Presence Of PI3-kinase Inhibitor Research Articles

GABA Coordinates with Insulin in Regulating Secretory Function in Pancreatic INS-1 β-Cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABAARs). We and others recently reported that islet β-cells also express GABAARs and that activation of GABAARs increases insulin release. Here we investigate the effects of insulin on the GABA-GABAAR system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 µM) suppressed the GABA-induced current (IGABA) by 43%. Zinc-free insulin also suppressed IGABA to the same extent of inhibition by regular insulin. The inhibition of IGABA occurs within 30 seconds after application of insulin. The insulin-induced inhibition of IGABA persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABAARs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 µM, p<0.01) and insulin (1 µM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABAARs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABAAR signaling presenting a feedback mechanism for fine-tuning β-cell secretion.

Effect of Hyperglycemia on Human Monocyte Activation

Our recent study defined the chemokine-induced human monocyte signaling under normoglycemic condition. To explore the hyperglycemia-induced monocyte signaling, we performed adhesion, migration, and transmigration assays on human monocytes obtained from...

Abstract 1207: Transactivation of EGFR/ErbB2 receptor by TNF abrogates TNF-induced apoptosis in bronchial epithelial cells and non-small cell lung cancer cells

Abstract [Background] Epidermal Growth Factor Receptor (EGFR) and other ErbB receptors; ErbB2-4, through cytokine and growth factor stimulation, are upregulated in pneumonitis and lung cancer, suggesting that the activation of ErbB receptors is response to pneumonitis and lung cancer in humans. TNF is a cytokine with many biological properties including both anti- and pro-apoptotic signaling pathways. However, the molecular switch, which determines TNF regulation of these two different functions is not well characterized. We previously presented that TNF transactivated both EGFR and ErbB2 via Src kinase activity that promote the survival response to TNF. In the present study, we tested the hypothesis that EGFR/ErbBs activity regulates TNF-mediated bronchial epithelial and non-small cell lung cancer cell survival. [Method] Bronchial epithelial, Beas-2B cells and non-small cell lung cancer, NCI-H292 cells were used for these studies. Cells were treated with TNF (100 ng/ml) in the presence of either PI 3-kinase inhibitor (wortmannin or LY294002), MAP kinase inhibitor (U0126), MMP inhibitor (GM6001 or TAPI-1 or −2) or EGFR tyrosine kinase inhibitor (Erlotinib). Apoptosis and signal transduction pathway activation were determined by TUNEL staining and Western blot analysis, respectively. [Results] In Beas-2B and NCI-H292 cells, TNF-induced apoptosis was significantly enhanced in the presence of EGFR tyrosine kinase inhibitor. Similarly, treatment of Beas-2B and NCI-H292 cells with TNF failed to increase apoptosis except in the presence of PI 3-kinase inhibitor, which increased the apoptotic response 10-fold. Treatment with TNF in the presence of MAP kinase inhibitor, did not increase apoptosis. In Beas 2B and NCI-H292 cells, TNF stimulated EGFR/ErbB2 phosphorylation within 5 min. EGFR/ErbB2 transactivation by TNF was blocked with GM6001 and TAPI-1, which inhibited MMP activity, especially TACE. Furthermore, in the presence of neutralizing antibodies for HB-EGF, TGF-α and EGF, EGFR/ErbB2 transactivation by TNF was inhibited, suggesting TNF-stimulated EGFR/ErbB2 transactivation via TACE cleavage of HB-EGF, TGF- α and EGF abrogates TNF-induced apoptosis in human bronchial epithelial cell and lung cancer cell. TNF stimulated AKT activation via EGFR tyrosine kinase activation. Akt was shown to be a downstream target of TNF-activated EGFR/ErbB2. [Conclusion] We demonstrate that TNF stimulates EGFR/ErbB2 transactivation via ADAM/MMP in a manner that requires HB-EGF, TGF- α and EGF with AKT as a critical downstream regulator of TNF-induced anti-apoptosis. This novel observation has significant implications for understanding the role of EGFR/ErbB2 in maintaining human bronchial epithelial cell homeostasis in an environment of inflammation, injury/repair, such as inflammation-associated carcinogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1207.

The PKB inhibitor Akti-1/2 potentiates PAR-1-mediated platelet function independently of its ability to block PKB.

The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small-molecule inhibitors and limiting genetic models. Recently, a selective, non-ATP-competitive PKB inhibitor, Akti-1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. To determine the effect of the PKB inhibitor Akti-1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Akti-1/2 potently inhibited thrombin-mediated PKB phosphorylation on Thr(308) and Ser(473) and phosphorylation of its downstream substrate GSK3beta, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti-1/2 strongly potentiated PAR-1-mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti-1/2 was associated with increased [Ca(++)](i), PKC activation and degranulation and was ablated by agents that antagonized this pathway. The PKB inhibitor Akti-1/2 increases PAR-1-mediated platelet responses in a PKB-independent, Ca(++)/PKC-dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti-1/2 and structurally related compounds.

Role of Cadherin-mediated Cell-Cell Adhesion in Pancreatic Exocrine-to-Endocrine Transdifferentiation

Although pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and beta-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic beta-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role.

