Protein Kinase C Delta Research Articles

Highlights of This Issue

Protein Kinase C-epsilon (PKCϵ) has recently emerged as a key regulator of several cell-cycle processes associated with DNA stress. Here, Martini and colleagues demonstrate the engagement of PKCϵ in the modulation of prophase-to-metaphase progression. Live imaging and immunofluorescence analyses reveal that the inhibition of PKCϵ results in a delay of centrosome migration and mitotic spindle organization. The close relationship between PKCϵ dependency for mitotic spindle organization and the impairment of the TOPO2A-dependent G2 cell cycle arrest, a hallmark of transformed cells, strongly indicated PKCϵ as a target in cancer therapy.Glioblastoma multiforme (GBM) is a highly aggressive cancer in the brain. Despite recent success using temozolomide, recurrence is common and survival rates remain poor. Therefore, additional treatment options are needed. One option is to target GBM subtypes or cell populations that may increase resistance or recurrence. To this end, patient-derived xenografts (PDXs) were screened with a small-molecule library to identify compounds with differential activity against GBM subtypes. Arsenic trioxide (ATO) was validated as a potent subtype-specific compound that activated the MNK1-eIF4E signaling axis. Examination of phase I/II clinical data of patients on ATO revealed subtype-specific clinical response. Subtype-specific resistance to ATO was mediated by activation of the MNK1-eIF4E signaling axis. Thus, targeting MNK1 may provide therapeutic benefit in response to ATO treatment of GBM.The chromatin remodeling gene EP300 and its paralog CREBBP are mutated in a third of bladder cancer patients. Knowing which tumor mutations are functionally relevant is critical, but determining this experimentally is time and cost intensive. Duex and colleagues determined which mutations had functional impact and investigated the correlation between this and bioinformatic prediction. Interestingly, the results of the prediction software correlated 100% with those of functional assessment. A gene signature reporting on bioinformatically predicted inactivating mutations correlated with advanced bladder cancer. This study highlights the potential of bioinformatics to leverage limited functional analysis to inform on patient outcomes and potential therapeutics.Pathways and mechanisms underlying dysregulation of growth factor shedding, which is involved in many pathologies including kidney disease and cancer, are at present poorly understood. Toward addressing this problem, a combined experimental and computational approach was pursued to uncover genetic mediators of the dysregulated tumor growth factor-alpha (TGFα). Wilson and colleagues used a foundational shRNA screen to identify genetic mediators of TGFα shedding, uncovered hidden information from this screen using a computational network approach, and validated these newly predicted regulators for their effects on shedding. Combined, this study highlights the utility of this integrative approach for novel therapeutic discovery.

Protein Kinase C-epsilon in Membrane Delivery during Phagocytosis.

During phagocytosis, internal membranes are recruited to the site of pathogen binding and fuse with the plasma membrane, providing the membrane needed for pseudopod extension and target uptake. The mechanism by which vesicles destined for the phagosome are generated, targeted, and fuse is unknown. We established that Golgi-associated protein kinase C-epsilon (PKC-ε) is necessary for the addition of membrane during FcyR-mediated phagocytosis. PKC-ε is tethered to the Golgi through interactions between its’ regulatory domain and the Golgi lipids PI4P and diacylglycerol; disruption of these interactions prevents PKC-ε concentration at phagosomes and decreases phagocytosis. The accumulated evidence suggests that PKC-ε orchestrates vesicle formation at the Golgi by a mechanism requiring lipid binding but not enzymatic activity. This review discusses how PKC-ε might mediate vesicle formation at the level of budding and fission. Specifically, we discuss PKC-ε binding partners, the formation of lipid subdomains to generate membrane curvature, and PKC-ε mediated links to the actin and microtubule cytoskeleton to provide tension for vesicle fission. Assimilating information from several model systems, we propose a model for PKC-ε mediated vesicle formation for exocytosis during phagocytosis that may be applicable to other processes that require directed membrane delivery and fusion.

