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-kB activation and blocks inflammation. However, the precise mechanism of NF-kB 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-kB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-kB 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-kB 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 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-kB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.Keywords: human lung microvascular endothelial cells; heat shock protein 90 inhibitor; LPS; NF-kB; cAMP response element binding protein binding protein Clinical RelevanceNF-kB is a master regulator of inflammation. We report on a new mechanism regulating NF-kB activity that could reveal new targets for the management of acute lung injury/acute respiratory distress syndrome and other pulmonary inflammatory disease.

Endotoxin‐ and Mechanical Stress–Induced Epigenetic Changes in the Regulation of the Nicotinamide Phosphoribosyltransferase Promoter

“…This result is consistent with other studies that have demonstrated LPS-mediated downregulation of H3K9 acetylation occurring during inflammation of the brain 21 and also in LPS-challenged HLMVECs mimicking lung infection. 18 To address the mechanisms driving downregulation of H3K9 acetylation, we utilized pharmacological inhibition of TLR4 receptor to establish the essential role of TLR4 pathway. The results demonstrated that CLI-095-mediated pharmacological inhibition of TLR4 receptor attenuated the rhNAMPT-induced decreases in H3K9 acetylation, which suggests that the changes in acetylation on the his- A, Human lung ECs were pretreated with either vehicle (Veh) or histone deacetylase (HDAC) class I and II-specific inhibitor TSA (400 nM and 1,000 nM) for 20 hours and were further unstimulated or stimulated with LPS (100 ng/mL) for 4 hours.…”

Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells

“…p65 acetylation at Ser276 and Ser536 enhances its DNA binding efficiency and inhibits its interaction with IKB␣ (32). Previously, we reported that, in human lung endothelial cells, the inhibition of NF-B by hsp90 inhibitors is independent of p65 Ser276 phosphorylation (31); a more direct mechanism appears to be involved. For example, the physical exclusion of NF-B from the promoter by glucocorticoid receptors effectively suppresses NF-B activation in dexamethasone-treated pancreatic cancer cells (20).…”

“…This allows rapid chromatin remodeling and eviction of nucleosomes from the enhancer region that unmasks obscured NF-B enhancer elements (19). We have demonstrated that inhibition of this process by hsp90 inhibitor suppresses gene expression (31).…”

Identification of RNA-binding Proteins in Macrophages by Interactome Capture

“…LPS-mediated macrophage activation not only involves cytoskeletal remodeling, but requires HSP90 activity that is essential for stabilization and maturation of protein factors involved in NFB activation and inflammation (81)(82)(83). Among the novel RBPs we identified the HSP90 cochaperone P23 (32) or cytoplasmic PTGES3 (33), which was therefore chosen for further analysis.…”