Dominant-Negative Hsp90 Reduces VEGF-Stimulated Nitric Oxide Release and Migration in Endothelial Cells

• DOI: 10.1161/atvbaha.107.155499
• Publication Date: 2007-11-02T00:59:05Z
• Authors: Robert Q. Miao; Jason Fontana; David Fulton; Michelle I. Lin; Kenneth D. Harrison; William C. Sessa
• Keywords: Vascular Endothelial Growth Factor A; Nitric Oxide Synthase Type III; Endothelial Cells; Nitric Oxide; Adenoviridae; Mice; 03 medical and health sciences; 0302 clinical medicine; Cell Movement; Mutagenesis, Site-Directed; Animals; Cattle; HSP90 Heat-Shock Proteins; Lung; Proto-Oncogene Proteins c-akt; Cells, Cultured; Signal Transduction

Objectives— Heat-shock protein 90 (Hsp90) coordinates the regulation of diverse signaling proteins. We try to develop a new tool explore regulatory functions Hsp90 in endothelial cells (ECs) instead existing chemical approaches. Methods and Results— designed dominant-negative construct by site-direct mutagenesis residue Asp-88 Asn (D88N-Hsp90) based on structure ATP/ADP-binding site. Recombinant wild-type binds ATP-Sepharose beads manner inhibited ATP or 17-AAG, specific inhibitor for Hsp90, however binding activity D88N-Hsp90 was markedly reduced inhibitory effects 17-AAG were negligible. The dimerization between endogenous Hsp90α exogenous HA-Hsp90β confirmed immunoprecipitation, association eNOS less than WT-Hsp90. Furthermore, adenoviral transduction bovine aortic ECs with suppressed VEGF-induced phosphorylation Akt, eNOS, NO release effect blocked okadaic acid. Moreover, abolished VEGF-stimulated Rac activation stress fiber formation. Transduction decreased growth medium mediated migration ECs, but not Akt1(−/−) suggesting that Akt is key target Hsp90. Conclusions— Our data demonstrate modulates cell mobility mainly through PP2A-mediated dephosphorylation activation.