A BNST-Centered Microcircuit Regulating Inflammation-Associated Anorexia and Feeding

Loss of appetite or anorexia associated with inflammation impairs quality of life and increases morbidity in many diseases. Compelling evidence suggests that inflammation-associated anorexia results from the actions of pro-inflammatory factors on the nervous system. However, the exact neural mechanism that mediates inflammation-associated anorexia is still poorly understood. Here we identified a specific population of neurons, marked by the expression of protein kinase C-delta, in the oval region of the bed nucleus of the stria terminalis (BNST) that mediates inflammation-associated anorexia. Importantly, our results demonstrated that these neurons mediate bidirectional control of general feeding behaviors. These neurons inhibit the lateral hypothalamus-projecting neurons in the ventral part of BNST to regulate feeding, receive inputs from other canonical feeding circuits. Our data therefore define a unique BNST-centered microcircuit that might function as a central hub for feeding regulation, which could offer therapeutic targets for feeding-related diseases such as anorexia and obesity.

Inhibition of A2A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455.

Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1, A2A, A2B, and A3 receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N5-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A2AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A2AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A2AAR agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A2AAR antagonist TP455, as well as by the reference A2AAR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A2AAR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by A2AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the A2AAR. In conclusion, the A2AAR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective A2AAR antagonists as potential new therapeutics.

A Central Amygdala-Substantia Innominata Neural Circuitry Encodes Aversive Reinforcement Signals

Aversive stimuli can impact motivation and support associative learning as reinforcers. However, the neural circuitry underlying the processing of aversive reinforcers has not been elucidated. Here, we report that a subpopulation of central amygdala (CeA) GABAergic neurons expressing protein kinase C-delta (PKC-δ+) displays robust responses to aversive stimuli during negative reinforcement learning. Importantly, projections from PKC-δ+ neurons of the CeA to the substantia innominata (SI) could bi-directionallymodulate negative reinforcement learning. Moreover, consistent with the idea that SI-projecting PKC-δ+ neurons of the CeA encode aversive information, optogenetic activation of this pathway produces conditioned place aversion, a behavior prevented by simultaneous ablating of SI glutamatergic neurons. Taken together, our data define a cell-type-specific neural circuitry modulating associative learning by encoding aversive reinforcement signals.

MicroRNA-143 promotes cardiac ischemia-mediated mitochondrial impairment by the inhibition of protein kinase Cepsilon.

The cardioprotection of protein kinase Cepsilon (PKCε) against myocardial infarction (MI) mediated by its anti-apoptotic property and underlying mechanism of targeted regulation by microRNA (miRNA) are not established. MI-induced injury, PKCε expression, and targeted regulation of miRNA-143 (miR-143) to PKCε have been evaluated using animal MI and cellular hypoxic models conjugated with series of state-of-art molecular techniques. The results demonstrated that PKCε significantly downregulated along with increased infarcted area and apoptotic and necrotic damage in MI model, and the targeted relationship and potential binding profile were established between miR-143 and PKCε. Both in vivo and in vitro ischemic tests showed that miR-143 induced apoptosis and necrosis, which was reversed by antagomiR-143 or AMO-143. The upregulation of miR-143 by transfection of miR-143 in vitro also induced cell loss, and this effect of miR-143 was completely reversed by co-transfection of miR-143 with AMO-143. The identically deleterious action of miR-143 on mitochondrial membrane potential and ATP synthesis was also observed in both animal MI and cellular hypoxic models, as well as miR-143 overexpressed models and converted by either antagomiR or AMO. Importantly, overexpression of miR-143 downregulated PKCε in all tested models and this downregulation was reversed in the presence of antagomiR or AMO. The direct targeted regulation of miR-143 on PKCε was confirmed by luciferase reporter and miRNA-masking tests. In conclusion, MI-mediated upregulation of miR-143 inhibits PKCε expression and consequently interference with the cardioprotection of PKCε to mitochondrial, and leads to mitochondrial membrane potential dissipation and myocardial death eventually.

Protein kinase C-delta (PKC\u03b4), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ−/− mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ−/− mice. Mechanistically, increased bacterial growth in macrophages from PKCδ−/− mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.

Activating mu-opioid receptors in the spinal cord mediates the cardioprotective effect of remote preconditioning of trauma.

