American Journal of Respiratory Cell and Molecular Biology
Heat shock protein (hsp) 90 inhibition attenuates NF-kappaB activation and blocks inflammation. However, the precise mechanism of NF-kappaB 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-kappaB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-kappaB 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 IKBalpha was accomplished regardless of the phosphorylation/ubiquitination state of the protein. Hence, 17-AAG did not block LPS-induced NF-kappaB 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 kappaB elements of the IKBalpha (an NF-kappaB-responsive gene) promoter. The effect of LPS on IKBalpha 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-kappaB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.
Original Publication Citation
Thangjam, G.S., Dimitropoulou, C., Joshi, A.D., Barabutis, N., Shaw, M.C., Kovalenkov, Y., . . . Catravas, J.D. (2014). Novel mechanism of attenuation of LPS-induced NF-kappaB activation by the heat shock protein 90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, in human lung microvascular endothelial cells. Am J Respir Cell Mol Biol, 50(5), 942-952. doi: 10.1165/rcmb.2013-0214OC
Thangjam, Gagan S.; Dimitropoulou, Christiana; Joshi, Atul D.; Barabutis, Nektarios; Shaw, Mary C.; Kovalenkov, Yevgeniy; Wallace, Christopher M.; Fulton, David J.; Patel, Vijay; and Catravas, John D., "Novel Mechanism of Attenuation of LPS-Induced NF-Kappab Activation by the Heat Shock Protein 90 Inhibitor, 17-N-Allylamino-17-Demethoxygeldanamycin, in Human Lung Microvascular Endothelial Cells" (2014). Bioelectrics Publications. 6.