Increase In PI 3-kinase Activity Research Articles

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

Temporal and Spatially Regulated Oncogenic KIT Expression and Loss of Dnmt3a Cooperate to Drive MPN Development: Role of PI3Kinase in Dnmt3a Loss Induced Hyperproliferation in Myeloid Cells

Recent studies, including studies from our group have shown that somatic mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with systemic mastocytosis (SM), myelodysplastic syndrome and AML. Importantly, the presence of this mutation is associated with poor prognosis and overall patient survival. We have shown that approximately 12% of patients with SM possess DNMT3A mutations, which are commonly co-expressed with an activating mutation of KIT (KITD816V in humans and KITD814V in mice). However, it is unclear how DNMT3A mutations contribute to myeloid lineage derived mast cell growth and differentiation either alone or in conjunction with KITD816V. Further, the mechanisms by which loss of DNMT3A impairs various mast cell functions either alone or in cooperation with KITD816V are poorly understood. Utilizing mice conditionally deficient in Dnmt3a in the hematopoietic compartment, we show that loss of Dnmt3a in vivo results in increased numbers of mast cells relative to controls. In vitro, loss of Dnmt3a results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors. Previous studies have shown that the level of expression of the transcription factor, Microthalamia (MITF) (high MITF expression drives mast cell commitment) and CEBPα (high C/EBPα drives basophil commitment) in Basophil-Mast cell common progenitor (BMPs) drives the fate of these cells towards either basophilic lineage or mast cell lineage. To assess why loss of Dnmt3a results in increased mast cell lineage commitment, we performed RNAseq analysis and found elevated expression of MITF and a profound repression in the expression of C/EBPα in BM precursors. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with the expression of MITF and C/EBPα. To our surprise, no appreciable differences in the expression of Gata-1, Gata-2 or PU.1 were noted in the absence of Dnmt3a relative to controls. These results demonstrate that early loss of C/EBPα in Dnmt3a null cells contributes to enhanced maturation of mast cells from their precursors. We next assessed the growth and survival potential of Dnmt3a mast cells and found a significant increase in SCF-mediated growth of these cells compared to controls. Biochemical analysis revealed greater PI3Kinase activation in Dnmt3a null cells, and these findings were supported by genome wide transcriptome analysis involving Ingenuity pathway analysis (IPA) and Gene set enrichment analysis (GSEA). Importantly, mast cells derived from Dnmt3a null BM lacking the expression of p85α regulatory subunit of PI3Kinase (Dnmt3a-/-:p85α-/-) or pharmacologic inhibition of PI3Kinase completely corrected SCF induced hyper proliferation in these cells to near normal levels. The increase in PI3Kinase activity in Dnmt3a null cells was associated with the loss of PTEN expression. To understand how loss of Dnmt3a and expression of an oncogenic form of KIT cooperate to drive MPN, we generated mice in which the expression of oncogenic KIT was temporally and spatially regulated in a cell type and tissue specific manner using the Mx-Cre system along with the expression of Dnmt3a. Consistent with earlier studies, we observed that KitD814V/+ mutation alone only resulted in the development of lymphoid neoplasms. In contrast, Dnmt3a-/- :KitD814V/+ mice manifested robust signs of MPN development including splenomegaly with increased numbers of neutrophils and monocytes in the peripheral blood associated with a significant reduction in lymphoid cells including B cells. Importantly, we noted a 5-fold increase in the fraction of LSKs and GMPs in the BM of Dnmt3a-/- :KitD814V/+ mice compared to controls and consequently an increase in the presence of mature Gr-1 and Mac-1 positive myeloid cells in the BM, spleen and in the PB of these mice relative to controls. The observed MPN was more aggressive in Dnmt3a-/- :KitD814V/+ mice compared to Dnmt3a+/- :KitD814V/+ mice and of stem cell origin as transplantation of BM cells from these mice recapitulated the development of MPN in recipient mice, similar to that observed in the original animals. The impact of PI3Kinase inhibition on mitigating oncogenic KIT and loss of Dnmt3a induced MPN and overall survival will be discussed. DisclosuresNo relevant conflicts of interest to declare.