Remote precoditioning of trauma (RPCT) confers cardioprotective effects against myocardial ischemia/reperfusion injury, which are mediated by spinal opioid receptors. The aim of this study was to identify the roles of opioid receptor subtypes in the cardioprotective effect of RPCT and possible mechanisms. In this study, 192 Sprague-Dawley rats were allocated to 12 groups. Except for the sham group, rats in all groups were subjected to myocardial ischemia reperfusion. Rats in the ischemia precondition (IPC) group were treated with IPC. In the RPCT groups, an abdominal incision was made 15 min before inducing ischemia. The selective delta-, kappa-, and mu-opioid receptor antagonists were administered to groups of animals receiving RPCT, respectively. Data were collected for myocardial infarct size, intercellular adhesion molecule 1 (ICAM-1), plasma cardiac troponin I (cTnI) concentrations, activation of protein kinase C epsilon (PKCe) in myocardial cell membranes, and adenosine release in the spinal cord. Compared with the control groups, infarct size, plasma concentrations of cTnI, and myocardial ICAM-1 expression were significantly lower, while adenosine release and PKCe activation were enhanced in the IPC and RPCT groups. Compared with the RPCT group, infarct size, plasma cTnI concentration, and myocardial ICAM-1 expression were greater and adenosine release and PKCe activation were reduced in the mu-opioid receptor antagonist plus RPCT group. The spinal mu-opioid receptor mediated the cardiac protective effect of RPCT. The mechanism may be enhanced by adenosine release in the spinal cord and PKCe activation in the myo-cardium, thereby inhibiting inflammation induced by ischemia/reperfusion injury.

Abstract 468: Smooth Muscle Cells Accelerate Endothelial Regeneration Through a Protein Kinase C-delta-Dependent Secretion of CXCR2 Ligands

Background: Efficient regeneration of denuded endothelial cells (ECs) is an important process that counteracts the pro-stenotic activities. Unfortunately, this natural healing process is frequently compromised by disease conditions such as diabetes. Protein kinase C-delta (PKCδ) plays a complex role in the arterial injury response by regulating apoptosis of vascular smooth muscle cells (SMCs) as well as production of chemokines capable of attracting resident and circulating cells. In this study, we explored whether SMCs participate in endothelial repair through a PKCδ-dependent paracrine mechanism. Methods and Results: Following balloon injury to the rat carotid, SMC-specific gene transfer of Prkcd accelerated reendothelialization compared to the empty vector, reflected by a larger area excluded from Evans blue measured 14 days post injury (59.60±5.01% vs 25.38±7.52%). In contrast, SMC-specific knockdown of endogenous Prkcd delayed reendothelialization compared to the non-targeting shRNA control (41.31±6.54% vs 70.31±5.97%). In vitro , media conditioned by AdPKCδ-infected SMCs increased endothelial wound healing without affecting their proliferation and viability. In addition, SMCs in a PKCδ-dependent fashion attracted circulating angiogenic cells (CACs), a cell population that promotes neovascularization via production of angiogenic factors. A PCR-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Blocking CXCL7 or CXCR2 significantly inhibited endothelial wound healing and CAC migration in response to AdPKCδ-infected SMC conditioned media. In vivo , PKCδ overexpression in SMCs following balloon injury increased CXCL7 production and stimulated CACs recruitment to injured arteries. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the negative effects of Prkcd knockdown on reendothelialization. Conclusions: Regeneration of denuded endothelium involves multiple cell types from the vascular wall as well as circulation. SMCs stimulate reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of ECs from uninjured endothelium and CACs from circulation.

Regulation of CCK-induced ERK1/2 activation by PKC epsilon in rat pancreatic acinar cells

