Phosphatidylinositol 3-kinase is required for adherens junction-dependent mammary epithelial cell spheroid formation

Adherens junctions facilitate and maintain epithelial cell-cell adhesion. This is true of mammary epithelial cells, both in two dimensional monolayers and in three-dimensional basement membrane cultures. Using the immortalized, functional mouse mammary epithelial scp2 cell line, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PI3-kinase) disrupted adherens junctions. In monolayers, this disruption was associated with decreased E-cadherin and beta-catenin at sites of cell-cell contact and decreased association of both proteins with the cytoskeleton. Changes in the distribution of f-actin after PI3-kinase inhibition suggest that this disruption of adherens junctions may be mediated by alterations to the cytoskeleton. In basement membrane cultures, PI3-kinase inhibition reversibly prevented adherens junction-dependent spheroid formation and differentiative milk protein gene expression, both in scp2 cells and in a second mouse mammary epithelial cell line, EpH4. Decreasing the calcium concentration in the culture medium produced similar, although less dramatic, phenotypic effects. These data indicate that adherens junctions contribute, at least in part, to the efficient induction of basement membrane-dependent differentiation of mammary epithelial cells.     

Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration

The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha, and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC(50) of approximately 5 microM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-alpha but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.     

Phosphatidylinositol 3-kinase activation is required for stress protocol-induced modification of hippocampal synaptic plasticity

Stress dramatically affects the induction of hippocampal synaptic plasticity; however, the molecular details of how it does so remain unclear. Phosphatidylinositol 3-kinase (PI3K) signaling plays a crucial role in promoting neuronal survival and neuroplasticity, but its role, if any, in stress-induced alterations of long term potentiation (LTP) and long term depression (LTD) is unknown. We found here that inhibitors of PI3K signaling blocked the effects of acute restraint-tail shock stress protocol on LTP and LTD. Therefore, the purpose of the present study is to explore the signaling events involving PI3K in terms of its role in mediating stress protocol-induced alterations of LTP and LTD. We found that stress protocol-induced PI3K activation can be blocked by various inhibitors, including RU38486 for glucocorticoid receptors, LY294002 for PI3K, and dl-2-amino-5-phosphonopentanoic acid for N-methyl-D-aspartate receptors or brain-derived neurotrophic factor antisense oligonucleotides. Also, immunoblotting analyses revealed that stress protocol induced a profound and prolonged phosphorylation of numbers of PI3K downstream effectors, including 3-phosphoinositide-dependent protein kinase-1, protein kinase B, mammalian target of rapamycin (mTOR), p70 S6 kinase, and eukaryotic initiation factor 4B in hippocampal CA1 homogenate, which was prevented by the PI3K inhibitor pretreatment. More importantly, we found that stress protocol significantly increased the protein expression of dendritic scaffolding protein PSD-95 (postsynaptic density-95), which is known to be involved in LTP and LTD, in an mTOR-dependent manner. These results identify a key role of PI3K signaling in mediating the stress protocol-induced modification of hippocampal synaptic plasticity and further suggest that PI3K may do so by invoking the protein expression of PSD-95.     

Phosphatidylinositol 3-kinase

Currently, a central question in biology is how signals from the cell surface modulate intracellular processes. In recent years phosphoinositides have been shown to play a key role in signal transduction. Two phosphoinositide pathways have been characterized, to date. In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphospate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators. The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake. Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking. In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined.     

Phosphatidylinositol 3-kinase is required for insulin-stimulated tyrosine phosphorylation of Shc in 3T3-L1 adipocytes

The interactions between the phosphatidylinositol 3-kinase (PI 3-kinase) and Ras/MAPK kinase pathways have been the subject of considerable interest. In the current studies, we find that epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) lead to rapid phosphorylation of Shc (maximum at 1-2 min), whereas insulin-mediated Shc phosphorylation is relatively delayed (maximum at 5-10 min), suggesting that an intermediary step may be necessary for insulin stimulation of Shc phosphorylation. The Src homology-2 (SH2) domain of Shc is necessary for PDGF- and EGF-mediated Shc phosphorylation, whereas the phosphotyrosine binding (PTB) domain is critical for the actions of insulin. Because the Shc PTB domain can interact with phospholipids, we postulated that PI 3-kinase might be a necessary intermediary step facilitating insulin-stimulated phosphorylation of Shc. In support of this, we found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked insulin-stimulated but not EGF- or PDGF-stimulated Shc phosphorylation. Furthermore, overexpression of a dominant negative PI 3-kinase construct (p85N-SH2) blocked insulin, but not EGF- or PDGF-induced Shc phosphorylation. All three growth factors cause localization of Shc to the plasma membrane, but only the effect of insulin was inhibited by wortmannin, supporting the view that PI 3-kinase-generated phospholipids mediate insulin-stimulated Shc phosphorylation. Consistent with this, expression of a constitutively active PI 3-kinase (p110(C)(AAX)) increased membrane localization of Shc, and this was completely blocked by wortmannin. A mutant Shc with a disrupted PTB domain (Shc S154) did not localize to the membrane in p110(C)(AAX)-expressing cells or after insulin stimulation and was not phosphorylated by insulin. In summary, 1) PI 3-kinase is a necessary early step in insulin-stimulated Shc phosphorylation, whereas the effects of EGF and PDGF on Shc phosphorylation are independent of PI 3-kinase. 2) PI 3-kinase-stimulated generation of membrane phospholipids can localize Shc to the plasma membrane through the Shc PTB domain facilitating phosphorylation by the insulin receptor.     