Adenosine A2A receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

Hypoxia and HIF-2α-dependent A2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A2A receptor. A2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A2A receptor-overexpressing HLMVECs. Adenoviral-mediated A2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A2A-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

Glycine-extended gastrin stimulates proliferation via JAK2- and Akt-dependent NF-κB activation in Barrett’s oesophageal adenocarcinoma cells

Glycine-extended gastrin (G-Gly) is a mitogen for several gastrointestinal tissues although the mechanisms responsible are ill-defined and it is unknown if G-Gly can influence signalling in Barrett's oesophagus. G-Gly stimulated proliferation in OE19 and OE33 cells in a dose-dependant manner. This was unaffected by a CCK2 receptor antagonist but abolished by COX-2 inhibitors. G-Gly induced proliferation, COX-2 mRNA abundance, and PGE2 secretion, were all abolished by inhibition of JAK2, PI3-kinase, Akt or NF-kappaB. G-Gly stimulated phosphorylation of JAK2 and increased PI3-kinase activity in JAK2 immunoprecipitates. G-Gly increased Akt phosphorylation and kinase activity and NF-kappaB reporter activity in a JAK2-, PI3-kinase- and Akt-sensitive manner. G-Gly increased COX-2 promoter transcription in an Akt and NF-kappaB-dependent manner and also reduced COX-2 mRNA degradation in an Akt-insensitive manner. We conclude that G-Gly induced signalling involves a JAK2/PI3-kinase/Akt/NF-kappaB sequence leading to COX-2 transcription. G-Gly also seems to stabilise COX-2 mRNA via a separate pathway.

Crosstalk between the AMP-activated kinase and insulin signaling pathways rescues murine blastocyst cells from insulin resistance

Maternal insulin resistance results in poor pregnancy outcomes. In vivo and in vitro exposure of the murine blastocyst to high insulin or IGF1 results in the down-regulation of the IGF1 receptor (IGF1R). This in turn leads to decreased glucose uptake, increased apoptosis, as well as pregnancy resorption and growth restriction. Recent studies have shown that blastocyst activation of AMP-activated protein kinase (AMPK) reverses these detrimental effects; however, the mechanism was not clear. The objective of this study was to determine how AMPK activation rescues the insulin-resistant blastocyst. Using trophoblast stem (TS) cells derived from the blastocyst, insulin resistance was recreated by transfecting with siRNA to Igf1r and down-regulating expression of the protein. These cells were then exposed to AMPK activators 5-aminoimidazole-4-carboxamide riboside and phenformin, and evaluated for apoptosis, insulin-stimulated 2-deoxyglucose uptake, PI3-kinase activity, and levels of phospho-AKT, phospho-mTor, and phospho-70S6K. Surprisingly, disrupted insulin signaling led to decreased AMPK activity in TS cells. Activators reversed these effects by increasing the AMP/ATP ratio. Moreover, this treatment increased insulin-stimulated 2-deoxyglucose transport and cell survival, and led to an increase in PI3-kinase activity, as well as increased P-mTOR and p70S6K levels. This study is the first to demonstrate significant crosstalk between the AMPK and insulin signaling pathways in embryonic cells, specifically the enhanced response of PI3K/AKT/mTOR to AMPK activation. Decreased insulin signaling also resulted in decreased AMPK activation. These findings provide mechanistic targets in the AMPK signaling pathway that may be essential for improved pregnancy success in insulin-resistant states.

Phosphatidylinositol kinases as regulators of GA‐stimulated α‐amylase secretion in barley (Hordeum vulgare)

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3-kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4-kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between alpha-amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [gamma-(32)P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the alpha-amylase secretion and protoplast cell vacuolation in a dose-dependent manner. Wortmannin showed a similar inhibitory effect on alpha-amylase secretion and PI4 kinase activity. GA evoked only a long-term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3-dimercapto-1-propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

The effect of cinnamon extract on insulin resistance parameters in polycystic ovary syndrome: a pilot study

Cinnamon extract has been shown to reduce insulin resistance in in vitro and in vivo studies by increasing phosphatidylinositol 3-kinase activity in the insulin signaling pathway and thus potentiating insulin action. Fifteen women with polycystic ovary syndrome (PCOS) were randomized to daily oral cinnamon and placebo for 8 weeks. Comparisons of post-treatment to baseline insulin sensitivity indices using fasting and 2-hour oral glucose tolerance tests showed significant reductions in insulin resistance in the cinnamon group but not in the placebo group. A larger trial is needed to confirm the findings of this pilot study and to evaluate the effect of cinnamon extract on menstrual cyclicity.

