Differential effects of heat shock protein 90 and serine 1179 phosphorylation on endothelial nitric oxide synthase activity and on its cofactors

Abstract

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. Previous studies showed that bovine eNOS serine 1179 (Serine 1177 for human eNOS) phosphorylation enhanced NO synthesis. Meanwhile, heat shock protein 90 (Hsp90) plays a critical role in maintenance of eNOS structure and function. However, the regulatory difference and importance between Serine 1179 phosphorylation and Hsp90 on eNOS activity have not been evaluated. In current studies, S1179D eNOS was employed to mimic phospho-eNOS and exhibited markedly increased enzyme activity than wild type eNOS (WT eNOS). Hsp90 showed a dose-dependent increase for both WT eNOS and S1179D eNOS activity at the presence of all eNOS cofactors, such as Calcium/Calmodulin (Ca2+ /CaM), BH4, and NADPH etc. The enhancement effects were abolished by dominant-negative mutant Hsp 90 protein. ENOS-cofactors dynamic assay showed that Hsp90 enhanced WT eNOS affinity to NADPH, L-arginine, and CaM but not to Ca2+ and BH4. The impact of eNOS Serine 1179 phosphorylation and Hsp90 on eNOS affinity to cofactors has also been compared. Different from the effect of Hsp90 on eNOS affinity to specific cofactors, Serine 1179 phosphorylation significantly increased eNOS affinity to all cofactors. Moreover, VEGF-induced eNOS phosphorylation in bovine aortic endothelial cells (BAECs) and more NO generation from eNOS compared to control. Inhibition of Hsp90 by geldanamycin decreased eNOS activity and decreased endothelial viability. In conclusion, by changing eNOS structure, Hsp90 profoundly affected eNOS functions, including change of affinity of eNOS to cofactors like Ca2+, L-arginine, BH4 and further affecting NO generation capability. These specific cofactors regulated by Hsp 90 could become potential therapeutic targets of the eNOS-related diseases in future.

New Insights Into Heat Shock Protein 90 in the Pathogenesis of Pulmonary Arterial Hypertension

“…Moreover, inhibition of Hsp90 or silencing of Hsp90 causes the eNOS dimer to dissociate into monomers and exposes Ser1179 and Thr497 on the monomers to various phosphatases, resulting in eNOS dephosphorylation and degradation. Structure studies showed that Hsp90 associates with the oxygenase domain of eNOS in which eNOS also associates with many kinds of different cofactors or substrates, such as Heme, tetrahydrobiopterin (BH4), and L-arginine (Balligand, 2002;Chen et al, 2017). In fact, Hsp90 uniquely regulates eNOS through specifically changing a few eNOS cofactor affinity, such as reduced nicotinamide adenine nucleotide phosphate and calcium (Ca 2+ )/calmodulin (Chen et al, 2017).…”

“…Structure studies showed that Hsp90 associates with the oxygenase domain of eNOS in which eNOS also associates with many kinds of different cofactors or substrates, such as Heme, tetrahydrobiopterin (BH4), and L-arginine (Balligand, 2002;Chen et al, 2017). In fact, Hsp90 uniquely regulates eNOS through specifically changing a few eNOS cofactor affinity, such as reduced nicotinamide adenine nucleotide phosphate and calcium (Ca 2+ )/calmodulin (Chen et al, 2017). Meanwhile, sufficient study data confirm that mitochondrial dysfunction in pulmonary arterial endothelial cells (PAECs) decreases the cellular ATP levels and Hsp90 chaperone activity, resulting in a reduced interaction between Hsp90 and eNOS in cardiovascular disorders (Sud et al, 2008).…”

Primary Cilia are Sensory Hubs for Nitric Oxide Signaling

“…eNOS activation is mainly calcium dependent, but some studies have shown that a calcium independent pathway exists, via heat shock protein 90 (HSP90). HSP90 is also known to localize to cilia axonemes, and may act as a signal transductor that interacts with eNOS in the vasculature [62,63]. HSP90 activation can lead to an increase in eNOS activity while calcium levels are high, and can also lead to more eNOS activity at low calcium levels due to its ability to directly bind to eNOS and increase the binding affinity for calmodulin [64,65].…”

The Roles of Primary Cilia in Cardiovascular Diseases

“…Though eNOS triggering is principally a Ca 2+ -dependent process, some studies have suggested a Ca 2+ -independent pathway in NO biosynthesis is also possible. This Ca 2+ -independent pathway depends on the heat shock protein 90 (HSP90) [129,130]. HSP90 is a molecular chaperone, but it may also act as a signal transduction agent concomitant with eNOS in several systems, including the cardiovascular system.…”