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.     

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na+, K+ -ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ETB), since application of the ETB-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.     

G-protein-coupled receptor kinase interactor-1 (GIT1) is a new endothelial nitric-oxide synthase (eNOS) interactor with functional effects on vascular homeostasis

Endothelial cell nitric-oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in the vasculature, undergoes extensive post-translational modifications that modulate its activity. Here we have identified a novel eNOS interactor, G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), which plays an unexpected role in GPCR stimulated NO signaling. GIT1 interacted with eNOS in the endothelial cell cytoplasm, and this robust association was associated with stimulatory eNOS phosphorylation (Ser(1177)), enzyme activation, and NO synthesis. GIT1 knockdown had the opposite effect. Additionally, GIT1 expression was reduced in sinusoidal endothelial cells after liver injury, consistent with previously described endothelial dysfunction in this disease. Re-expression of GIT1 after liver injury rescued the endothelial phenotype. These data emphasize the role of GPCR signaling partners in eNOS function and have fundamental implications for vascular disorders involving dysregulated eNOS.     

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.     

Endothelial Nitric-oxide Synthase (eNOS) Is Activated through G-protein-coupled Receptor Kinase-interacting Protein 1 (GIT1) Tyrosine Phosphorylation and Src Protein

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ET_B) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser¹¹⁷⁷), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ET_B receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production.     

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.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr⁸⁰¹, on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.     

A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.     

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.     

Phosphorylation of tyrosine 801 of vascular endothelial growth factor receptor-2 is necessary for Akt-dependent endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells

Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-gamma in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGF-stimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.     

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na⁺,K⁺-ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ET_B), since application of the ET_B-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.     

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.     

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.     

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.     

Glomerular endothelial PI3 kinase-α couples to VEGFR2, but is not required for eNOS activation

Vascular endothelial growth factor (VEGF)-dependent signals are central to many endothelial cell (EC) functions, including survival and regulation of vascular tone. Akt and endothelial nitric oxide synthase (eNOS) activity are implicated to mediate these effects. Dysregulated signaling is characteristic of endothelial dysfunction that sensitizes the glomerular microvasculature to injury. Signaling intermediates that couple VEGF stimulation to eNOS activity remain unclear; hence, we examined the PI3 kinase isoforms implicated to regulate these enzymes. Using a combination of small molecule inhibitors and RNAi to study responses to VEGF in glomerular EC, we observed that the PI3 kinase p110α catalytic isoform is coupled to VEGFR2 and regulates the bulk of Akt activity. Coimmunoprecipitation experiments support a physical association of p110α with VEGFR2. Downstream, Akt-mediated FOXO1 phosphorylation in EC is regulated by p110α. The p110δ isoform contributes a minor amount of VEGF-stimulated Akt activation. However, we observe no effect of p110α or p110δ to regulate VEGF-stimulated eNOS activation via Akt-mediated phosphorylation on eNOS Ser1177, or NO-mediated vasodilation of the afferent arteriole ex vivo. VEGFR2-stimulated eNOS activation and NO production are inhibited by Compound C, an inhibitor of AMP-stimulated kinase, independent of PI3 kinase signaling. PI3 kinase-α/δ-mediated signaling downstream of VEGFR2 activation regulates Akt-dependent survival signals, but our data suggest it is not required to activate eNOS or to elicit NO production in glomerular EC.     

Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.     

Renal activity of Akt kinase in obese Zucker rats

Insulin resistance (IR) and consequent hyperinsulinemia are hallmarks of Type 2 diabetes (DM2). Akt kinase (Akt) is an important molecule in insulin signaling, implicated in regulation of glucose uptake, cell growth, cell survival, protein synthesis, and endothelial nitric oxide (NO) production. Impaired Akt activation in insulin-sensitive tissues contributes to IR. However, Akt activity in other tissues, particularly those affected by complications of DM2, has been less studied. We hypothesized that hyperinsulinemia could have an impact on activity of Akt and its effectors involved in regulation of renal morphology and function in DM2. To address this issue, renal cortical Akt was determined in obese Zucker rats (ZO), a model of DM2, and lean controls (ZL). We also studied expression and phosphorylation of the mammalian target of rapamycin (mTOR) and endothelial NO synthase (eNOS), molecules downstream of Akt in the insulin signaling cascade, and documented modulators of renal injury. Akt activity was measured by a kinase assay with GSK-3 as a substrate. Expression of phosphorylated (active) and total proteins was measured by immunoblotting and immunohistochemistry. Renal Akt activity was increased in ZO as compared to ZL rats, in parallel with progressive hyperinsulinemia. No differences in Akt were observed in the skeletal muscle. Corresponding to increases in Akt activity, ZO rats demonstrated enhanced phosphorylation of renal mTOR. Acute PI3K inhibition with wortmannin (100 mug/kg) attenuated renal Akt and mTOR activities in ZO, but not in ZL rats. In contrast to mTOR, eNOS phosphorylation was similar in ZO and ZL rats, despite higher total eNOS expression. In conclusion, ZO rats demonstrated increases in renal Akt and mTOR activity and expression. However, eNOS phosphorylation did not follow this pattern. These data suggest that DM2 is associated with selective IR in the kidney, allowing pro-growth signaling via mTOR, whereas potentially protective effects mediated by eNOS are blunted.     

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.