Mechanisms Regulating Tumor Angiogenesis by 12-Lipoxygenase in Prostate Cancer Cells

12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

Mechanism of Constitutive Phosphoinositide 3-Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.

46. In Vivo Adenoviral Gene Transfer of Activated PI3-Kinase Rescues Cardiac Dysfunction and Injury Induced by Ischemia-Reperfusion after Chronic Cardiac Akt Activation

Acute activation of the serine-threonine kinase, Akt, is cardioprotective and reduces both infarction and dysfunction after ischemia-reperfusion injury (IRI). However, Akt is paradoxically and chronically activated in human advanced heart failure suggesting it may become maladaptive. To test the effects of chronic Akt activation, we generated transgenic mice (TG) with cardiac specific expression of either activated Akt (myr-Akt) or dominant-negative (dn) Akt and studied their response to global IRI in the ex vivo Langendorff perfusion model. We have previously reported that after IRI (20min Ischemia, 40min Reperfusion), none of the myr-Akt TG hearts recovered any function while littermate control (NTG) hearts recovered to 53.5|[plusmn]|3.5% of the baseline LV developed pressure (LVDP) (n=11 each, p<0.01). dn-Akt manifested a moderate reduction in functional recovery (34.4|[plusmn]|7.5%, n=5, p<0.001), consistent with a cardioprotective role for endogenous Akt. TTC staining and CPK release into the perfusate demonstrated dramatically increased infarct size in myr-Akt TG hearts (p<0.01). Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of upstream activation of PI3Kinase (PI3K) through both proteasome-dependent degradation of insulin-receptor substrate-1 (IRS-1) and inhibition of IRS transcription. QRT-PCR demonstrated mRNA levels for both IRS-1 and IRS-2 were significantly reduced in myr-Akt TG hearts (68.7%, p<0.001 and 59.9%, p<0.005, respectively). To understand the functional significance of this, we performed in vivo cardiac gene transfer of constitutively active PI3K (Ad.BD110) that does not depend on IRS-1 for activation or control virus in myr-Akt TG mice. Three days after transfection, expression of BD110 was documented by immunoblotting and associated with a significant increase in PI3K activity (2.6 fold, p<0.05). No functional recovery was seen in myr-Akt TG mice transduced with control virus (Ad.GFP) after IRI. However, after Ad.BD110 infection, myr-Akt TG mice demonstrated significant functional recovering achieving a maximal LVDP of 34.5|[plusmn]|1.8% (n=7, p<0.01 compared to myr-Akt TG without BD110). CPK release from myr-Akt TG hearts was also dramatically reduced after IRI (0.015 fold, p<0.01 vs myr-Akt TG alone, p=NS vs NTG). Taken together, these data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. These findings demonstrate a mechanism by which chronic Akt activation can become maladaptive in heart failure and suggest the importance of therapeutic strategies that target multiple PI3K-dependent pathways simultaneously.

Insulin-like growth factors inhibit podocyte apoptosis through the PI3 kinase pathway

Abnormal podocyte development and progressive podocyte injury have been implicated in a number of human kidney diseases. Factors necessary for regulating development and maintenance of this cell type are only beginning to emerge. To study the role of the insulin-like growth factor (IGF) system in regulating podocyte survival, we induced human fetal podocytes to undergo apoptosis. We demonstrated a significant increase in apoptosis when these cells were incubated in the presence of etoposide, as measured by DNA fragmentation and nuclear membrane condensation and blebbing. Podocyte apoptosis was reduced to control levels when the cells were coincubated in the presence of IGF-1. We showed that the protective effect of IGFs in this cell type was mediated through the activation of the phosphatidylinositol 3'-kinase (PI3K) pathway. IGF-1 stimulation resulted in the formation of the insulin receptor substrate (IRS)-1-p85 complex, an increase in PI3 kinase activity, and activation of protein kinase B (AKT/PKB) and the bcl-2 family member bad. Incubation of the podocytes with inhibitors of the PI3 kinase pathway resulted in a loss of this IGF-1 protective effect. These data demonstrate an important role for the IGF system in fetal podocyte survival in vitro, and suggest potential mediators to slow or alleviate the loss or damage of the podocyte in progressive renal disease.

