Targeting of PED/PEA-15 molecular interaction with phospholipase D1 enhances insulin sensitivity in skeletal muscle cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.     

Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.     

PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization.     

Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg _ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.     

Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action

The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser(116). In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser(116) PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser(116). In addition, a mutant of PED/PEA-15 featuring the substitution of Ser(116)-->Gly (PED(S116-->G)) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser(116). Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PED(S116-->G) compared to that in PED/PEA-15(WT) cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PED(S116-->G) mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.     

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can trigger apoptosis in some tumor cells but not other tumor cells. To explore the signal transduction events in TRAIL-triggered apoptosis and its modulation in nontransfected tumor cells, we analyzed TRAIL-induced death-inducing signaling complex (DISC) in TRAIL-sensitive and -resistant glioma cells. Caspase-8 and caspase-10 were recruited to the DISC, where they were proteolytically activated to initiate apoptosis in TRAIL-sensitive glioma cells. Caspase-8 and caspase-10 were also recruited to the DISC in TRAIL-resistant cells, but their further activation was inhibited by two antiapoptotic proteins termed cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15kDa (PED/PEA-15). Both long and short forms of c-FLIP were recruited to the DISC, where the long form c-FLIP was cleaved to produce intermediate fragments. Of the three isoforms of PED/PEA-15 proteins, only the doubly phosphorylated form was expressed and recruited to the DISC in TRAIL-resistant cells, indicating that the phosphorylation status of PED/PEA-15 determines its recruitment in the cells. Treatment with calcium/calmodulin-dependent protein kinase inhibitor rescued TRAIL sensitivity in TRAIL-resistant cells, providing a potential new approach to sensitize the cells to TRAIL-induced apoptosis.     

PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus.

We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non-diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA-15. The PED gene maps on human chromosome 1q21-22. Transfection of PED/PEA-15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin-stimulated glucose transport and cell-surface recruitment of Glut4, the major insulin-sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/PEA-15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.     

Dose-related cytoprotective effect of α-lipoic acid on hydrogen peroxide-induced oxidative stress to pancreatic beta cells

α-Lipoic acid (α-LA), an antioxidant used for diabetic polyneuropathy, was reported to induce AMP-activated protein kinase activation and reductions in insulin secretion in pancreatic beta-cells at high concentrations (≥ 500 µmol/l). This study investigated whether α-LA has a protective role under oxidative stress in beta-cells and its effect is dose-related. In INS-1 cells treated with α-LA (150-1200 µmol/l) for 24 h, α-LA itself (≥300 µmol/l) induced apoptotic death dose-dependently. However, pre-treatment with 150 and 300 µmol/l α-LA reduced the hydrogen peroxide-induced apoptosis in INS-1 cells and isolated islets. α-LA alleviated hydrogen peroxide-induced reactive oxygen species production, mitochondrial membrane depolarization and c-JNK activation in beta-cells. α-LA induced phosphoinositide 3-kinase-dependent Akt phosphorylation in INS-1 cells. While α-LA is harmful to beta-cells at high concentrations in vitro, it has potential cytoprotective effects on beta-cells under oxidative stress as in diabetes by its antioxidant properties and possibly by Akt phosphorylation at clinically relevant concentrations.     

Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human beta-cells from hyperglycemia-induced apoptosis

Studies in vivo indicate that IRS2 plays an important role in maintaining functional beta-cell mass. To investigate if IRS2 autonomously affects beta-cells, we have studied proliferation, apoptosis, and beta-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that beta-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a beta-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of beta-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human beta-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve beta-cell function. Our results indicate that IRS2 acts autonomously in beta-cells in maintenance and expansion of functional beta-cell mass in vivo.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

Multiple members of the mitogen-activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function.

293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H(2)O(2) or anisomycin. In PED-expressing 293 cells (293(PED)), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293(PED) cells, decreased apoptosis induced by anisomycin and H(2)O(2) was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293(PED) cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293(PED) cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED.     

Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro,and Defective Red Blood Cell Alloimmune Response In Vivo

TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4⁺ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response.     

The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells

The NADH shuttle system, which transports reducing equivalents from the cytosol to the mitochondria, is essential for the coupling of glucose metabolism to insulin secretion in pancreatic beta cells. Aralar1 and citrin are two isoforms of the mitochondrial aspartate/glutamate carrier, one key constituent of the malate-aspartate NADH shuttle. Here, the effects of Aralar1 overexpression in INS-1E beta cells and isolated rat islets were investigated for the first time. We prepared a recombinant adenovirus encoding for human Aralar1 (AdCA-Aralar1), tagged with the small FLAG epitope. Transduction of INS-1E cells and isolated rat islets with AdCA-Aralar1 increased aralar1 protein levels and immunostaining revealed mitochondrial localization. Compared with control INS-1E cells, overexpression of Aralar1 potentiated metabolism secretion coupling stimulated by 15 mm glucose. In particular, there was an increase of NAD(P)H generation, of mitochondrial membrane hyperpolarization, ATP levels, glucose oxidation, and insulin secretion (+45%, p < 0.01). Remarkably, this was accompanied by reduced lactate production. Rat islets overexpressing Aralar1 secreted more insulin at 16.7 mm glucose (+65%, p < 0.05) compared with controls. These results show that aspartate-glutamate carrier capacity limits glucose-stimulated insulin secretion and that Aralar1 overexpression enhances mitochondrial metabolism.     

Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC

Activation of the initiator-caspase, caspase-8 is under tight control of multiple antiapoptotic regulators including ARC, cFlip_S, cFlip_L and PED/PEA-15. Since there is little data regarding the expression of caspase-8 and its antiapoptotic regulators in human tumours in vivo, we analysed their expression in renal cell carcinomas (RCCs) to identify which of these genes might be crucial for the well known impaired apoptosis and—as a result—resistance towards chemotherapy and ionizing radiation of RCCs. Caspase-8, cFlip_S, cFlip_L and PED/PEA-15 mRNA expression was significantly increased only in early stages of RCCs compared to non-neoplastic renal tissue. In contrast, ARC mRNA expression was significantly increased in RCCs of all stages without differences between the tumour stages and grades. Importantly, the relative mRNA expression ratio between ARC and caspase-8 was significantly increased during carcinogenesis and tumour progression. In contrast, the relative mRNA expression ratio between cFlip_S, cFlip_L or PED/PEA-15 and caspase-8 remained constant during all tumour stages. In conclusion, our analysis revealed that ARC is the only caspase-8 inhibiting regulator being constantly overexpressed in RCCs. Furthermore, the balance between antiapoptotic ARC and proapoptotic caspase-8 is the only one to be disturbed during carcinogenesis and tumour progression of RCCs. This inhibition of Caspase-8 might therefore be one example for the multiple antiapoptotic functions of ARC in RCCs possibly contributing to the marked resistance of RCCs towards radio- and chemotherapy and reflects a shift of gene expression towards a more antiapoptotic context in RCCs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Akt down-regulates ERK1/2 nuclear localization and angiotensin II-induced cell proliferation through PEA-15

Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.