The extracellular signal-regulated kinase ERK1/2 is activated in pancreatic acinar cells by cholecystokinin (CCK) and other secretagogues with this activation mediated primarily by protein kinase C (PKC). To identify the responsible PKC isoform, we utilized chemical inhibitors, cell permeant inhibitory peptides and overexpression of individual PKC dominant negative variants by means of adenoviral vectors. While the broad-spectrum PKC inhibitor GF109203X strongly inhibited ERK1/2 activation induced by 100 pM CCK, Go6976 which inhibits the classical PKC isoforms (alpha, beta and gamma), as well as Rottlerin, a specific PKC delta inhibitor, had no inhibitory effect. To test the role of PKC epsilon, we used specific cell permeant peptide inhibitors which block PKC interaction with their intracellular receptors or RACKs. Only PP93 (PKC epsilon peptide inhibitor) inhibited CCK-induced ERK1/2 activation, while PP95, PP101 and PP98, which are PKC alpha, delta and zeta peptide inhibitors respectively, had no effect. We also utilized adenovirus to express dominant negative PKC isoforms in pancreatic acini. Only PKC epsilon dominant negative inhibited CCK-induced ERK1/2 activation. Dominant negative PKC epsilon expression similarly blocked the effect of carbachol and bombesin to activate ERK1/2. Immunoprecipitation results demonstrated that CCK can induce an interaction of c-Raf-1 and PKC epsilon, but not that of other isoforms of Raf or PKC. We conclude that PKC epsilon is the isoform of PKC primarily involved with CCK-induced ERK1/2 activation in pancreatic acinar cells.

Regulation of protein kinase C-epsilon and its age-dependence

Protein kinase C (PKC) is an important mediator in the cardioprotection of ischemic preconditioning and has been shown to translocate to mitochondria upon activation. However, little is known about the cellular signaling underlying the translocation of PKC isoforms to mitochondria and its age-dependence. The present study aimed to explore whether adenosine-induced translocation of PKCε to mitochondria is mediated by caveolin-3 and/or adenosine A2B receptor/PI3 kinase mediated signaling, and whether the mitochondrial targeting of PKCε is age-related. Immunofluorescence imaging of isolated mitochondria from cardiomyocytes and H9c2 cells showed that while adenosine-induced increase in mitochondrial PKCε was inhibited by adenosine A1 receptor blocker, pretreatment with adenosine A2B receptor specific inhibitor MRS 1754 or PI3K inhibitor Wortmannin did not significantly reduce adenosine-mediated increase in mitochondrial PKCε. Interestingly, adenosine-induced increase in mitochondrial translocation of PKCε was significantly blocked by suppressing caveolin-3 expression with specific siRNA. When compared to that in young adult rat hearts, the level of mitochondrial PKCε in middle-aged rat hearts was significantly lower at the basal condition and in response to adenosine treatment, along with largely decreased mitochondrial HSP90 and TOM70 protein expression. We demonstrate that adenosine-induced translocation of PKCε to mitochondria is mediated by a caveolin-3-dependent mechanism and this process is age-related, possibly in part, through regulation of HSP90 and TOM70 expression. These results point out a novel mechanism in regulating PKC function in mitochondria.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake.

Thyroid hormones are powerful modulators of heart function and susceptibility to arrhythmias via both genomic and non-genomic actions. We aimed to explore expression of electrical coupling protein connexin-43 (Cx43) in the heart of rats with altered thyroid status and impact of omega-3 polyunsaturated fatty acids (omega-3) supplementation. Adult male Lewis rats were divided into following six groups: euthyroid controls, hyperthyroid (treated with T(3)) and hypothyroid (treated with methimazol) with or without six-weeks lasting supplementation with omega-3 (20 mg/100 g/day). Left and right ventricles, septum and atria were used for immunoblotting of Cx43 and protein kinase C (PKC). Total expression of Cx43 and its phosphorylated forms were significantly increased in all heart regions of hypothyroid rats compared to euthyroid controls. In contrast, the total levels of Cx43 and its functional phosphorylated forms were decreased in atria and left ventricle of hyperthyroid rats. In parallel, the expression of PKC epsilon that phosphorylates Cx43, at serine 368, was increased in hypothyroid but decreased in hyperthyroid rat hearts. Omega-3 intake did not significantly affect either Cx43 or PKC epsilon alterations. In conclusion, there is an inverse relationship between expression of cardiac Cx43 and the levels of circulating thyroid hormones. It appears that increased propensity of hyperthyroid while decreased of hypothyroid individuals to malignant arrhythmias may be in part attributed to the changes in myocardial Cx43.