EGF stimulates mesangial cell mitogenesis via PI3-kinase-mediated MAPK-dependent and AKT kinase-independent manner: involvement of c-fosand p27Kip1

Epidermal growth factor (EGF) is a potent mitogen for mesangial cells. The mechanism by which EGF induces DNA synthesis is not precisely understood. We investigated the role of phosphatidylinositol (PI)3-kinase in regulating mitogenesis. EGF increased PI3-kinase activity resulting in stimulation of PDK-1 and Akt kinase activities. Blocking of PI3-kinase activity using LY-294002 or adenoviral expression of PTEN, which dephosphorylates PI3,4,5-tris-phosphate and thus inactivates PI3-kinase signaling, significantly inhibits EGF-induced DNA synthesis. Expression of dominant-negative Akt kinase, however, had no effect on DNA synthesis. But it inhibited EGF-induced phosphorylation of FoxO3a transcription factor, thus demonstrating its functional consequences. These data indicate that EGF increases the DNA synthesis in a PI3-kinase-dependent but Akt-independent manner. In addition to activating PI3-kinase signaling, EGF increased Erk1/2 MAPK activity, leading to transcriptional activation of its nuclear target Elk-1 and resulting in c-fos expression. Inhibition of MAPK activity by MEK inhibitor U-0126 abolished EGF-induced DNA synthesis. Because EGF activates PI3-kinase, which also regulates DNA synthesis, the effect of PI3-kinase on MAPK activity was also examined. Inhibition of PI3-kinase signaling blocked EGF-induced MAPK activity as well as Elk-1-dependent reporter transcription and c-fos gene transcription. To further determine the mechanism of EGF-induced DNA synthesis, we investigated the effect of EGF on the cyclin-dependent kinase inhibitor p27(Kip1). EGF reduced the expression of p27(Kip1). Inhibition of PI3-kinase action or MAPK activity abolished the reduction in p27(Kip1) expression induced by EGF. These data provide the evidence that a linear signal transduction pathway involving PI3-kinase-dependent MAPK regulates EGF-induced DNA synthesis in mesangial cells by regulating c-fos and p27(Kip1) expression.

Increased PI3-Kinase in Presympathetic Brain Areas of the Spontaneously Hypertensive Rat

Existing evidence led us to hypothesize that increases in p85alpha, a regulatory subunit of PI3-kinase, in presympathetic brain areas contribute to hypertension. PI3-kinase p85alpha, p110alpha, and p110delta mRNA was 1.5- to 2-fold higher in the paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHR) compared with their controls, Wistar Kyoto rats (WKY). The increase in p85alpha/p110delta was attenuated in SHR treated with captopril, an angiotensin (Ang)-converting enzyme inhibitor, from in utero to 6 months of age. In the rostral ventrolateral medulla (RVLM), p110delta mRNA was approximately 2-fold higher in SHR than in WKY. Moreover, the increases in mRNA were associated with higher PI3-kinase activity in both nuclei. The functional relevance was studied in neuronal cultures because SHR neurons reflect the augmented p85alpha mRNA and PI3-kinase activity. Expression of a p85 dominant-negative mutant decreased norepinephrine (NE) transporter mRNA and [3H]NE uptake by approximately 60% selectively in SHR neurons. In summary, increased p85alpha/p110delta expression in the PVN and RVLM is associated with increased PI3-kinase activity in the SHR. Furthermore, normalized PI3-kinase p85alpha/p110delta expression within the PVN might contribute to the overall effect of captopril, perhaps attributable to a consequent decrease in NE availability.

Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial Cells through Phosphoinositide 3-Kinase

Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways.

Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents.

Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.

Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells are known to express D-type cyclins at very low levels and these levels increase dramatically during in vitro and in vivo differentiation. Here, we investigate some of the signalling pathways regulating expression of cyclin D1 and progression to S phase, the Ras/Extracellular signal-regulated protein kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. We demonstrate that ERK phosphorylation is fully dispensable for the regulation of cyclin D1 level and for the progression from G1 to S phase in ES cells. By contrast, PI3-kinase activity is required for both. Differentiation induced by retinoic acid results in the gain of ERK-dependent control of cyclin D1 expression and of S phase progression. Differentiation is also paralleled by an increase in PI3-kinase activity. This leads (a) to an increase in the p70 S6 kinase-dependent regulation of the steady-state level of cyclin D1, and (b) to a concomitant decrease in the GSK3beta-dependent rate of cyclin D1 degradation. Altogether, these multiple pathways account for the dramatic increase in the level of cyclin D1 protein which parallels ES cell differentiation. Our studies suggest that PI3-kinase is an important regulator of the ES cell cycle and that its activity is not regulated by mitogen stimulation.

Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats.

Arteries from deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184+/-5 mm Hg] compared with sham rats (SBP=111+/-2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85alpha, p110alpha, p110beta, and p110delta but not p110gamma PI3-kinase subunits; p110delta protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110beta protein density, but neither L-NNA nor DOCA-salt had differences in p85alpha and p110alpha. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 micromol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110delta density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.

Troglitazone inhibits growth and improves insulin signaling by suppression of angiotensin II action in vascular smooth muscle cells from spontaneously hypertensive rats

Troglitazone, a thiazolizidinedione, has recently been reported to possess anti-arteriosclerotic properties. To evaluate mechanisms underlying the anti-arteriosclerotic effects of troglitazone, we examined the effect of troglitazone on growth, expression of growth factors, and insulin signaling in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) which produce angiotensin II (Ang II) in a homogeneous culture. Troglitazone inhibited basal and serum-stimulated DNA synthesis and inhibited increases in the number of VSMC from SHR and normotensive Wistar–Kyoto (WKY) rats. Its inhibition was greater in VSMC from SHR. Troglitazone abolished DNA synthesis in response to Ang II in VSMC from both rat strains and markedly inhibited DNA synthesis in response to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AA in VSMC from SHR. Troglitazone did not alter the expression of transforming growth factor (TGF)-β1, PDGF A-chain, or basic fibroblast growth factor (bFGF) mRNAs in VSMC from WKY rats, but it markedly decreased expression of these growth factor mRNAs in VSMC from SHR. Troglitazone markedly decreased basal and Ang II-stimulated expression of extracellular signal-regulated kinase proteins in VSMC from both rat strains. Troglitazone abolished Ang II-induced suppression of phosphatidilinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from WKY rats. Basal PI3-kinase activity, tyrosine phosphorylation of IRS-1, and IRS-1 associated p85 levels were lower in VSMC from SHR than in cells from WKY rats. Troglitazone significantly increased PI3-kinase activity, IRS-1 associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from SHR. These results indicate that troglitazone produce its anti-arteriosclerotic effects through suppression of the action of growth-promoting factors including Ang II, and that troglitazone inhibits Ang II-induced suppression of insulin signaling in VSMC from SHR, suggesting that tissue Ang II may lead to insulin resistance and to arteriosclerosis in hypertension. Troglitazone may be useful in the treatment of insulin resistance as well as of hypertensive vascular diseases.

Muscarinic Receptors Mediate Phospholipase C-dependent Activation of Protein Kinase B via Ca2+, ErbB3, and Phosphoinositide 3-Kinase in 1321N1 Astrocytoma Cells

In 1321N1 astrocytoma cells, heterotrimeric G-protein-coupled receptors that activate phosphoinositide-specific phospholipase Cbeta (PLCbeta) isoforms via G(q), induced a prolonged activation of protein kinase B (PKB) after a short delay. For example, the effect of carbachol acting on M3 muscarinic receptors is blocked by wortmannin, suggesting it is mediated via a phosphoinositide 3-kinase (PI 3-kinase). In support of this, carbachol increased PI 3-kinase activity in PI 3-kinase (p85) immunoprecipitates. The pathway linking PLC-coupled receptors to PI 3-kinase was deduced to involve phosphoinositide hydrolysis and Ca2+-dependent ErbB3 transactivation but not protein kinase C on the basis of the following evidence: (i) inhibition of carbachol stimulated PLC by pretreatment with the phorbol ester phorbol 12-myristate 13-acetate concomitantly reduced PKB activity, whereas stimulation of other PLC-coupled receptors also activated PKB; (ii) Ca2+ ionophores and thapsigargin stimulated PKB activity in a wortmannin-sensitive manner, whereas bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked carbachol-stimulated PKB activity; (iii) phorbol 12-myristate 13-acetate alone did not activate PKB, whereas a protein kinase C inhibitor did not prevent the activation of PKB by carbachol; and (iv) carbachol stimulated ErbB3-tyrosine phosphorylation and association with p85, and both these and PKB activity were blocked by tyrphostin AG1478, an epidermal growth factor receptor-tyrosine kinase inhibitor. These experiments define a novel pathway linking G(q)-coupled G-protein-coupled receptors to the activation of PI 3-kinase and PKB.