Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635

Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.     

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.     

Acute activation and phosphorylation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, provide beneficial effects independent of their lipid-lowering effects. One beneficial effect appears to involve acute activation of endothelial nitric oxide (NO) synthase (eNOS) and increased NO release. However, the mechanism of acute statin-stimulated eNOS activation is unknown. Therefore, we hypothesized that eNOS activation may be coupled to altered eNOS phosphorylation. Bovine aortic endothelial cells (BAECs), passages 2-6, were treated with either lovastatin or pravastatin from 0 to 30 min. eNOS phosphorylation was examined by Western blot by use of phosphospecific antibodies for Ser-1179, Ser-635, Ser-617, Thr-497, and Ser-116. Statin stimulation of BAECs increased eNOS phosphorylation at Ser-1179 and Ser-617, which was blocked by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitor wortmannin, and at Ser-635, which was blocked by the protein kinase A (PKA) inhibitor KT-5720. Statin treatment of BAECs transiently increased NO release by fourfold, measured by cGMP accumulation, and was attenuated by N-nitro-l-arginine methyl ester, wortmannin, and KT-5720 but not by mevalonate. In conclusion, these data demonstrate that eNOS is acutely activated by statins independent of HMG-CoA reductase inhibition and that in addition to Ser-1179, eNOS phosphorylation at Ser-635 and Ser-617 through PKA and Akt, respectively, may explain, in part, a mechanism by which eNOS is activated in response to acute statin treatment.     

Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.     

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.     

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

Regulation of endothelial nitric-oxide synthase activity through phosphorylation in response to epoxyeicosatrienoic acids

Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.     

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction path-ways involved

Vascular endothelial cells play crucial roles in regulating cardiovascular function, maintaining car-diovascular homeostasis and preventing the occur-rence of cardiac and cerebral vascular diseases. All these protective effects are fulfilled through various vasoactive products secreted by endothelium including nitric oxide (NO), prostacyclin (PGI2) and endothe-lium-derived hyperpolarizing factor (EDHF). NO, pro-duced from L-arginine by endothelial nitric-oxide synthase (eNOS), is an impor…     

Endothelial Nitric Oxide Synthase Dimerization Is Regulated by Heat Shock Protein 90 Rather than by Phosphorylation

Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways

Recently, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARgamma ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase ( approximately 3.1-fold) was achieved with 20 microm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 microm treatment for 12 h) significantly increased both the phosphorylation of Ser(1179) of eNOS (eNOS-Ser(1179)) and the dephosphorylation of eNOS-Ser(116) but did not alter eNOS-Thr(497) phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser(1179) with no alteration in eNOS-Ser(116) dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARgamma antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation but with no alteration in eNOS-Ser(116) dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARgamma-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation and PPARgamma-independent, eNOS-Ser(116) dephosphorylation.     

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction pathways involved

Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regulation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS activity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) signaling pathway in eNOS expression and its phosphorylation in response to EETs via direct addition of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno-associated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to produce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)¹¹⁷⁹ but had no effect on the change of eNOS-Thr(P)⁴⁹⁷, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphorylation level at eNOS-Ser¹¹⁷⁹ via PI3K/Akt and eNOS-Thr⁴⁹⁷ via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.     

Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells

During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF(165) on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF(165) activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGF(165), whereas the VEGFR-2 kinase inhibitor blocks VEGF(165)-stimulated eNOS activation, suggesting VEGF(165) predominantly activates eNOS via VEGFR-2. Although VEGF(165) also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominantly via VEGFR-2. The lack of VEGF(165)-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Ser(1179)-eNOS. Although VEGF(165)-stimulated eNOS phosphorylation is observed at Ser(617) and Ser(635), pregnancy does not significantly alter this response. Our finding that VEGF(165) activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt.     

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.     

Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase

We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions.     

Epinephrine regulation of the endothelial nitric-oxide synthase: roles of RAC1 and beta3-adrenergic receptors in endothelial NO signaling

beta-Adrenergic receptors (betaAR) play an important role in vasodilation, but the mechanisms whereby adrenergic pathways regulate the endothelial isoform of nitric-oxide synthase (eNOS) are incompletely understood. We found that epinephrine significantly increases eNOS activity in cultured bovine aortic endothelial cells (BAEC). Epinephrine-dependent eNOS activation was accompanied by an increase in phosphorylation of eNOS at Ser(1179) and with decreased eNOS phosphorylation at the inhibitory phosphoresidues Ser(116) and Thr(497). Epinephrine promoted activation of the small G protein Rac1 and also led to the activation of protein kinase A. All of these responses to epinephrine in BAEC were blocked by the beta(3)AR blocker SR59230A. We transfected and validated duplex small interfering RNA (siRNA) constructs to selectively "knock down" specific signaling proteins in BAEC. siRNA-mediated knockdown of Rac1 completely blocked all beta(3)AR signaling to eNOS and also abrogated epinephrine-dependent cAMP-dependent protein kinase (PKA) and Akt activation. However, siRNA-mediated knockdown of PKA did not affect Rac1 activation by epinephrine but did attenuate Akt activation by epinephrine. These findings indicate that Rac1 is an upstream regulator of beta(3)AR signaling to PKA and to eNOS and identify a novel beta(3)AR --> Rac1 --> PKA --> Akt pathway in endothelium. We exploited the p21-activated kinase pulldown assay to identify proteins associated with activated Rac1 and found that epinephrine stimulated the association of eNOS with Rac1; epinephrine-stimulated eNOS-Rac1 interactions were blocked by the beta(3)AR antagonist SR59230A. Co-transfection of eNOS cDNA with constitutively active Rac1 enhanced beta(3)AR-promoted eNOS-Rac1 association; co-transfection of eNOS with dominant negative Rac1 completely blocked the eNOS-Rac1 association. We also found that epinephrine-induced Rac1 --> PKA --> Akt pathway mediates beta(3)AR-mediated endothelial cell migration. Taken together, our data establish that the small G protein Rac1 is a key regulator of beta(3)AR signaling in cultured aortic endothelial cells with potentially important implications for the pathways involved in adrenergic modulation of eNOS pathways in the vascular wall.