Hsp90 Inhibition Suppresses Lps-Mediated Nfkb Target Gene Expression Without Affecting Nfkb Nuclear Translocation In Human Lung Microvascular Endothelial Cells

Heat shock protein 90 (hsp90) regulates the stability and function of several kinases, including IKB kinases (IKKs). We investigated the mechanisms by which hsp90 inhibitors prevent induction of NFkB‐dependent genes. The hsp90 inhibitor, 17‐AAG, significantly suppressed lipopolysaccharide (LPS)‐mediated IKK‐beta activation and phosphorylation of IKB‐alpha. However, 17‐AAG did not restore IKB‐alpha protein level and was unable to block p65 nuclear translocation and total DNA binding, as reflected by ELISA, in LPS‐stimulated human lung microvascular endothelial cells (HLMVEC). Conversely, prolonged 17‐AAG treatment (>16h) blocked LPS‐mediated induction of NFkB regulated genes, IKB‐alpha, IL‐8 and MMP‐9. Interestingly, binding of p65 to the IKB‐alpha promoter, as reflected by ChIP assay, was reduced by 17‐AAG. Selective pharmacologic inhibition of several key steps in the toll‐like receptor 4 (TLR‐4) signaling pathway revealed that the regulation of NFkB target genes by LPS is dependent on the cAMP‐dependent kinase (PKA) and MAPK pathways. Thus, LPS stimulated p65 (Ser276) phosphorylation in a PKA‐dependent manner; this was significantly inhibited by 17‐AAG and by IKK inhibitors (IKK16 and BAY 11‐7082). We conclude that hsp90 inhibition suppresses NFkB signaling and target gene expression by affecting p65 post‐translational modifications. (Supported by NIH grants: HL093460 and HL101902)