Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCε). We recently identified aldehyde dehydrogenase 2 (ALDH2) as an PKCε substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCε knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCε and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCε, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCε knockout hearts. Our data suggest that ALDH2 is downstream of PKCε in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCε to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.
Activation of PKCε confers protection against neuronal ischemia/reperfusion. Since activation of PKCε leads to its translocation to multiple intracellular sites, a mitochondrial-selective PKCε activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKCε. PKCε can regulate key cytoprotective mitochondrial functions including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondrial selective activation of PKCε to protect primary brain cell cultures or mice subjected to ischemic stroke. Pre-treatment with either general PKCε activator peptide, ψεRACK, or mitochondrial-selective PKCε activator, ψεHSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both ψεRACK and ψεHSP90 were blocked by the PKCε antagonist, εV1–2, indicating protection requires PKCε interaction with its anchoring protein, εRACK. Further supporting a mitochondrial mechanism for PKCε, neuroprotection by ψεHSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, ψεHSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hours of reperfusion. Thus selective activation of mitochondrial PKCε preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.
mitochondria; astrocytes; acute stroke; cell culture; animal models
Although functional coupling between protein kinase Cε (PKCε) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCε remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCε and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCε with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCε complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCε can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCε resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCε in mice, which is cardioprotective, greatly increased interaction of PKCε with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCε did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCε transgenesis. Collectively, these data demonstrate that PKCε forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCε-induced cardioprotection.
mitochondria; signal transduction; permeability transition pore; cardioprotection
We applied a combined proteomic and metabolomic approach to obtain novel mechanistic insights in PKCε-mediated cardioprotection. Mitochondrial and cytosolic proteins from control and transgenic hearts with constitutively active or dominant negative PKCε were analyzed using difference in-gel electrophoresis (DIGE). Among the differentially expressed proteins were creatine kinase, pyruvate kinase, lactate dehydrogenase, and the cytosolic isoforms of aspartate amino transferase and malate dehydrogenase, the two enzymatic components of the malate aspartate shuttle, which is required for the import of reducing equivalents from glycolysis across the inner mitochondrial membrane. These enzymatic changes appeared to be dependent on PKCε activity, as they were not observed in mice expressing inactive PKCε. High-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy confirmed a pronounced effect of PKCε activity on cardiac glucose and energy metabolism: normoxic hearts with constitutively active PKCε had significantly lower concentrations of glucose, lactate, glutamine and creatine, but higher levels of choline, glutamate and total adenosine nucleotides. Moreover, the depletion of cardiac energy metabolites was slower during ischemia/reperfusion injury and glucose metabolism recovered faster upon reperfusion in transgenic hearts with active PKCε. Notably, inhibition of PKCε resulted in compensatory phosphorylation and mitochondrial translocation of PKCδ. Taken together, our findings are the first evidence that PKCε activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCδ and PKCε in cardioprotection.
proteomics; metabolism; cardioprotection; protein kinase C
Foetal nicotine exposure results in decreased protein kinase C epsilon (PKCε) expression and increased cardiac vulnerability to ischaemia and reperfusion injury in adult rat offspring. The present study tested the hypothesis that maternal nicotine administration causes increased promoter methylation of the PKCε gene resulting in PKCε repression in the heart.
Methods and results
Nicotine treatment of pregnant rats starting at day 4 of gestation increased the methylation of the Egr-1 binding site at the PKCε gene promoter and decreased PKCε protein and mRNA abundance in near-term foetal hearts. Methylation of the Egr-1 binding site reduced Egr-1 binding to the PKCε promoter in the heart. Site-specific deletion of the Egr-1 binding site significantly decreased PKCε promoter activity. The effects of nicotine were sustained in the heart of adult offspring. Ex vivo studies found no direct effect of nicotine on PKCε gene expression. However, maternal nicotine administration increased norepinephrine content in the foetal heart. Treatment of isolated foetal hearts with norepinephrine resulted in the same effects of increased methylation of the Egr-1 binding site and PKCε gene repression in the heart. 5-Aza-2′-deoxycytidine inhibited the norepinephrine-induced increase in methylation of the Egr-1 binding site and restored Egr-1 binding and PKCε gene expression to the control levels.
This study demonstrates that prolonged nicotine exposure increases the sympathetic neurotransmitter release in the foetal heart and causes programming of PKCε gene repression through promoter methylation, linking maternal smoking to pathophysiological consequences in the offspring heart.
