Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells

Robert Q. Miao, Jason Fontana, David J Fulton, Michelle I. Lin, Kenneth D. Harrison, William C. Sessa

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

OBJECTIVES - 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 Hsp90α and exogenous HA-Hsp90β 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.

Original languageEnglish (US)
Pages (from-to)105-111
Number of pages7
JournalArteriosclerosis, thrombosis, and vascular biology
Volume28
Issue number1
DOIs
StatePublished - Jan 1 2008

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HSP90 Heat-Shock Proteins
Vascular Endothelial Growth Factor A
Nitric Oxide
Endothelial Cells
tanespimycin
Adenosine Triphosphate
Okadaic Acid
Stress Fibers
Dimerization

Keywords

  • ATP-binding
  • Akt
  • Dominant-negative
  • Hsp90
  • Migration

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells. / Miao, Robert Q.; Fontana, Jason; Fulton, David J; Lin, Michelle I.; Harrison, Kenneth D.; Sessa, William C.

In: Arteriosclerosis, thrombosis, and vascular biology, Vol. 28, No. 1, 01.01.2008, p. 105-111.

Research output: Contribution to journalArticle

Miao, Robert Q. ; Fontana, Jason ; Fulton, David J ; Lin, Michelle I. ; Harrison, Kenneth D. ; Sessa, William C. / Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells. In: Arteriosclerosis, thrombosis, and vascular biology. 2008 ; Vol. 28, No. 1. pp. 105-111.
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abstract = "OBJECTIVES - 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 Hsp90α and exogenous HA-Hsp90β 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.",
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AB - OBJECTIVES - 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 Hsp90α and exogenous HA-Hsp90β 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.

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