Phosphatidylinositol 3-kinase activation is required to form the NKG2D immunological synapse

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP₃. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.     

Phosphatidylinositol 4-kinase β is required for the ciliogenesis of zebrafish otic vesicle

The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.     

Rhinovirus activates interleukin-8 expression via a Src/p110beta phosphatidylinositol 3-kinase/Akt pathway in human airway epithelial cells

Rhinovirus (RV) is responsible for the majority of common colds and triggers exacerbations of asthma and chronic obstructive lung disease. We have shown that RV serotype 39 (RV39) infection activates phosphatidylinositol 3 (PI 3)-kinase and the serine threonine kinase Akt minutes after infection and that the activation of PI 3-kinase and Akt is required for maximal interleukin-8 (IL-8) expression. Here, we further examine the contributions of Src and PI 3-kinase activation to RV-induced Akt activation and IL-8 expression. Confocal fluorescent microscopy of 16HBE14o- human bronchial epithelial cells showed rapid (10-min) colocalization of RV39 with Src, p85alpha PI 3-kinase, p110beta PI 3-kinase, Akt and Cit-Akt-PH, a fluorescent Akt pleckstrin homology domain which binds PI(3,4,5)P(3). The chemical Src inhibitor PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine} and the PI 3-kinase inhibitor LY294002 each inhibited Akt phosphorylation and the colocalization of RV39 with Akt. Digoxigenin-tagged RV coprecipitated with a Crosstide kinase likely to be Akt, and inhibition of Src blocked kinase activity. Digoxigenin-tagged RV39 colocalized with the lipid raft marker ceramide. In 16HBE14o- and primary mucociliary differentiated human bronchial epithelial cells, inhibition of Src kinase activity with the Src family chemical inhibitor PP2, dominant-negative Src (K297R), and Src small interfering RNA (siRNA) each inhibited RV39-induced IL-8 expression. siRNA against p110beta PI 3-kinase also inhibited IL-8 expression. These data demonstrate that, in the context of RV infection, Src and p110beta PI 3-kinase are upstream activators of Akt and the IL-8 promoter and that RV colocalizes with Src, PI 3-kinase, and Akt in lipid rafts.     

Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes

Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.     

PIKE-mediated PI3-kinase activity is required for AMPA receptor surface expression

AMPAR (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor) is an ion channel involved in the formation of synaptic plasticity. However, the molecular mechanism that couples plasticity stimuli to the trafficking of postsynaptic AMPAR remains poorly understood. Here, we show that PIKE (phosphoinositide 3-kinase enhancer) GTPases regulate neuronal AMPAR activity by promoting GluA2/GRIP1 association. PIKE-L directly interacts with both GluA2 and GRIP1 and forms a tertiary complex upon glycine-induced NMDA receptor activation. PIKE-L is also essential for glycine-induced GluA2-associated PI3K activation. Genetic ablation of PIKE (PIKE(-/-)) in neurons suppresses GluA2-associated PI3K activation, therefore inhibiting the subsequent surface expression of GluA2 and the formation of long-term potentiation. Our findings suggest that PIKE-L is a critical factor in controlling synaptic AMPAR insertion.     

Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.     

Phosphatidylinositol 3-kinase regulates the CD4/CD8 T cell differentiation ratio

The signaling pathways that control T cell differentiation have only begun to be elucidated. Using T cell lines, it has been shown that class IA phosphatidylinositol 3-kinase (PI3K), a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, is activated after TCR stimulation. Nonetheless, the contribution of p85/p110 PI3K isoforms in T cell development has not been described. Mice deficient in the other family of class I PI3K, p110gamma, which is regulated by G protein-coupled receptors, exhibit reduced thymus size. Here we examine T cell development in p110gamma-deficient mice and in mice expressing an activating mutation of the p85 regulatory subunit, p65(PI3K), in T cells. We show that p110gamma-deficient mice have a partial defect in pre-TCR-dependent differentiation, which is restored after expression of the p65(PI3K) activating mutation. Genetic alteration of both PI3K isoforms also affects positive selection; p110gamma deletion decreased and p65(PI3K) expression augmented the CD4(+)/CD8(+) differentiation ratio. Finally, data are presented showing that both PI3K isoforms influenced mature thymocyte migration to the periphery. These observations underscore the contribution of PI3K in T cell development, as well as its implication in determining the CD4(+)/CD8(+) T cell differentiation ratio in vivo.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Grepafloxacin inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in human airway epithelial cells

We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.     

Phosphatidylinositol 3-kinase/Akt auto-regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts

It has been reported that platelet-derived growth factor (PDGF)-BB stimulates the synthesis of interleukin (IL)-6 in osteoblasts. In the present study, we investigated whether the phosphatidylinositol 3-kinase (PI3K)/Akt is involved in the PDGF-BB-induced IL-6 synthesis in osteoblast-like MC3T3-E1 cells. PDGF-BB markedly induced the phosphorylation of Akt and GSK-3beta. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, significantly amplified the synthesis of IL-6 by PDGF-BB. The PDGF-BB-induced GSK-3beta phosphorylation was suppressed by the Akt inhibitor. The IL-6 synthesis stimulated by PDGF-BB was markedly enhanced by LY294002 and wortmannin, inhibitors of PI3K. Wortmannin and LY294002 suppressed the PDGF-BB-induced phosphorylation of Akt and GSK-3beta. Taken together, these results strongly suggest that PI3K/Akt negatively regulates the PDGF-BB-stimulated IL-6 synthesis in osteoblasts.     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.     

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.     

PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.