Abstract 940: The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines

Abstract Background: Hepatocellular carcinoma (HCC) is the third largest contributor to cancer mortality in the world. Alphafetoprotein (AFP) is synthetized and secreted by the majority of HCCs. Despite its controversial value as classical HCC diagnostic and follow-up marker, the AFP was lately shown to correlate with the volume of liver cancer. It is also useful as a predictive marker for radiotherapy treatment response and patients prognosis. On the molecular level, AFP has a proliferative role in HCC since it acts on its own receptor on the surface of hepatocytes; however, its mechanism of action remains unclear. Our laboratory have previously demonstrated that telomerase and protein kinase C (PKC) modulate AFP secretion in HCC cell lines and this effect is additive. The aim of this study is to elucidate the PKC isoforms involved in AFP secretion modulation and telomerase expression. Methods: Two AFP secretory cell lines, HepG2/C3a and PLC/PRF/5, and one non-secretory AFP cell line, SNU-387, were cultured in DMEM (RPMI-1640 for SNU-387) media with 10% FBS and 1% Penicillin/Streptomycin and incubated in humid 5% CO2 incubator. The RNA was extracted; the RT-PCR was performed to characterize the PKC isoforms and the modulation of hTERT by these isoforms. The role of each isoform on AFP secretion was elucidated using different PKC isoform modulators; the AFP concentration was measured using ELISA technique. The viability and toxicity of cells were assessed using WST-1. Results: The three cell lines express the PKC alpha, delta and epsilon isoforms. Whereas the other isoforms Beta 1 and 2 are slightly expressed in C3a, the gamma and beta isoforms are slightly expressed in PLC and SNU respectively. Dose-dependent inhibition of PKC alpha and beta by GO6976 decreased AFP secretion by 40% at 10 uM by the HepG2/C3a cell line. The inhibition of PKC delta by Rottlerin dose-dependently decreased AFP secretion by 30% at 5 uM. The PKC epsilon modulator FR236924 dose-dependently inhibited AFP secretion by 50% at 1 uM. Pan PKC inhibitor, GO6983, decreased AFP secretion by 45% at 5 uM. The same profile was obtained for PLC/PRF/5 with a maximum 30% of AFP down-regulation except for Rottlerin which showed a 75% of AFP inhibition. The catalytic subunit expression of telomerase, hTERT, was only inhibited by Rottlerin at 5 uM. Our experiments did not show any cell death nor cell proliferation modulation when incubating the three cell lines with PKC inhibitors. Conclusion: Combining the above results with our previous ones, we suggest for the first time, that PKC delta modulates AFP secretion through telomerase however the mechanism by which PKC alpha and epsilon modulate AFP secretion is to be elucidated. However, further experiments are needed to support this statement. Citation Format: Raia Doumit, Roula Tahtouh, Rita Ammoury, Riad Sarkis, Nada Alaaddine, George Hilal. The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 940.

Distinctive roles of PKC delta isozyme in platelet function

Platelet activation is a complex balance of positive and negative signaling pathways. Several protein kinase C (PKC) isoforms are expressed in human platelets. They are a major regulator of platelet granule secretion, activation and aggregation activity. One of those isoforms is the PKCδ isozyme, it has a central yet complex role in platelets such as opposite signaling functions depending on the nature of the agonist, it concentration and pathway. In fact, it has been shown that PKCδ has an overall negative influence on platelet function in response to collagen, while, following PAR stimulation, PKCδ has a positive effect on platelet function. Understanding the crucial role of PKCδ in platelet functions is recently emerging in the literature, therefore, further investigations should shed light into its specific role in hemostasis. In this review, we focus on the different roles of PKCδ in platelet activation, aggregation and thrombus formation.

