Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium‐dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCα, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCα can be co‐immunoprecipitated with EAAC1. When the glial glutamate transporter GLT‐1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester‐activated subtypes of PKC was examined in C6 glioma transfected with GLT‐1. Of the classical subtypes, only PKCα was detected, and of the non‐classical subtypes, PKCδ and PKCε were detected. In this system, phorbol ester‐dependent internalization of GLT‐1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCδ (rottlerin). PKCα immunoreactivity was found in GLT‐1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCα in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCα may be required for the regulated redistribution of GLT‐1.     

TNFα activation of PKCδ, mediated by NFκB and ER stress,cross-talks with the insulin signaling cascade

TNFα plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCδ, a novel PKC isoform, is known to participate in proximal TNFα signaling events. However, it has remained unclear whether PKCδ plays a role in distal TNFα signaling events. Here we demonstrate that PKCδ is activated by TNFα in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCδ and JNK, we used pharmacological and genetic inhibitors of NFκB. We found that inhibition of NFκB attenuated PKCδ and JNK activations. Further analysis revealed that ER stress contributes to TNFα-stimulated PKCδ and JNK activations. To investigate the role of PKCδ in TNFα action, we used 29-mer shRNAs to silence PKCδ expression. A reduction of ~90% in PKCδ protein levels reduced TNFα-stimulated stress kinase activation, including JNK. Further, PKCδ was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCδ was necessary for induction of the UPR. Indeed, a reduction in PKCδ protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCδ does not globally regulate the UPR. Next, the role of PKCδ in TNFα mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCδ expression. We found that a reduction in PKCδ protein levels reversed the TNFα-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFα-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFκB and ER stress contribute in part to PKCδ activation; 2) PKCδ plays a key role in the propagation of the TNFα signal; and 3) PKCδ contributes to TNFα-induced inhibition of insulin signaling events.     

Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage-induced apoptosis

Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage-induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner.     

Acidic extracellular pH induces p120-catenin-mediated disruption of adherens junctions via the Src kinase-PKCδ pathway

An acidic microenvironment induces disruption of adherens junctions (AJs) of hepatoma cells. This study investigated the impact of an acidic extracellular pH (pHe) on p120-catenin (p120-ctn) serine phosphorylation. pH 6.6 treatment increased intracellular calcium levels, activated protein kinase C (PKC)α and PKCδ, and decreased serine phosphorylation of p120-ctn. Further knockdown of PKCα and δ by small interference RNA (siRNA) prevented the pH 6.6-induced downregulation of p120-ctn at AJ and the serine dephosphorylation of p120-ctn. Moreover, PP2 pretreatment and siRNA of c-Src abrogated the pH 6.6-induced PKCδ activation. Together, the c-Src-PKCδ cascade and PKCα regulate the acidic pHe-induced AJ disruption.     

The Protective Effects of Riluzole on Manganese-Induced Disruption of Glutamate Transporters and Glutamine Synthetase in the Cultured Astrocytes

Chronic exposure to excessive manganese (Mn) can lead to manganism, a type of neurotoxicity accomplished with extracellular glutamate (Glu) accumulation. To investigate this accumulation, this study focused on the role of astrocyte glutamate transporters (GluTs) and glutamine synthetase (GS), which have roles in Glu transport and metabolism, respectively. And the possible protective effects of riluzole (a glutamatergic modulator) were studied in relation to Mn exposure. At first, the astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2) for 24 h, and 100 μM riluzole was pretreated to astrocytes for 6 h before 500 μM MnCl(2) exposure. Then, [(3)H]-glutamate uptake was measured by liquid scintillation counting; Na(+)-K(+) ATPase and GS activities were determined by a colorimetric method; glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and GS mRNA expression were determined by RT-PCR and protein levels were measured by western blotting. The results showed that Mn inhibited Glu uptake, Na(+)-K(+) ATPase and GS activities, GLAST, GLT-1, and GS mRNA, and protein in a concentration-dependent manner. And they were significantly higher for astrocytes pretreated with 100 μM riluzole than the group exposed to 500 μM MnCl(2). The results suggested that Mn disrupted Glu transport and metabolism by inhibiting GluTs and GS. Riluzole activated protective effects on enhancing GluTs and GS to reverse Glu accumulation. In conclusion, Mn exposure results in the disruption of GLAST, GLT-1, and GS expression and function. Furthermore, riluzole attenuates this Mn toxicity.     

Protein quality control disruption by PKCβII in heart failure; rescue by the selective PKCβII inhibitor, βIIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words).     

Protein kinase C-δ negatively regulates T cell receptor-induced NF-κB activation by inhibiting the assembly of CARMA1 signalosome

T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-κB and JNK activation. In this report, we identified protein kinase Cδ (PKCδ) as a CARMA1-associated protein by a biochemical affinity purification approach. PKCδ interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKCδ inhibited CARMA1-mediated NF-κB activation, whereas knockdown of PKCδ potentiated TCR-triggered NF-κB activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKCδ kinase-dead mutant indicated that the kinase activity of PKCδ was dispensable for its ability to inhibit TCR-triggered NF-κB activation. Furthermore, we found that PKCδ inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKCθ, Bcl10, or MALT1. These observations suggest that PKCδ is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome.     

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT<Subscript>1A</Subscript> Receptor and Upregulation of Nrf2-Dependent GSH Synthesis

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT_1A receptor, but not 5-HT_2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT_1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT_1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT_1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT_1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT_1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT_1A receptor and upregulation of Nrf2-dependent GSH synthesis.     

Complementary neuronal and glial expression of two high-affinity glutamate transporter GLT1/EAAT2 forms in rat cerebral cortex

The glutamate transporter GLT1 is essential in limiting transmitter signaling and restricting harmful receptor overstimulation. It has been shown recently that GLT1 exists in two forms, the generic GLT1 and a 3'-end-spliced variant of GLT1 (GLT1v), both with similar transport characteristics. To differentiate clearly the cellular distribution of both GLT1 forms in the cortex, specific cRNA probes for non-radioactive in situ hybridization were generated and applied to adult rat brain sections. The results were complemented by western and northern blot analyses and by immunocytochemical investigations using specific peptide antibodies against both GLT1 forms. The study confirmed that generic GLT1 mRNA was expressed predominantly in astrocytes and, to a small extent, in neurons, whereas GLT1 protein was detected only in cell membranes of astrocytes. On the other hand, GLT1v mRNA and protein were demonstrated predominantly in neurons and in non-astrocytic glial cells irrespective of the cortical areas studied. A cytoplasmic granular staining of neurons and astrocytes predominated in the demonstration of GLT1v protein. It is concluded that the cellular expression of the two GLT1 forms is complementary. The cytoplasmic vesicular distribution of GLT1v may represent an endogenous protective mechanism to limit glutamate-induced excitotoxicity.     

Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.     

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.     

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.