pp. 942-952.
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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 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.",
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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.

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