Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells.
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We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10(-7) M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 +/- 8.5% at 30 s and 175.8 +/- 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 microM) resulted in a significant inhibition of both C2 ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 microM), inhibited only C2 ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110alpha and p110gamma indicated that both endothelin and C2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110alpha and p110gamma, whereas C2 ceramide interacted only with the catalytic subunit p110alpha. In summary, C2 ceramide activated PI 3-kinase p110alpha subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2 ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110alpha and p110gamma subunits (type IA and IB) of PI 3-kinase. (+info)
New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.
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The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the dominantly inherited disease Cowden syndrome, which is characterized by multiple hamartomas and a high proclivity for developing cancer. A series of publications over the past year now suggest a mechanism by which PTEN loss of function results in tumors. PTEN appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell growth and survival by dephosphorylating the 3 position of phosphoinositides. (+info)
Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle.
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1. Development of the pacemaker system in the small intestine depends upon signalling via tyrosine kinase (Kit) receptors. The downstream pathways initiated by Kit in interstitial cells of Cajal (ICC) have not been investigated. Wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibitors of phosphatidylinositol 3'-kinase (PI3-kinase), were used to test the involvement of this pathway in the development and maintenance of ICC and electrical rhythmicity in the murine small intestine. 2. ICC and electrical slow waves were present in the murine jejunum at birth. ICC and electrical rhythmicity continued to develop in neonates such that adult activity was recorded after 1 week. Development of ICC and rhythmicity were maintained in organ culture. 3. Wortmannin or LY 294002 inhibited the development of slow waves and blocked rhythmicity within 2-4 days. Loss of slow waves was preceded by disappearance of Kit-positive cells from the myenteric (IC-MY) and deep muscular plexus (IC-DMP) regions. Wortmannin or LY 294002 had no acute effect on slow waves. 4. Muscles from older animals (day 10-day 30) developed resistance to wortmannin treatment, but when the exposure to wortmannin was increased to 35 days, damage to ICC networks and electrical dysrhythmias were observed. 5. PI3-kinase appears to be a critical downstream signalling element linking Kit receptors to ICC development and maintenance of phenotype. ICC are more sensitive to Kit or PI3-kinase blockade at birth, but the importance of the PI3-kinase signalling in the maintenance of ICC persists into adulthood. Interference with PI3-kinase signalling in immature or adult animals could result in disruption of ICC and gastrointestinal dysrhythmias. (+info)
Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas.
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CPP32/apopain (Caspase-3), a protease of the Ced-3/ICE family, is a central mediator in the apoptosis induced by TNF or anti-Fas. In this study we demonstrate that wortmannin, an inhibitor of PI-3K, enhances the activation of CPP32 (Caspase-3) and DNA fragmentation in TNF-treated U937 cells and anti-Fas-treated Jurkat cells. Caspase-3-like activity, Ac-DEVD-MCA cleavage activity, is enhanced by wortmannin in the range of the concentration (1 - 100 nM) specifically inhibiting PI-3K. LY294002, another PI-3K inhibitor, also enhances Caspase-3-like activity, but inhibitors for myosin light chain kinase and calmodulin dependent kinase do not have any effect on the Caspase-3-like activity. Wortmannin (1 - 100 nM) enhances the processing of Caspase-3 (32K) into active form (17K) in TNF- or anti-Fas-treated cells, but not in untreated cells. These observations suggest that inhibition of PI-3K induces the activation of processing enzyme of Caspase-3 or increases the susceptibility of Caspase-3 to the processing enzyme. PI-3K seems to protect the cells from apoptosis by suppressing the activation of Caspase-3. (+info)
Role of PI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1 in Baf-3 cells.
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In Baf-3 cells, IL-3 and IGF-1 both inhibit cell death. These growth factors act at least on two different pathways involved in the inhibition of apoptosis. They both upregulate Bcl-X at the mRNA and protein levels and also activate a pathway which inhibits apoptosis in the absence of protein synthesis. Recently, these two growth factors have been shown to activate the PI3-kinase-AKT pathway which leads to the phosphorylation of the pro-apoptotic Bcl-XL regulator Bad. In this study, we have investigated the role of PI3-kinase in the regulation of Bcl-X expression and in the survival of Baf-3 cells. We show that PI3-kinase activation is involved in the upregulation of Bcl-X mRNA induced by both IL-3 and IGF-1. Moreover, PI3-kinase activity is also necessary for inhibition of apoptosis and caspase regulation by IGF-1 but not IL-3. (+info)
Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton's tyrosine kinase.
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Bruton's tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases. (+info)
Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors.
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Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway. (+info)
Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.
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Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity. (+info)