Possible Association Between DHEA and PKCε in Hepatic Encephalopathy Amelioration: A Pilot Study

Objective: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome caused by liver failure and by an impaired neurotransmission and neurological function caused by hyperammonemia (HA). HE, in turn, decreases the phosphorylation of protein kinase C epsilon (PKCε), contributing to the impairment of neuronal functions. Dehydroepiandrosterone (DHEA) exerts a neuroprotective effect by increasing the GABAergic tone through GABAA receptor stimulation. Therefore, we investigated the protective effect of DHEA in an animal model of HE, and the possible modulation of PKCε expression in different brain area.Methods: Fulminant hepatic failure was induced in 18 male, Sprague–Dawley rats by i.p. administration of 3 g/kg D-galactosamine, and after 30 min, a group of animals received a subcutaneous injection of 25 mg/kg (DHEA) repeated twice a day (3 days). Exploratory behavior and general activity were evaluated 24 h and 48 h after the treatments by the open field test. Then, brain cortex and cerebellum were used for immunoblotting analysis of PKCε level.Results: DHEA administration showed a significant improvement of locomotor activity both 24 and 48 h after D-galactosamine treatment (****p < 0.0001) but did not ameliorate liver parenchymal degeneration. Western blot analysis revealed a reduced immunoreactivity of PKCε (*p < 0.05) following D-galactosamine treatment in rat cortex and cerebellum. After the addition of DHEA, PKCε increased in the cortex in comparison with the D-galactosamine-treated (***p < 0.001) and control group (*p < 0.05), but decreased in the cerebellum (*p < 0.05) with respect to the control group. PKCε decreased after treatment with NH4Cl alone and in combination with DHEA in both cerebellum and cortex (****p < 0.0001). MTS assay demonstrated the synergistic neurotoxic action of NH4Cl and glutamate pretreatment in cerebellum and cortex along with an increased cell survival after DHEA pretreatment, which was significant only in the cerebellum (*p < 0.05).Conclusion: An association between the DHEA-mediated increase of PKCε expression and the improvement of comatose symptoms was observed. PKCε activation and expression in the brain could inhibit GABA-ergic tone counteracting HE symptoms. In addition, DHEA seemed to ameliorate the symptoms of HE and to increase the expression of PKCε in cortex and cerebellum.

Abstract P509: Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH 4 ) is oxidized to dihydrobiopterin (BH 2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor ( myr -PKCε–; N- myr -EAVSLKPT) and activator ( myr -PKCε+; N- myr -HDAPIGYD). We hypothesize that myr -PKCε– with uncoupled eNOS (i.e., bound to BH 2 ) should reduce leukocyte-endothelial interactions in postcapillary venules and H 2 O 2 levels after SWL. Myr -PKCε+ should promote similar effects when eNOS is coupled (i.e., bound to BH 4 ). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH 2 and test solutions (Krebs’ buffer, 10 μM myr -PKCε+, or 10 μM myr -PKCε–) with or without 100 μM BH 4 . Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail ( myr -PKCε+ [0.9 mg/kg]/ myr -PKCε- [0.8 mg/kg] with BH 2 [2mg/kg] or BH 4 [0.8 mg/kg]) was given i.v. NO or H 2 O 2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student-Newman-Keuls test. BH 2 -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr -PKCε+ ( n = 6) and attenuated with myr- PKCε– ( n = 5) compared to control (n=5) at 120 min (p<0.01). Following SWL, all treatments except myr -PKCε+ with BH 2 significantly reduced H 2 O 2 ( n = 5, p < .01) and increased NO levels ( n = 5, p < .01) compared to control. NO and H 2 O 2 levels following administration of myr -PKCε+ with BH 2 were similar to SWL control. Results suggest that myr -PKCε– attenuates uncoupled eNOS activity and H 2 O 2 release in rat renal veins, after SWL. Promoting coupled eNOS activity with BH 4 yields similar results. Promoting eNOS coupling with BH 4 or inhibiting uncoupled eNOS with myr- PKCε– attenuates oxidative stress and endothelial dysfunction following SWL and BH 2 -induced inflammation.

