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, Vijay 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 - May 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, Vijay; Catravas, John D.

In: American journal of respiratory cell and molecular biology, Vol. 50, No. 5, 05.2014, 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, Vijay ; 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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