Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids.

Apoptotic cell death following injury of vascular endothelium is assumed to play an important role in the pathogenesis of atherosclerosis. In this report, we demonstrate that high density lipoproteins (HDL), a major anti-atherogenic lipoprotein fraction, protect endothelial cells against growth factor deprivation-induced apoptosis. HDL blocked the mitochondrial pathway of apoptosis by inhibiting dissipation of mitochondrial potential (Deltapsi(m)), generation of reactive oxygen species, and release of cytochrome c into the cytoplasm. As a consequence, HDL prevented activation of caspases 9 and 3 and apoptotic alterations of the plasma membrane such as increase of permeability and translocation of phosphatidylserine. Treatment of endothelial cells with HDL induced activation of the protein kinase Akt, an ubiquitous transducer of anti-apoptotic signals, and led to phosphorylation of BAD, a major Akt substrate. Suppression of Akt activity both by wortmannin and LY-294002 or by a dominant negative Akt mutant abolished the anti-apoptotic effect of HDL. Two bioactive lysosphingolipids present in HDL particles, sphingosylphosphorylcholine and lysosulfatide, fully mimicked the survival effect of HDL by blocking the mitochondrial pathway of apoptosis and potently activating Akt. In conclusion, the present study identifies HDL as a carrier of endogenous endothelial survival factors and suggests that inhibition of endothelial apoptosis by HDL-associated lysosphingolipids may represent an important and novel aspect of the anti-atherogenic activity of HDL.     

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

Carbon monoxide differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phosphatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during anoxia-reoxygenation injury

Carbon monoxide (CO), previously considered a toxic waste product of heme catabolism, is emerging as an important gaseous molecule. In addition to its important role in neurotransmission, exogenous CO protects against vascular injury, transplant rejection, and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. We have recently shown that CO attenuates endothelial cell apoptosis during anoxia-reoxygenation injury by activating MKK3/p38alpha mitogen-activated protein kinase (MAPK) pathways. Our current study is the first to demonstrate that CO can differentially modulate STAT1 and STAT3 activation and, specifically, that STAT3 activation by CO is responsible for the anti-apoptotic effect in endothelial cells. In addition, we show that the anti-apoptotic effects of CO depend upon both phosphatidylinositol 3-kinase/Akt and p38 MAPK signaling pathways in endothelial cells, whereas previous reports have implicated only the MKK3/p38 MAPK pathway. Using chemical inhibitors and dominant negative constructs, we show that CO enhances STAT3 activation via phosphatidylinositol 3-kinase/Akt and p38 MAPK pathways with subsequent attenuation of Fas expression and caspase 3 activity. These data highlight the anti-apoptotic signaling mechanisms of CO and, importantly, delineate potential therapeutic strategies to prevent ischemia-reperfusion or anoxia-reoxygenation injury in the vasculature.     

HIV-1-Tat protein activates phosphatidylinositol 3-kinase/ AKT-dependent survival pathways in Kaposi's sarcoma cells

In this study we found that Tat protected vincristine-treated Kaposi's sarcoma cells from apoptosis and from down-regulation of several anti-apoptotic genes such as AKT-1, AKT-2, BCL2, BCL-XL, and insulin-like growth factor I and induced the de novo expression of the interleukin-3 gene. Moreover, we found that Tat enhanced phosphorylation of AKT and BAD proteins. The inhibition of phosphatidylinositol 3-kinase with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated both the anti-apoptotic effect and the phosphorylation of AKT induced by Tat. After treatment with Tat, the AKT enzymatic activity showed a biphasic increase: an early activation (15 min), independent from protein synthesis; and a delayed activation (24 h), which was significantly decreased upon blockage of protein synthesis. Experiments with a function blocking anti-vascular endothelial cell growth factor receptor-2 antibody suggested that both the early and delayed AKT activation and the protection from apoptosis were triggered by the interaction of Tat with vascular endothelial cell growth factor receptor-2. Moreover, experiments with function-blocking antibodies directed against insulin-like growth factor I/insulin-like growth factor I receptor or interleukin-3 indicated their involvement in the delayed activation of AKT and their contribution to the anti-apoptotic effect of Tat on vincristine-treated Kaposi's sarcoma cells.     

A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.     

Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.     

Akt signaling mediates VEGF/VPF vascular permeability in vivo

VEGF is an endothelial cell cytokine that promotes angiogenesis and enhances microvascular permeability. Recently, it has been shown that the protein kinase Akt functions in a key intercellular signaling pathway downstream of VEGF. Here, we employed adenovirus-mediated gene transfer in conjunction with the Miles assay in hairless albino guinea pigs to assess the role of Akt signaling in vascular permeability. VEGF-induced vascular permeability was blocked by the transduction of a dominant negative mutant of Akt. Conversely, transduction of a constitutively active form of Akt promoted vascular permeability in a manner similar to VEGF protein administration. This Akt-mediated increase in vascular permeability was inhibited by the eNOS inhibitor L-NAME. These data show that Akt signaling is both necessary and sufficient for vascular permeability in an in vivo model.     

Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

Vaspin, an adipocytokine recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. However, the exact mechanism underlying this action has not been fully elucidated. Furthermore, the specific function of vaspin is largely unknown, especially in vascular cells. We examined whether vaspin affects the insulin-signaling pathway in cultured endothelial cells and is capable of preventing free fatty acid (FFA)-induced apoptosis in endothelial cells through its insulin sensitizing effect, specifically, through its stimulatory effect on PI3-kinase/Akt signaling pathways. Vaspin significantly increased Akt phosphorylation and prevented the impairment of Akt phosphorylation by linoleic acid (LA) in insulin-stimulated endothelial cells, which effects were abolished by pretreatment with the PI3-kinase inhibitor, Wortmannin. Moreover, pretreatment with vaspin prevented LA-induced apoptosis in insulin-stimulated endothelial cells; this anti-apoptotic effect of vaspin was also eliminated by pretreatment with Wortmannin. The present study indicates that vaspin protects vascular endothelial cells from FFA-induced apoptosis through upregulation of the PI3-kinase/Akt signaling pathway. Our study is the first to demonstrate that vascular cells can be targets of vaspin. Our results further suggest that vaspin could have beneficial effects on the atherosclerosis.     

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.     

Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.     

p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein.     

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.     

Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

Isoflavone genistein protects human vascular endothelial cells against tumor necrosis factor-α-induced apoptosis through the p38β mitogen-activated protein kinase

Isoflavone genistein may have beneficial effects on vascular function, but the mechanism is unclear. Here, we investigated whether genistein protects vascular endothelial cells against apoptosis induced by tumor necrosis factor-α. We show that genistein significantly inhibited TNF-α-induced apoptosis in human aortic endothelial cells as determined by caspase-3 activation, 7-amino actinomycin D staining, in situ apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of Bcl-2 protein and its promoter activity. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptors had no effect on the cytoprotective effect of genistein. However, inhibition of p38 mitogen-activated protein kinase (p38) completely abolished this genistein effect. Accordingly, stimulation of HAECs with genistein resulted in rapid activation of p38β, but not p38α. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Preservation of the functional integrity of the endothelial monolayer may represent an important mechanism by which genistein exerts its vasculoprotective effect.     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

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.     

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.