Variants in PRKCE and KLC1, Potential Negative Regulators of Type I Psoriasis

Background: Psoriasis is a multifactorial disease with a complex genetic predisposition. The pathophysiology of psoriasis is associated with genetic variants, especially in negative regulatory genes. To better characterize gene variants in psoriasis and identify the relationship between clinical characteristics and variant genes in its pathogenesis. DNA was extracted from 282 type Ⅰ psoriasis patients and purified, and 13 variable genes were amplified and sequenced using the Sanger method. Results: Among the 13 investigated genes, the variants frequencies of protein kinase C epsilon (PRKCE) (c.240T>C, 35.9% vs 47.7%, P< .05) and kinesin light chain 1 (KLC1) (c.216A>G, 2.9% vs 98.1%, P< .01) were significantly lower in patients than in normal Asian individuals. Additionally, we found considerable differences in the relationship between variants in genes CADM2, JPH2, SPTLC3 and clinical characteristics stratified by medical history and family history. Moreover, MEGF6 gene variants (39.52% vs. 22.50%, χ2=3.83, p < .05) showed a stronger association with the group as mild (PASI≤10) than with the group as heavy. Conclusions: Our results provide a comprehensive correlation analysis of negative regulatory genes that are regulated in psoriasis. This integrated analysis offers novel insight into the pathogenic mechanisms involved in psoriasis.

mTORC and PKCε in Regulation of Alcohol Use Disorder

Alcohol use disorder (AUD) is characterized by compulsive binge alcohol intake, leading to various health and social harms. Protein Kinase C epsilon (PKCε), a specific family of PKC isoenzyme, regulates binge alcohol intake, and potentiates alcohol-related cues. Alcohol via upstream kinases like the mammalian target to rapamycin complex 1 (mTORC1) or 2 (mTORC2), may affect the activities of PKCε or vice versa in AUD. mTORC2 phosphorylates PKCε at hydrophobic and turn motif, and was recently reported to be associated with alcohol-seeking behavior, suggesting the potential role of mTORC2-PKCε interactions in the pathophysiology of AUD. mTORC1 regulates translation of synaptic proteins involved in alcohol-induced plasticity. Hence, in this article, we aimed to review the molecular composition of mTORC1 and mTORC2, drugs targeting PKCε, mTORC1, and mTORC2 in AUD, upstream regulation of mTORC1 and mTORC2 in AUD and downstream cellular mechanisms of mTORCs in the pathogenesis of AUD.

Mechanisms of protein kinase C epsilon down-regulation by transforming growth factor-beta in lung cancer cells

ABSTRACTProtein kinase C epsilon (PKCε), a diacylglycerol (DAG)/phorbol ester-regulated PKC isoform, has been widely linked to oncogenesis and metastasis. PKCε plays important roles in the regulation of motility and invasiveness in non-small cell lung cancer (NSCLC). We previously reported that this kinase becomes prominently down-regulated upon TGF-β-induced epithelial-to-mesenchymal transition (EMT), which leads to prominent phenotypic changes. While the phorbol ester PMA causes down-regulation of PKCα, δ and ε within hours, TGF-β requires at least 4 days to reduce the expression levels of PKCε without affecting the expression of other PKCs, an effect that parallels the acquisition of a mesenchymal phenotype. Despite the prominent transcriptional component involved in EMT, we found that PKCε down-regulation does not involve changes in PKCε mRNA levels and was entirely independent of transcriptional activation of the PRKCE gene. Further mechanistic analysis revealed that the reduction in PKCε expression is dependent on proteasomal and endolysosomal pathways, but independent of autophagy processing mechanisms. Site-directed mutagenesis of Lys312 and Lys321 in PKCε prevented its down-regulation in response to either TGF-β or the phorbol ester PMA. The shift in PKCε isozyme levels depending on cell plasticity underscores relevant functional consequences by modulating the expression of this oncogenic/metastatic kinase and highlights key roles of protein stability mechanisms in the control of PKCε phenotypic outcomes.

Bidirectional sex-dependent regulation of α6 and β3 nicotinic acetylcholine receptors by protein kinase Cε

Nicotine and alcohol are the most commonly abused substances worldwide, and comorbid nicotine and alcohol addiction is highly prevalent. Nicotinic acetylcholine receptors (nAChRs) containing the α6 and β3 subunits are expressed in neural reward circuits and are critical for both nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKCε), which impact transcription of α6 and β3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKCε−/− mice, who also consume less nicotine and alcohol compared to wild-type (WT) littermates. Here, we show that female PKCε−/− mice have enhanced expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior, as female but not male PKCε−/− mice exhibit locomotor hypersensitivity to nicotine. Female PKCε−/− mice show no differences in alcohol-induced sedation compared to WT littermates, while male PKCε−/− have enhanced sedation compared to WT mice, a phenotype that has previously been reported in α6−/− mice. Female PKCε−/− mice also show reduced depression-like behavior in response to systemic injections of varenicline compared to WT littermates, and this effect was absent in male mice. Additionally, we found that female PKCε−/− mice show altered alcohol and nicotine consumption patterns in chronic voluntary two bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKCε, and highlight the importance of studying both sexes in preclinical animal models.

The Central Role of Protein Kinase C Epsilon in Cyanide Cardiotoxicity and Its Treatment

In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.