Cardioprotective Effects Induced by Preconditioning with Halogenated Anesthetics

Abstract Background: Numerous studies discuss the protective effects of halogenated anesthetics on myocyte injury induced by the ischemia-reperfusion syndrome of the heart. This mechanism is known as pharmacological preconditioning. Aim of the study: The objective of this study was to identify the effects of two volatile anesthetics frequently used in current clinical practice, Isoflurane and Sevoflurane, on the in situ heart. The study was performed on laboratory animals with induced brain death. Material and methods: The animals were divided into 3 groups, the control group (n = 8), IZO-PRE group (n = 8) and SEVO-PRE group (n = 8), on which the experimental protocol established for this study was applied. From a molecular point of view, the expressions of protein kinase C-epsilon (PKCε) and of glycogen synthase kinase – 3 beta (GSK-3β) were investigated. Results: Following the statistical analysis, we observed a significant reduction in the size of the infarcted area in the IZO-PRE group compared to the control group (p <0.0001). Regarding the SEVO-PRE group, no reduction was observed (p >0.05). The expression of GSK-3β is more pronounced in case of the SEVO-PRE group, at 5 minutes after reperfusion, and the effect disappears after 15 minutes. The expression of PKCε as a total form of the enzyme, occurs at 5 minutes after reperfusion in the SEVO-PRE group and late in the IZO-PRE group (after 15 minutes). Conclusions: Both anesthetics that were applied showed a cardioprotective effect. Isoflurane provided a better structural and morphological protection, but Sevoflurane resulted in a more effective protection in terms of functionality, significantly reducing the incidence of extremely severe life-threatening arrhythmias.

High Glucose Concentration Attenuates Insulin‐Stimulated Protein Kinase B Levels In Cultured Astrocytes

BACKGROUNDGlucoregulatory abnormalities have been implicated to contribute to the pathophysiology of Alzheimer‐type dementia. The brain requires insulin for supply of energy critical for processing of memory and cognition. Insulin acts on the insulin receptor initiating signaling cascades that activate protein kinase B (Akt) through phosphorylation (p‐Akt) in cultured astrocytes. However, it is not yet clear whether the insulin induced production of p‐Akt could be attenuated by high glucose concentrations through stimulation of an inhibitory signaling pathway involving activation of protein kinase C (PKC).OBJECTIVEThe objective of our study is to examine the phosphorylation of Akt in cultured neonatal astrocytes following insulin stimulation and to determine if this response could be attenuated by culturing astrocytes in high glucose media via activation of PKC.METHODSNeonatal astrocyte cultures were derived from rat hippocampus and cultured for three passages in normal (5.5 mM) or high glucose (25 mM) media. Astrocyte cultures were stimulated with increasing concentrations of insulin (10−11 to 10−6 M) and Western immunoblotting analysis was used to determine protein levels of p‐Akt and insulin receptor. Protein levels of PKC isoforms (alpha, gamma, and delta) were determined in whole cell lysates from separate astrocyte cultures grown in normal or high glucose media.RESULTSInsulin‐induced a dose‐dependent increase in p‐Akt protein level in astrocytes cultured in normal glucose. However, insulin stimulation of astrocytes cultured in high glucose media resulted in an attenuated protein level of p‐Akt (100 nM insulin: 3.6 ± 1.0 vs. 8.9 ± 1.6 fold increase compared to control), requiring more insulin to increase the protein levels of p‐Akt (interaction effect: p = 0.024). The protein levels of insulin receptor in cultured astrocytes also increased in response to stimulation with insulin. Astrocytes cultured in high glucose media exhibited increased protein levels of PKC alpha (1.5 fold increase, p < 0.05) and gamma (2 fold increase, p < 0.05) but not of the PKC delta isoform compared to astrocytes cultured in normal glucose media.CONCLUSIONSInsulin‐dependent intracellular signaling involving phosphorylation of Akt is attenuated in astrocytes cultured in high glucose media. Astrocytes respond to increased environmental glucose with isoform specific increases in PKC protein levels which may have resulted in negative regulation of astrocyte insulin receptor signaling.Support or Funding InformationFunding from NIH (NHLBI) T35 HL072483, R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award.

PKC epsilon involvement in Th17 in vitro differentiation: implications in psoriasis pathogenesis

