Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.     

Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols

Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPKalpha attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPKalpha also blunted Akt activation in response to black tea polyphenols, suggesting that p38alpha MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.     

Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

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.     

Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase

Both lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are platelet-derived phospholipids that elicit diverse biological responses. In endothelial cells, S1P stimulates the EDG-1 receptor-mediated activation of the endothelial isoform of nitric oxide synthase (eNOS), but the role of LPA in eNOS regulation is less well understood. We now report that LPA treatment of bovine aortic endothelial cells (BAEC) activates eNOS enzyme activity in a pathway that involves phosphorylation of eNOS on serine 1179 by protein kinase Akt. In contrast to the cellular responses elicited by S1P in COS-7 cells, LPA can stimulate the activation of eNOS and Akt independently of EDG-1 receptor transfection. LPA-stimulated enzyme activation was significantly attenuated in an eNOS mutant lacking the site that is phosphorylated by kinase Akt (eNOS S1179A). In BAEC, activation of eNOS by LPA is completely blocked by pertussis toxin, by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), and by the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, but is unaffected by U0126, an inhibitor of mitogen-activated protein (MAP) kinase pathways. Analysis of the LPA dose response for eNOS activation reveals an EC(50) of approximately 40 nM, a concentration well below the potency of LPA at the EDG-1 receptor. Taken together, these results indicate that LPA potently activates eNOS in BAEC in a pathway distinct from the EDG-1 receptor, but mediated by a similar receptor-mediated pathway dependent on pertussis toxin-sensitive G proteins and involving activation of the PI3-K/Akt pathway. These studies have identified a role for the phospholipid LPA in eNOS activation, and point out the complementary role of distinct platelet-derived lipids in endothelial signaling pathways.     

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (D-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance.     

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.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration. The protein kinase Akt activates the endothelial NO synthase (eNOS) by phosphorylation of Ser-1177. Therefore, we investigated the contribution of Akt-mediated eNOS phosphorylation to VEGF-induced EC migration. Inhibition of NO synthase or overexpression of a dominant negative Akt abrogated VEGF-induced cell migration. In contrast, overexpression of constitutively active Akt was sufficient to induce cell migration. Moreover, transfection of an Akt site phospho-mimetic eNOS (S1177D) potently stimulated EC migration, whereas a non-phosphorylatable mutant (S1177A) inhibited VEGF-induced EC migration. Our data indicate that eNOS activation via phosphorylation of Ser-1177 by Akt is necessary and sufficient for VEGF-mediated EC migration.     

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…     

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.     

Cyclosporin A inhibits flow-mediated activation of endothelial nitric-oxide synthase by altering cholesterol content in caveolae

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-mediated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 mum CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress=dynes/cm2). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo, CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased cholesterol content in caveolae and displaced eNOS from caveolae, which may be caused by CsA disrupting the association of caveolin-1 and cyclophilin A. The magnitude of the cholesterol depleting effect was similar to that of beta-cyclodextrin, a cholesterol-binding molecule, and beta-cyclodextrin had a similar inhibitory effect on flow-mediated eNOS activation. Treating bovine aortic endothelial cells for 24 h with 30 mug/ml cholesterol blocked the CsA effect and restored eNOS phosphorylation in response to flow. These data suggest that decreasing cholesterol content in caveolae by CsA is a potentially important pathogenic mechanism for CsA-induced endothelial dysfunction and hypertension.     

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.     

Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS)

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity.

Bovine carotid artery endothelial (BAE) cells are resistant to tumor necrosis factor-alpha (TNF), like most other cells. We examined if mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase/Akt pathways are involved in this effect. In BAE cells, TNF activates MAP kinase in a MAP kinase kinase 1 (MEK1) manner and Akt in PI3-kinase-dependent manner. Pretreatment with either the MEK1 inhibitor U0126 or PI3-kinase inhibitor LY294002 sensitized BAE cells to TNF-induced apoptosis. Neither U0126 nor LY294002 pretreatment affected TNF-induced activation of NF-kappaB, suggesting that the MAP kinase or PI3-kinase/Akt-mediated anti-apoptotic effect induced by TNF was not relevant to NF-kappaB activation. Both MAP kinase and PI3-kinase/Akt -mediated signaling could prevent cytochrome c release and mitochondrial transmembrane potential (Deltapsi) decrease. PI3-kinase/Akt signaling attenuated caspase-8 activity, whereas MAP kinase signaling impaired caspase-9 activity. These results suggest that TNF-induced MAP kinase and PI3-kinase/Akt signaling play important roles in protecting BAE cells from TNF cytotoxicity.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.     

Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase

Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC). Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-(32)P]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro (32)P phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.