The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells

The Notch family of transmembrane receptors have been implicated in a variety of cellular decisions in different cell types. Here we investigate the mechanism underlying Notch-1-mediated anti-apoptotic function in T cells using model cell lines as the experimental system. Ectopic expression of the intracellular domain of Notch-1/activated Notch (AcN1) increases expression of anti-apoptotic proteins of the inhibitors of apoptosis (IAP) family, the Bcl-2 family, and the FLICE-like inhibitor protein (FLIP) and inhibits death triggered by multiple stimuli that activate intrinsic or extrinsic pathways of apoptosis in human and murine T cell lines. Numb inhibited the AcN1-dependent induction of anti-apoptotic proteins and anti-apoptotic function. Using pharmacological inhibitors and dominant-negative approaches, we describe a functional role for phosphatidylinositol 3-kinase (PI3K)-dependent activation of the serine-threonine kinase Akt/PKB in the regulation of AcN1-mediated anti-apoptotic function and the expression of FLIP and IAP family proteins. Using a cell line deficient for the T cell-specific, Src family protein, the tyrosine kinase p56(lck) and by reconstitution approaches we demonstrate that p56(lck) is required for the Notch-1-mediated activation of Akt/PKB function. Furthermore, the Src tyrosine kinase inhibitor, PP2, abrogated ectopically expressed AcN1-mediated anti-apoptotic function and phosphorylation of p56(lck). We present evidence that endogenous Notch-1 associates with p56(lck) and PI3K but that Akt/PKB does not co-immunoprecipitate with the Notch1.p56(lck).PI3K complex. Finally, we demonstrate that the Notch1.p56(lck).PI3K complex is present in primary T cells that have been activated in vitro and sustained in culture with the cytokine interleukin-2.     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.     

Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts

Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G(q)-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human alpha(1A)-adrenergic receptor. Treatment of the cells with phenylephrine, a specific alpha(1)-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the alpha(1)-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca(2+) concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca(2+) does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the alpha(1A)-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.     

CT-1 mediated cardioprotection against ischaemic re-oxygenation injury is mediated by PI3 kinase, Akt and MEK1/2 pathways.

Cardiotrophin-1 protects cardiac myocytes from ischaemic re-oxygenation (IR) injury. CT-1 activates MEK1/2,p42/44MAPK as well as the phosphatidylinositol (PI) 3-OH kinase (PI3) protein kinase B (PKB/Akt) pathway. In this study we investigate the signalling pathways that mediate the anti-apoptotic cell survival effect of CT-1 in IR. Dominant negative gene based inhibitors of MEK1/2, PI3-kinase and Akt inhibited CT-1 mediated cardioprotection in re-oxygenation as did chemical inhibitors of the PI3-kinase pathway. Hence the PI3-kinase/Akt pathway is required in addition to MEK1/2 to mediate CT-1 cardioprotection in IR.     

Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.     

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.