Psoriasis is a noncontagious, arytematous-squamose dermatitits affecting both sexes and all races. Although its exact etiology is largely unknown, it is now recognized as one of the most common immune-mediated disorders and several studies demonstrate an impairment of regulatory T-cells (Tregs) function and an up-regulation of IL-17 levels produced by T-helper 17 lymphocytes (Th17)(1,2). Protein kinase C epsilon (PKCe) is a serine/threonine kinase which plays a key role in the proliferation and differentiation of epidermal cells. We have previously demonstrated a role for PKCe in the pathogenesis of the autoimmune disease Hashimoto’s thyroiditis (3). PKCe is over-expressed in CD4+ T lymphocytes isolated from PBMC fraction in patients affected by this pathology and its forced down-modulation primed the TGF-mediated in vitro Treg polarization of human T CD4+ cells. Since it has been demonstrated that PKC-signalling is altered in psoriatic keratinocytes (4), we investigated the involvement of PKCe in Th17 in vitro differentiation and its potentially implication in immune response correlated to psoriasis. Using western blot and real time PCR, we have observed that PKCe protein levels and mRNA increase during Th17-lineage in vitro differentiation from naive CD4+ T cells with a similar trend of Th17 markers of differentiation STAT3 and RoRyT. Moreover, PKCe overexpression significantly increases STAT3 and phosphorylated STAT3 levels, suggesting that PKCe boosts Th17 polarization. Thereafter, we sought to investigate PKCe expression in CD4+ lymphocytes obtained from peripheral blood of psoriatic patients and we observed that PKCe expression levels are significantly higher compared with healthy donors. Intriguingly, we observed a closely correlation of PKCe expression with PASI index, suggesting an involvement of the kinase with the severity of the disease. Collectively these data suggest that PKCe might be involved in Th17 differentiation, that it could be a key factor to regulate Th17 pathological expansion and therefore a potential psoriatic pharmacological target.

The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats

Event Abstract Back to Event The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats Jaya Kumar1, 2*, Hermizi Hapidin3, Yvonne Tee Get Bee3 and Zalina Ismail2 1 Universiti Kebangsaan Malaysia, Department of Physiology, UKM Medical Centre, Malaysia 2 Universiti Sains Malaysia, BRAINetwork Centre for Neurocognitive Sciences, Malaysia 3 Universiti Sains Malaysia, School of Health Sciences, Malaysia Cessation from prolonged and excessive ethanol intake can lead to hyperexcitability of glutamatergic neurotransmission in the amygdala, which induces an anxiety-like syndrome. Most alcoholics that suffer from such symptoms frequently depend on habitual drinking as self-medication to alleviate their symptoms. Metabotropic glutamate receptors 5 (mGlu5) and Protein Kinase C (PKC) epsilon have been related to ethanol consumption and anxiety-related behaviours. mGlu5 signalling has been associated with PKC activities and the epsilon isoform of PKC has been suggested as a crucial signalling target for modulation of ethanol consumption by mGlu5. This study explores the changes in mGlu5 and PKC epsilon at protein and gene-levels in the amygdala following acute ethanol challenge during EW induced anxiety in rats. Male Wistar rats were fed a modified liquid diet containing low fat cow milk, sucrose, and maltodextrin, with a gradual introduction of 2.4%, 4.8% and 7.2% ethanol for 20 days. Six hours into EW, the rats were intraperitoneally injected with normal saline and ethanol (2.5?g/kg, 20% v/v), and exposed to open field and elevated plus maze tests. Then, amygdala tissue was dissected from the rat brain for Western blot and gene expression studies. EW-induced anxiety was accompanied by a significant increment of mGlu5, total PKC epsilon and phosphorylated PKC epsilon protein levels and also of mRNA of mGlu5 (GRM5) in the amygdala. Acute administration of ethanol significantly attenuated EW-induced anxiety as well as an EW-induced increase in GRM5. The acute challenge of ethanol to EW rats had little effect on the phosphorylated and total protein levels of PKC epsilon in the amygdala. Our results show that amygdala PKC epsilon may not be directly involved in the development of anxiety following EW. Keywords: Amygdala, hyperexcitability, PKC, mGlu5, ethanol challenge Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: YIC05: Young Investigator Colloquium 5 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kumar J, Hapidin H, Tee Get Bee Y and Ismail Z (2016). The effects of acute ethanol challenge on amygdala protein and gene expression of mglu5 and pkc epsilon during ethanol withdrawal induced anxiety in rats. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00069 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Jaya Kumar, Universiti Kebangsaan Malaysia, Department of Physiology, UKM Medical Centre, Cheras, Kuala Lumpur, Malaysia, jayakumar@ukm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Google Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Google Scholar Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail PubMed Jaya Kumar Hermizi Hapidin Yvonne Tee Get Bee Zalina Ismail Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ginsenoside Re protects methamphetamine-induced dopaminergic neurotoxicity via modulating PKC delta gene

