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.     

Survivin down-regulation plays a crucial role in 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor-induced apoptosis in cancer

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-X(L), revealed that only survivin expression was decreased by lovastatin. Survivin down-regulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin down-regulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.     

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.     

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.     

蛋白激酶B/Akt抑制剂

磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路在细胞生长与存活中起着关键作用,PI3K/Akt通路的过度激活在多种肿瘤中常见。Akt激酶本身以及Akt激酶上游调节分子,例如PTEN和PI3K,在超过50%的人类肿瘤中均有异常变化。因此Akt成为肿瘤预防和肿瘤靶向治疗的热点之一。许多小分子化合物通过不同机制抑制Akt活性,根据小分子抑制剂与激酶的结合部位和化学结构不同,主要分为ATP竞争性抑制剂、Akt变构抑制剂和磷脂酰肌醇类似物抑制剂。本文综述了PI3K/Akt通路与肿瘤的关系和Akt抑制剂的研究现状,为新型抗癌药物的设计研究提供参考。     

Activation of NF-kappa B by bradykinin through a Galpha(q)- and Gbeta gamma-dependent pathway that involves phosphoinositide 3-kinase and Akt

Recent work has suggested a role for the serine/threonine kinase Akt and IkappaB kinases (IKKs) in nuclear factor (NF)-kappaB activation. In this study, the involvement of these components in NF-kappaB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, was suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-kappaB activation. BK-induced NF-kappaB activation and IKK2 activity were markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Galpha(q), and by two Gbetagamma scavengers. Co-expression of Galpha(q) potentiated BK-induced NF-kappaB activation, whereas co-expression of either an activated Galpha(q)(Q209L) or Gbeta(1)gamma(2) induced IKK2 activity and NF-kappaB activation without BK stimulation. BK-induced NF-kappaB activation was partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Galpha(q) and Gbeta(1)gamma(2) for NF-kappaB activation. Furthermore, BK could activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibited BK-induced NF-kappaB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Galpha(q), Gbeta(1)gamma(2), PI3K, Akt, and IKK for NF-kappaB activation.     

Dose-dependent activation of antiapoptotic and proapoptotic pathways by ethanol treatment in human vascular endothelial cells: differential involvement of adenosine

Moderate but not heavy drinking has been found to have a protective effect against cardiovascular morbidity. We investigated the effects of ethanol (EtOH) treatment on the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cultured human umbilical vein endothelial cells (HUVEC). Exposure of cells to 2-20 mm EtOH resulted in rapid (<10 min) induction of Akt phosphorylation that could be prevented by pertussis toxin or the PI3K inhibitors wortmannin and LY294002. Among the downstream effectors of PI3K/Akt, p70S6 kinase, glycogen synthase kinase 3alpha/beta, and IkappaB-alpha were phosphorylated, the latter resulting in 3-fold activation of NF-kappaB. EtOH also activated p44/42 mitogen-activated protein kinase in a PI3K-dependent manner. Low concentrations of EtOH increased endothelial nitric-oxide synthase activity, which could be blocked by transfection of HUVEC with dominant-negative Akt, implicating the PI3K/Akt pathway in this effect. The adenosine A1 receptor antagonist 1,3-dipopylcyclopentylxanthine prevented the phosphorylation of Akt observed in the presence of EtOH, adenosine, or the A1 agonist N(6)-cyclopentyladenosine. Incubation of HUVEC with 50-100 mm EtOH resulted in mitochondrial permeability transition and caspase-3 activation followed by apoptosis, as documented by DNA fragmentation and TUNEL assays. EtOH-induced apoptosis was unaffected by DPCPX and was potentiated by wortmannin or LY294002. We conclude that treatment with low concentrations of EtOH activates the cell survival promoting PI3K/Akt pathway in endothelial cells by an adenosine receptor-dependent mechanism and activation of the proapoptotic caspase pathway by higher concentrations of EtOH via an adenosine-independent mechanism can mask or counteract such effects.     

Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells

Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Akt and RhoA inhibition promotes anoikis of aggregated B16F10 melanoma cells

In the highly metastatic B16F10 melanoma cell line, activation of the signalling molecules that promote cell proliferation and survival on conventional adhesive culture dishes may also be responsible for the growth and resistance to anoikis of aggregates on a non-adhesive substratum. We have examined the influence of bacterial ADP-ribosyltransferases C3-like exoenzymes, which selectively modify RhoA, B and C proteins and inhibit signal pathways controlled by them. RNA interference [siRNA (small interfering RNA) Akt (also known as protein kinase B)] and a PI3K (phosphoinositide 3-kinase) inhibitor were used to analyse the changes caused by inhibiting the PI3K/Akt pathway. Inhibiting the activation of RhoA, B, C and Akt expression resulted in a decrease of the number of cells cultured in aggregates, and caspase 3 activation. RhoA activation and RhoB and RhoC expression were controlled by Akt, but not RhoA expression. Inhibiting Akt and RhoA reduced the expression of α5 integrin, and inactivated FAK (focal adhesion kinase) in B16F10 cells cultured as aggregates. Thus, inhibiting Rho subfamily proteins and Akt expression inactivates the FAK pathway and induces anoikis in anoikis-resistant cells. The activation of RhoA in melanoma cells can depend on PI3K/Akt activation, suggesting that PI3K/Akt is a suitable target for new therapeutic approaches.     

ApoA-I/SR-BI modulates S1P/S1PR2-mediated inflammation through the PI3K/Akt signaling pathway in HUVECs

Endothelial dysfunction plays a vital role during the initial stage of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) induces vascular endothelial injury and vessel wall inflammation. Sphingosine-1-phosphate (S1P) exerts numerous vasoprotective effects by binding to diverse S1P receptors (S1PRs; S1PR1-5). A number of studies have shown that in endothelial cells (ECs), S1PR2 acts as a pro-atherosclerotic mediator by stimulating vessel wall inflammation through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Scavenger receptor class B member I (SR-BI), a high-affinity receptor for apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL), inhibits nuclear factor-κB (NF-κB) translocation and decreases the plasma levels of inflammatory mediators via the PI3K/Akt pathway. We hypothesized that the inflammatory effects of S1P/S1PR2 on ECs may be regulated by apoA-I/SR-BI. The results showed that ox-LDL, a pro-inflammatory factor, augmented the S1PR2 level in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. In addition, S1P/S1PR2 signaling influenced the levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10, aggravating inflammation in HUVECs. Moreover, the pro-inflammatory effects induced by S1P/S1PR2 were attenuated by SR-BI overexpression and enhanced by an SR-BI inhibitor, BLT-1. Further experiments showed that the PI3K/Akt signaling pathway was involved in this process. Taken together, these results demonstrate that apoA-I/SR-BI negatively regulates S1P/S1PR2-mediated inflammation in HUVECs by activating the PI3K/Akt signaling pathway.     

Early phosphatidylinositol 3-kinase/Akt pathway activation limits poliovirus-induced JNK-mediated cell death

Poliovirus (PV)-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dysfunction mediated by early JNK activation in IMR5 neuroblastoma cells. We showed here that PV simultaneously activates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signaling pathway in these cells, limiting the extent of JNK activation and thereby cell death. JNK inhibition is associated with PI3K-dependent negative regulation of the apoptosis signal-regulating kinase 1, which acts upstream from JNK in PV-infected IMR5 cells. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.     

A truncated kringle domain of human apolipoprotein(a) inhibits the activation of extracellular signal-regulated kinase 1 and 2 through a tyrosine phosphatase-dependent pathway

Most proangiogenic factors exert their biological effects primarily by activating extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt signaling pathways. These pathways appear to play a critical role in endothelial cell migration, because selective inhibition of either ERK or PI3-K/Akt signaling almost completely prevented endothelial cell migration. Recently, we demonstrated that a truncated kringle domain of human apolipoprotein(a), termed rhLK68, inhibits endothelial cell migration in vitro. However, its mechanism of action was not well defined. In this study, we determined the effects of rhLK68 on ERK1/2 and PI3-K/Akt signaling pathways to explore the molecular mechanism of rhLK68-mediated inhibition of endothelial cell migration. Treatment with rhLK68 inhibited ERK1/2 phosphorylation but did not influence Akt activation. Interestingly, an inhibitor of protein-tyrosine phosphatase, sodium orthovanadate, dose-dependently reversed both rhLK68-induced dephosphorylation of ERK1/2 and decreased migration of endothelial cells, whereas rhLK68 showed no significant effects on MEKs phosphorylation. In conclusion, these results indicate that inhibition of endothelial cell migration by rhLK68 may be achieved by interfering with ERK1/2 activation via a protein-tyrosine phosphatase-dependent pathway.