LPS induces pp60^c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung

Abstract

Heat shock protein 90 (Hsp90) inhibitors were initially developed as anticancer agents; however, it is becoming increasing clear that they also possess potent anti-inflammatory properties. Posttranslational modifications of Hsp90 have been reported in tumors and have been hypothesized to affect client protein- and inhibitor-binding activities. In the present study we investigated the posttranslational modification of Hsp90 in inflammation. LPS, a prototypical inflammatory agent, induced concentration- and time-dependent tyrosine (Y) phosphorylation of Hsp90α and Hsp90β in bovine pulmonary arterial and human lung microvascular endothelial cells (HLMVEC). Mass spectrometry identified Y309 as a major site of Y phosphorylation on Hsp90α (Y300 of Hsp90β). LPS-induced Hsp90 phosphorylation was prevented by the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin (17-AAG) in vitro as well as in lungs from LPS-treated mice, in vivo. Furthermore, 17-AAG prevented LPS-induced pp60src activation. LPS-induced Hsp90 phosphorylation was also prevented by the pp60src inhibitor PP2. Additionally, Hsp90 phosphorylation was induced by infecting cells with a constitutively active pp60src adenovirus, whereas either a dominant-negative pp60src adenovirus or reduced expression of pp60src by a specific siRNA prevented the LPS-induced Y phosphorylation of Hsp90. Transfection of HLMVEC with the nonphosphorylatable Hsp90β Y300F mutant prevented LPS-induced Hsp90β tyrosine phosphorylation but not pp60src activation. Furthermore, the Hsp90β Y300F mutant showed a reduced ability to bind the Hsp90 client proteins eNOS and pp60src and HLMVEC transfected with the mutant exhibited reduced LPS-induced barrier dysfunction. We conclude that inflammatory stimuli cause posttranslational modifications of Hsp90 that are Hsp90-inhibitor sensitive and may be important to the proinflammatory actions of Hsp90.

Histone deacetylase inhibitors prevent pulmonary endothelial hyperpermeability and acute lung injury by regulating heat shock protein 90 function

“…Western blot analyses and immunoprecipitation experiments were performed as described previously (9,31). Densitometry was performed using Image Studio version 3.1 from Licor and plotted as fold change from vehicle.…”

“…Src -dependent Y-300 phosphorylation of Hsp90 (9). Inhibitors of Hsp90 block LPS-induced Y-300 phosphorylation in cultured human lung microvascular endothelial cells (HLM-VEC) (9) and prolong survival, attenuate inflammation, and reduce lung injury in a murine model of LPS-induced ALI/ ARDS (15).…”

“…Inhibitors of Hsp90 block LPS-induced Y-300 phosphorylation in cultured human lung microvascular endothelial cells (HLM-VEC) (9) and prolong survival, attenuate inflammation, and reduce lung injury in a murine model of LPS-induced ALI/ ARDS (15). Long-term treatment (Ͼ8 h) with small molecule inhibitors of Hsp90 causes destabilization and ubiquitin-mediated degradation of numerous client proteins, such as protein kinase B (Akt) and pp60…”

Novel regulators of endothelial barrier function

“…Inhibitors of Hsp90 also prevented LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling (18). In a recent study, Barabutis and coworkers (9) showed that LPS causes phosphorylation of HSP90 on the tyrosine Y309 residue in a Src-dependent manner. Expression of a nonphosphorylatable Hsp90 mutant reduced LPS-induced barrier dysfunction indicating HSP90 inhibition.…”

P53: “The Wall Watcher”

“…Although Hsp90 inhibitors were initially developed as antineoplastic drugs, an emerging body of evidence suggests that their anti-inflammatory activity enable them to have a beneficial role in the wider spectrum of human pathology [13][14][15]. We recently remonstrate that p53 is an Hsp90 client protein and that the anti-inflammatory and vascular barrier protective effects of Hsp90 inhibitors are due-at least in part-to p53 mediated actions [16].…”