Toxoplasma gondiitriggers Gi-dependent PI 3-kinase signaling required for inhibition of host cell apoptosis

Infection with the intracellular parasite Toxoplasma gondii renders cells resistant to multiple pro-apoptotic signals, but underlying mechanisms have not been delineated. The phosphoinositide 3-kinase (PI 3-kinase) pathway and the immediate downstream effector protein kinase B (PKB/Akt) play important roles in cell survival and apoptosis inhibition. Here, we show that Toxoplasma infection of mouse macrophages activates PKB/Akt in vivo and in vitro. In a mixed population of infected and non-infected macrophages, activation is only observed in parasite-infected cells. The PI 3-kinase inhibitors wortmannin and LY294002 block parasite-induced PKB phosphorylation. PKB activation occurs independently of Toll-like receptor adaptor protein MyD88 but uncoupling of Gi-protein-mediated signaling with pertussis toxin prevents PKB phosphorylation. Moreover, in the presence of PI 3-kinase inhibitors or pertussis toxin, not only PKB activation but also ERK1/2 activation during T. gondii infection is defective. Most importantly, the parasite's ability to induce macrophage resistance to pro-apoptotic signaling is prevented by incubation with PI 3-kinase inhibitors. This study demonstrates that T. gondii exploits host Gi-protein-dependent PI 3-kinase signaling to prevent induction of apoptosis in infected macrophages.

Role of PI 3-kinase and PIP 3 in submandibular gland branching morphogenesis

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser 473 by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP 3), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP 3 pathway.

Mechanism of phosphatidylinositol 3-kinase-dependent increases in BAC1.2F5 macrophage-like cell density in response to M-CSF: phosphatidylinositol 3-kinase inhibitors increase the rate of apoptosis rather than inhibit DNA synthesis.

To determine the role of phosphatidylinositol 3-kinase (PI 3-kinase) in macrophagecolony stimulating factor (M-CSF)-induced macrophage proliferation. The M-CSF-dependent BAC1.2F5 murine macrophage cell line was used. PI 3-kinase activity, Protein kinase B activation, increased cell numbers, induction of DNA synthesis and apoptosis were measured in response to serum, M-CSF and PI 3-kinase inhibitors. Wortmannin or LY294002 inhibited M-CSF-stimulated increases in BAC1.2F5 cell density. Further analysis showed that inhibition of PI 3-kinase had an insignificant effect on DNA synthesis, but significantly induced apoptosis. Other co-factors in serum mediated cell survival and prevented programmed cell death, in a PI 3-kinase-dependent manner. Stimulation of BAC1.2F5 macrophages with M-CSF induced phosphorylation of PKB/Akt as detected by activation-specific antibodies. Activation of PKB/Akt correlated with PI 3-kinase activation, suggesting that the protection from apoptosis in these cells is mediated by PKB/Akt. These results indicate that the lack of increase in cell numbers when cells are stimulated with M-CSF in the presence of PI 3-kinase inhibitors is due to a preferential PI 3-kinase requirement for protection against apoptosis, rather than a requirement for PI 3-kinase activation during the proliferation signal.

Epidermal Growth Factor Impairs the Cytochrome C/Caspase-3 Apoptotic Pathway Induced by Transforming Growth Factor β in Rat Fetal Hepatocytes Via a Phosphoinositide 3-Kinase–Dependent Pathway

Transforming growth factor β (TGF-β)–mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-β–induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-β disrupts the mitochondrial transmembrane potential (Δψm ) and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase–dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-β produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-xL , an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-xL levels even in the presence of TGF-β. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-β–induced bcl-xL down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-β–induced death by acting upstream from the mitochondrial changes,i.e., preventing bcl-xL down-regulation, cytochrome c release, and activation of caspase-3.(Hepatology 2000;32:528-535.)

A phosphatidylinositol 3-kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation.

ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45-65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy.

A phosphatidylinositol 3‐kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation

ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45–65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy. Int. J. Cancer 78:642–647, 1998. © 1998 Wiley-Liss, Inc.

Insulin-Like Growth Factor and Potassium Depolarization Maintain Neuronal Survival by Distinct Pathways: Possible Involvement of PI 3-Kinase in IGF-1 Signaling

Cultured cerebellar granule neurons die by apoptosis when switched from a medium containing an elevated level of potassium (K+) to one with lower K+ (5 mM). Death resulting from the lowering of K+ can be prevented by insulin-like growth factor (IGF-1). To understand how IGF-1 inhibits apoptosis and maintains neuronal survival, we examined the role of phosphoinositide 3-kinase (PI 3-kinase). Activation of PI 3-kinase has been shown previously to be required for NGF-mediated survival in the PC12 pheochromocytoma cell line. We find that in primary neurons, IGF-1 treatment leads to a robust activation of PI 3-kinase, as judged by lipid kinase assays and Western blot analysis. Activation of PI 3-kinase is likely to occur via tyrosine phosphorylation of the insulin receptor substrate protein. Treatment with two chemically distinct inhibitors of PI 3-kinase, wortmannin and LY294002, reduces PI 3-kinase activation by IGF-1 and inhibits its survival-promoting activity, suggesting that PI 3-kinase is necessary for IGF-1-mediated survival. Death resulting from PI 3-kinase blockade is accompanied by DNA fragmentation, a hallmark of apoptosis. Furthermore, neurons subjected to PI 3-kinase blockade can be rescued by transcriptional and translation inhibitors, suggesting that IGF-1-mediated activation of PI 3-kinase leads to a suppression of "killer gene" expression. In sharp contrast to IGF-1, elevated K+ does not activate PI 3-kinase and can maintain neuronal survival in the presence of PI 3-kinase inhibitors. Therefore, survival of granule neurons can be maintained by PI 3-kinase dependent (IGF-1-activated) and independent (elevated K+-activated) pathways.