Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.     

Histone deacetylase 3 mediates allergic skin inflammation by regulating expression of MCP1 protein

We have shown the induction of histone deacetylase 3 (HDAC3) in antigen-stimulated rat basophilic leukemia cells via NF-κB. We investigated the role of HDAC3 in allergic skin inflammation. We used a BALB/c mouse model of triphasic cutaneous anaphylaxis (triphasic cutaneous reaction; TpCR) and passive cutaneous anaphylaxis (PCA) to examine the role of HDAC3 in allergic skin inflammation. Triphasic cutaneous reaction involved induction of HDAC3 and was mediated by HDAC3. HDAC3 showed an interaction with FcεRIβ. Trichostatin A (TSA), an inhibitor of HDAC(s), disrupted this interaction. Cytokine array analysis showed that the down-regulation of HDAC3 led to the decreased secretion of monocyte chemoattractant protein 1 (MCP1). FcεRI was necessary for induction of HDAC3 and MCP1. ChIP assays showed that HDAC3, in association with Sp1 and c-Jun, was responsible for induction of MCP1 expression. TSA exerted a negative effect on induction of MCP1. HDAC3 exerted a negative regulation on expression of HDAC2 via interaction with Rac1. The down-regulation of HDAC3 or inactivation of Rac1 induced binding of HDAC2 to MCP1 promoter sequences. TSA exerted a negative effect on HDAC3-mediated TpCR. The BALB/c mouse model of PCA involved induction of HDAC3 and MCP1. HDAC3 and MCP1 were necessary for PCA that involved ear swelling, enhanced vascular permeability, and angiogenesis. Recombinant MCP1 enhanced β-hexosaminidase activity and histamine release and also showed angiogenic potential. TSA exerted a negative effect on PCA. Our data show HDAC3 as a valuable target for the development of allergic skin inflammation therapeutics.     

Thrombin induces NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells by a Rac1-dependent PI3K/Akt pathway

We previously showed that thrombin induces interleukin (IL)-8/CXCL8 expression via the protein kinase C (PKC)α/c-Src-dependent IκB kinase α/β (IKKα/β)/NF-κB signaling pathway in human lung epithelial cells. In this study, we further investigated the roles of Rac1, phosphoinositide 3-kinase (PI3K), and Akt in thrombin-induced NF-κB activation and IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by a PI3K inhibitor (LY294002), an Akt inhibitor (1-L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), and the dominant negative mutants of Rac1 (RacN17) and Akt (AktDN). Treatment of cells with thrombin caused activation of Rac and Akt. The thrombin-induced increase in Akt activation was inhibited by RacN17 and LY294002. Stimulation of cells with thrombin resulted in increases in IKKα/β activation and κB-luciferase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. Treatment of cells with thrombin induced Gβγ, p85α, and Rac1 complex formation in a time-dependent manner. These results imply that thrombin activates the Rac1/PI3K/Akt pathway through formation of the Gβγ, Rac1, and p85α complex to induce IKKα/β activation, NF-κB transactivation, and IL-8/CXCL8 expression in human lung epithelial cells.     

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.     

Histone deacetylase 9 activates gamma-globin gene expression in primary erythroid cells

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.     

Dual effects of histone deacetylase inhibition by trichostatin A on endothelial nitric oxide synthase expression in endothelial cells

Inhibition of histone deacetylases by trichostatin A (TSA) has pleiotropic effects on gene expression. We demonstrated that at low dose (0.1 microg) TSA increased the eNOS mRNA levels, which was followed by a time- and dose-dependent down-regulation. Cycloheximide, a protein synthesis inhibitor, completely abolished TSA-induced decrease in eNOS expression, indicating that new protein synthesis is required for the inhibiting effect. Mevastatin--an inhibitor HMG-CoA reductase and geranylgeranylation reaction dose-dependently antagonized TSA-induced reduction. This mevastatin-mediated antagonism was completely abolished by geranylgeranylpyrophosphate, suggesting that geranylgeranyl modification is needed to activate the eNOS mRNA destabilizing factor--a mechanism responsible for statin-mediated eNOS upregulation.     

A phosphodiesterase 3B-based signaling complex integrates exchange protein activated by cAMP 1 and phosphatidylinositol 3-kinase signals in human arterial endothelial cells

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based "signalosome" that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.     

LC3B-II deacetylation by histone deacetylase 6 is involved in serum-starvation-induced autophagic degradation

Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation.     

Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP): a new player in cell signaling

Precise balance between phosphorylation, catalyzed by protein kinases, and dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Deregulation of this balance leads to pathophysiological states that drive diseases such as cancer, heart disease, and diabetes. The recent discovery of the PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) family of Ser/Thr phosphatases adds a new player to the cast of phosphate-controlling enzymes in cell signaling. PHLPP isozymes catalyze the dephosphorylation of a conserved regulatory motif, the hydrophobic motif, on the AGC kinases Akt, PKC, and S6 kinase, as well as an inhibitory site on the kinase Mst1, to inhibit cellular proliferation and induce apoptosis. The frequent deletion of PHLPP in cancer, coupled with the development of prostate tumors in mice lacking PHLPP1, identifies PHLPP as a novel tumor suppressor. This minireview discusses the structure, function, and regulation of PHLPP, with particular focus on its role in disease.     

Phosphoinositide 3-kinase regulates plasma membrane targeting of the Ras-specific exchange factor RasGRP1

Receptor-induced targeting of exchange factors to specific cellular membranes is the predominant mechanism for initiating and compartmentalizing signal transduction by Ras GTPases. The exchange factor RasGRP1 has a C1 domain that binds the lipid diacylglycerol and thus can potentially mediate membrane localization in response to receptors that are coupled to diacylglycerol-generating phospholipase Cs. However, the C1 domain is insufficient for targeting RasGRP1 to the plasma membrane. We found that a basic/hydrophobic cluster of amino acids within the plasma membrane-targeting domain of RasGRP1 is instead responsible for plasma membrane targeting. This basic/hydrophobic cluster binds directly to phospholipid vesicles containing phosphoinositides via electrostatic interactions with polyanionic phosphoinositide headgroups and insertion of a tryptophan into the lipid bilayer. B cell antigen receptor ligation and other stimuli induce plasma membrane targeting of RasGRP1 by activating the phosphoinositide 3-kinase signaling pathway, which generates phosphoinositides within the plasma membrane. Direct detection of phosphoinositides by the basic/hydrophobic cluster of RasGRP1 provides a novel mechanism for coupling and co-compartmentalizing phosphoinositide 3-kinase and Ras signaling and, in coordination with diacylglycerol detection by the C1 domain, gives RasGRP1 the potential to serve as an integrator of converging signals from the phosphoinositide 3-kinase and phospholipase C pathways.     

PKCepsilon is essential for gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells

Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects. In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces gelsolin mRNA independently of the de novo protein synthesis. Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression. Furthermore, inhibition of PKCepsilon by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCepsilon is the major isoform for this process. In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity. Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin.     

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.