Faculty Collaboration Database

Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells. Arterioscler Thromb Vasc Biol 2008 Jan;28(1):105-11

Date

11/03/2007

Pubmed ID

17975117

DOI

10.1161/ATVBAHA.107.155499

Scopus ID

2-s2.0-37549072585 (requires institutional sign-in at Scopus site)   57 Citations

Abstract

OBJECTIVE: Heat-shock protein 90 (Hsp90) coordinates the regulation of diverse signaling proteins. We try to develop a new tool to explore the regulatory functions of Hsp90 in endothelial cells (ECs) instead of the existing chemical approaches.

METHODS AND RESULTS: We designed a dominant-negative Hsp90 construct by site-direct mutagenesis of residue Asp-88 to Asn (D88N-Hsp90) based on the structure of the ATP/ADP-binding site. Recombinant wild-type Hsp90 protein binds ATP-Sepharose beads in manner inhibited by ATP or 17-AAG, a specific inhibitor for Hsp90, however the binding activity of D88N-Hsp90 was markedly reduced and the inhibitory effects of ATP or 17-AAG were negligible. The dimerization between endogenous Hsp90alpha and exogenous HA-Hsp90beta was confirmed by immunoprecipitation, however the association between eNOS and D88N-Hsp90 was less than WT-Hsp90. Furthermore, adenoviral transduction of bovine aortic ECs with D88N-Hsp90 suppressed VEGF-induced phosphorylation of Akt, eNOS, and NO release and the inhibitory effect was blocked by okadaic acid. Moreover, D88N-Hsp90 abolished VEGF-stimulated Rac activation and suppressed VEGF-induced stress fiber formation. Transduction with D88N-Hsp90 decreased growth medium mediated migration of wild-type ECs, but not Akt1(-/-) ECs suggesting that Akt is key target of Hsp90.

CONCLUSIONS: Our data demonstrate that dominant-negative Hsp90 modulates endothelial cell mobility mainly through PP2A-mediated dephosphorylation of Akt and Rac activation.

Author List

Miao RQ, Fontana J, Fulton D, Lin MI, Harrison KD, Sessa WC

MESH terms used to index this publication - Major topics in bold

Adenoviridae
Animals
Cattle
Cell Movement
Cells, Cultured
Endothelial Cells
HSP90 Heat-Shock Proteins
Lung
Mice
Mutagenesis, Site-Directed
Nitric Oxide
Nitric Oxide Synthase Type III
Proto-Oncogene Proteins c-akt
Signal Transduction
Vascular Endothelial Growth Factor A