High glucose promotes cell proliferation and enhances GDNF and RET expression in pancreatic cancer cells

Phosphoinositide 3-kinases (PI3Ks) are key enzymes that activate intracellular signaling molecules when a number of different growth factors bind to cell surface receptors. PI3Ks are divided into three classes (I, II, III), and enzymes of each class have different tissue specificities and physiological functions. The α-isoform (PI3K-C2α) of class II PI3Ks is considered ubiquitous and preferentially activated by insulin. Our previous study showed that suppression of PI3K-C2α leads to apoptotic cell death. The aim of this study is to determine whether depletion of PI3K-C2α affects ERK or PKB/Akt activity following stimulation with serum and insulin growth factors in Chinese hamster ovary cells expressing human insulin receptors (CHO-IR) and human HepG2 liver cells. Different antisense oligonucleotides (ODNs), which were designed based on the sequence of the C2 domain of the human PI3K-C2α gene, were transfected into cells to inhibit PI3K-C2α expression. Insulin- or serum-induced stimulation of ERK was significantly suppressed by depletion of PI3K-C2α, whereas phosphorylation of IRS-1 and the stimulation of PKB/Akt by insulin were not affected. The number of apoptotic cells was also increased by depletion of PI3K-C2α protein levels. Taken together, our data indicate that PI3K-C2α may be a crucial factor in the stimulation of ERK activity in response to serum or insulin, whereas it is less important for the stimulation of PKB/Akt activity in response to insulin.     

The α-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2alpha isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2alpha activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2alpha required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2alpha is a candidate for participation in insulin-specific intracellular signalling.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.     

Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms

Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in?vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.     

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.     

Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.     

A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication

Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.     

Class II Phosphoinositide 3-Kinases Contribute to Endothelial Cells Morphogenesis

The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.     

Overexpression of phosphoinositide-3-kinase class II alpha enhances mesenchymal stem cell survival in infarcted myocardium

The efficacy of mesenchymal stem cell (MSC) therapy for myocardial regeneration is limited by the poor survival of stem cells after transplantation into the infarcted heart. To improve the cell survival of MSCs in the infarcted heart, MSCs were genetically engineered to overexpress phosphoinositide-3-kinase class II alpha (PI3K-C2α). PI3K-C2α overexpression increased PI3K expression and the cell viability of MSCs. Furthermore, levels of survival-related phosphorylation were elevated in PI3K-C2α-MSCs. But, the level of apoptotic proteins downregulated and the number of PI-positive cells decreased in PI3K-C2α-MSCs compared to hypoxic MSCs. Nine rats per group had 1 × 10⁶ cells (20 μl PBS) transplanted after myocardial infarction. One week after transplantation, infarct size and area of fibrosis were reduced in the PI3K-C2α-MSC-transplanted group. The number of TUNEL positive cells declined, while the mean microvessel count per field was higher in the PI3K-C2α-MSC group than the MSC-injected group. Heart function was improved in the PI3K-C2α-MSCs group as assessed using a Millar catheter at 3 weeks after transplantation. These findings suggest that overexpression of PI3K-C2α in MSCs can assist cell survival and enhance myocardial regeneration.     

Class II phosphoinositide 3-kinase C2alpha: what we learned so far

More than fifteen years after the first identification of a class II isoform of phosphoinositide 3-kinase (PI3K) in Drosophila melanoǵaster this subfamily remains the most enigmatic among all PI3Ks. What are the functions of these enzymes? What are their mechanisms of activation? Which downstream effectors are specifically regulated by these isoforms? Are class I and class II PI3Ks redundant or do they control different intracellular processes? And, more important, do class II PI3Ks have a role in human diseases? The recent increased interest on class II PI3Ks has started providing some answers to these questions but still a lot needs to be done to completely uncover the contribution of these enzymes to physiological processes and possibly to pathological conditions. Here we will summarise the recent findings on the alpha isoform of mammalian class II PI3Ks (PI3K-C2α ) and we will discuss the potential involvement of this enzyme in human diseases.     

Individual phosphoinositide 3-kinase C2alpha domain activities independently regulate clathrin function

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.     

Class II phosphoinositide 3-kinase is activated by insulin but not by contraction in skeletal muscle.

While the role of the class IA phosphoinositide 3-kinase (PI 3-kinase) in insulin signaling is well established, little is known about the role of the class II PI 3-kinases. We show that insulin stimulation of intact rat soleus and epitrochlearis muscles causes a 3- to 4-fold increase in the activity of the wortmannin-resistant alpha isoform of the class II PI 3-kinase (PI3K-C2alpha). This activation is rapid and parallels the insulin-induced activation of the class IA PI 3-kinase associated with IRS-1 in these muscles. However, while contraction activated p38 Map kinase, it did not stimulate the activity of the class II PI 3-kinase. Therefore, activation of class II PI 3-kinase is unlikely to provide a mechanism that explains the fact that exercise-induced activation of glucose uptake is not blocked by wortmannin. However, the results suggest that activation of class II PI 3-kinase is likely to play a role in insulin signaling pathways in skeletal muscle.     

Involvement of Class II Phosphoinositide 3-Kinase α-Isoform in Antigen-Induced Degranulation in RBL-2H3 Cells

In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca²⁺ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P₂ was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P₂ may play roles in the secretory process.     

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2α) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2α mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2α was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2α can modulate HCC cell growth.