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(Circulation. 2005;111:3473-3480.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Estradiol-Mediated Endothelial Nitric Oxide Synthase Association With Heat Shock Protein 90 Requires Adenosine Monophosphate-Dependent Protein Kinase

Eberhard Schulz, MD^*; Elad Anter, MD^*; Ming-Hui Zou, MD, PhD; John F. Keaney, Jr, MD

From the Evans Memorial Department of Medicine (E.S., E.A., J.F.K.), Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass; and the Vascular Research Laboratory (M.-H.Z.), Graduate School of Medicine, University of Tennessee, Knoxville.

Correspondence to John F. Keaney, Jr, MD, Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St, Boston, MA 02118. E-mail jkeaney{at}bu.edu

Received March 2, 2005; revision received March 17, 2005; accepted March 21, 2005.

Background— Estradiol activates endothelial nitric oxide synthase (eNOS) by mechanisms that involve estrogen receptor- (ER), protein kinase B/Akt, mitogen-activated protein kinases, and heat shock protein 90 (HSP90). Recently, AMP-activated protein kinase (AMPK), an enzyme that plays a crucial role in cellular adaptation to metabolic stress, has been implicated in physiological eNOS activation by the hormones adiponectin and insulin. We therefore investigated whether AMPK is activated by estradiol in endothelial cells and plays a role in estradiol-induced eNOS activation.

Methods and Results— Porcine aortic endothelial cells exhibited time- and concentration-dependent AMPK activation as determined by phosphorylation of AMPK and its downstream target acetyl coenzyme A carboxylase in response to estradiol (1 nmol/L to 10 µmol/L, 1 to 30 minutes). AMPK activation by estradiol was independent of both AMP levels and ER but required estradiol conversion to its catechol metabolites. Estradiol treatment increased eNOS catalytic activity, an effect that was largely reversed when endothelial cells were infected with an AMPK dominant-negative adenovirus. However, inhibition of AMPK did not alter estradiol-induced eNOS phosphorylation at serine 1177 or threonine 495 but decreased eNOS interaction with HSP90. Consistent with this observation, blood vessels from ₁-AMPK-null mice exhibited defective eNOS-mediated NO production in response to estradiol.

Conclusions— Taken together, these data indicate that AMPK activity is essential for estradiol-induced eNOS activation via the promotion of eNOS interaction with HSP90. These data point to a novel role for AMPK in modulating endothelial cell NO bioactivity and HSP90 function.

Key Words: endothelium • nitric oxide synthase • signal transduction • adenylate kinase • heat shock proteins

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