Event Abstract Back to Event Ginsenoside Re protects methamphetamine-induced dopaminergic neurotoxicity via modulating PKC delta gene Eun-Joo Shin1, Sung Kwon Ko2, Ji Hoon Jeong3, Choon-Gon Jang4 and Hyoung-Chun Kim1* 1 Kangwon National University, Neuropsychopharmacology & Toxicology Program, College of Pharmacy, Korea, South 2 Semyung University, Department of Oriental Medical Food & Nutrition, Republic of Korea 3 Chung-Ang University, Department of Pharmacology, College of Medicine, Korea, South 4 Sungkyunkwan University, Department of Pharmacology, School of Pharmacy, Republic of Korea Ginsenoside Re, one of the main constituents of Panax ginseng, possesses novel antioxidant and anti-inflammatory properties. However, the pharmacological mechanism of ginsenoside Re in dopaminergic degeneration remains elusive. We suggested that protein kinase C (PKC) δ mediates methamphetamine (MA)-induced dopaminergic toxicity. Treatment with Re significantly attenuated MA-induced dopaminergic degeneration in vivo by inhibiting impaired enzymatic antioxidant systems, mitochondrial oxidative stress, mitochondrial translocation of PKCδ, mitochondrial dysfunction, pro-inflammatory microglial activation and apoptosis. These protective effects were comparable to those observed with genetic inhibition of PKCδ in PKCδ knockout (-/-) mice and with PKCδ antisense oligonucleotides, and Re did not provide any additional protective effects in the presence of PKCδ inhibition. We also examined effects of Re against MA toxicity in the SH-SY5Y neuroblastoma cells. Treatment with Re resulted in significant attenuations against decrease in the activity of glutathione peroxidase (GPx) and increase in the activity of superoxide dismutase (SOD) in the cytosolic and mitochondrial fraction. The changes in GSH paralleled those in GPx. Re treatment exhibited significant protections against mitochondrial oxidative damage, mitochondrial translocation of PKCδ, mitochondrial dysfunction, apoptotic events, and a reduction in the tyrosine hydroxylase (TH) expression and TH activity induced by MA. These protective effects of Re were comparable to those of PKCδ antisense oligonucleotide (ASO). However, Re did not significantly provide additional protective effects mediated by genetic inhibition of PKCδ in SH-SY5Y neuroblastoma cells. Our results suggest that PKCδ is a critical target for ginsenoside Re-mediated protective activity in response to dopaminergic degeneration induced by MA in vivo and in vitro. Acknowledgements This study is supported by a grant (Project No.S111415L020100) of the ‘Forestry Technology Projects’ provided by the Korea Forest Service, and in part by a grant (14182MFDS979) from the Korea Food and Drug Administration, Republic of Korea. Keywords: Methamphetamine, Superoxide Dismutase, antioxidant, ginseng, neuroblastoma cells Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 5: Boosting Brain Function with Nutraceuticals: What is the Evidence and how might they Work Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Shin E, Ko S, Jeong J, Jang C and Kim H (2016). Ginsenoside Re protects methamphetamine-induced dopaminergic neurotoxicity via modulating PKC delta gene. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00023 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Hyoung-Chun Kim, Kangwon National University, Neuropsychopharmacology & Toxicology Program, College of Pharmacy, Chunchon, Kangwon-do, Korea, South, kimhc@kangwon.ac.kr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Eun-Joo Shin Sung Kwon Ko Ji Hoon Jeong Choon-Gon Jang Hyoung-Chun Kim Google Eun-Joo Shin Sung Kwon Ko Ji Hoon Jeong Choon-Gon Jang Hyoung-Chun Kim Google Scholar Eun-Joo Shin Sung Kwon Ko Ji Hoon Jeong Choon-Gon Jang Hyoung-Chun Kim PubMed Eun-Joo Shin Sung Kwon Ko Ji Hoon Jeong Choon-Gon Jang Hyoung-Chun Kim Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.