Nicotine; Heart; Norepinephrine; Protein kinase C; DNA methylation
Protein kinase Cε (PKCε) plays a pivotal role in cardioprotection during cardiac ischemia and reperfusion injury. Recent studies demonstrated that prenatal cocaine exposure caused a decrease in PKCε expression and increased heart susceptibility to ischemic injury in adult offspring, suggesting an in utero programming of PKCε gene expression pattern in the heart. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for cocaine-mediated repression of the PKCε gene in the heart. Pregnant rats were administered either saline or cocaine intraperitoneally (15 mg/kg) twice daily from days 15 to 20 of gestational age, and term fetal hearts were studied. Cocaine treatment significantly decreased PKCε mRNA and protein levels in the heart. CpG dinucleotides found in cAMP response element-binding protein (CREB), CREB/c-Jun1, and CREB/c-Jun2 binding sites at the proximal promoter region of the PKCε gene were densely methylated and were not affected by cocaine. In contrast, methylation of CpGs in the activator protein 1 (AP-1) binding sites was low but was significantly increased by cocaine. Reporter gene assays showed that the AP-1 binding site played a strong stimulatory role of PKCε gene transcription. Methylation of the AP-1 binding sites significantly decreased AP-1 binding to the PKCε promoter. Supershift analyses implicated c-Jun homodimers binding to the AP-1 binding sites. Cocaine did not affect nuclear c-Jun levels or the binding of c-Jun to the unmethylated AP-1 binding sites. The results indicate a role for DNA methylation in cocaine-mediated PKCε gene repression in the developing heart and suggest an epigenetic mechanism affecting this gene linked with vulnerability of ischemic injury in the heart of adult offspring.
The epsilon isoform of protein kinase C (PKCε) has important roles in the function of the cardiac, immune and nervous systems. As a result of its diverse actions, PKCε is the target of active drug discovery programs. A major research focus is to identify signaling cascades that include PKCε and the substrates that PKCε regulates. In this review we will identify and discuss those proteins that have been conclusively shown to be direct substrates of PKCε by the best currently available means. We will also describe binding partners that anchor PKCε near its substrates. We review the consequences of substrate phosphorylation and discuss cellular mechanisms by which target specificity is achieved. We begin with a brief overview of the biology of PKCε and methods for substrate identification, and proceed with a discussion of substrate categories to identify common themes that emerge and how these may be used to guide future studies.
The ε isoform of protein kinase C (PKCε) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCε signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCε orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCε. In cardiac cells, PKCε interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCε or by ischemic preconditioning enhances the formation of PKCε-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCε-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCε-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCε-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.
Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCε) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCε (DNPKCε) as a strategy to demonstrate that PKCε is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCε to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in-vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS-treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCε is essential for normal TRPV1 responses in vitro and in vivo.
capsaicin; desensitization; HSV; modulation; nociception; PKC; TRPV1
Mitochondria are central players in the pathophysiology of ischemia-reperfusion. Activation of plasma membrane G-coupled receptors or the Na, K-ATPase trigger cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome-mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K+ channel (mitoKATP). MitoKATP opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCε), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.
mitochondrial KATP channel; protein kinase C; reactive oxygen species; permeability transition; signaling pathways
We have recently implicated mitochondrial mechanisms in models of neuropathic and inflammatory pain, in some of which a role of protein kinase Cε (PKCε) has also been implicated. Since mitochondria contain several proteins that are targets of PKCε, we evaluated the role of mitochondrial mechanisms in mechanical hyperalgesia induced by proinflammatory cytokines that induce PKCε-dependent nociceptor sensitization, and by a direct activator of PKCε (ψεRACK), in the rat. Prostaglandin E2 (PGE2)-induced hyperalgesia is short lived in naïve rats, while it is prolonged in ψεRACK pre-treated rats, a phenomenon referred to as priming. Inhibitors of two closely-related mitochondrial functions, electron transport (complexes I-V) and oxidative stress (reactive oxygen species), attenuated mechanical hyperalgesia induced by intradermal injection of ψεRACK. In marked contrast, in a PKCε-dependent form of mechanical hyperalgesia induced by prostaglandin E2 (PGE2), inhibitors of mitochondrial function failed to attenuate hyperalgesia. These studies support the suggestion that at least two downstream signaling pathways can mediate the hyperalgesia induced by activating PKCε.
Hyperalgesia; Mitochondria; Priming; Protein kinase Cε; Pain
PKCε is central to cardioprotection. Sub-proteome analysis demonstrated co-localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia-inducible factor 1α (HIF-1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF-1α. However, there is no established mechanistic link between PKCε, p-GSK3β and HIF1-α. Here we hypothesized that cardiac-restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF-1α-dependent mechanism. aPKCε and wild-type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O2) and cardiac metabolism, functional parameters, p-GSK3β/HIF-1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP-dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P< 0.005, n = 8). Electron microscopy revealed a hypoxia-associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P<0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p-GSK3β and HIF1-α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p-GSK3β/HIF-1α-mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia.
Members of the protein kinase C (PKC) family have long been studied for their contributions to oncogenesis. Among the ten different isoforms of this family of serine/threonine kinases, protein kinase Cε (PKCε) is one of the best understood for its role as a transforming oncogene. In vitro, overexpression of PKCε has been demonstrated to increase proliferation, motility, and invasion of fibroblasts or immortalized epithelial cells. In addition, xenograft and transgenic animal models have clearly shown that overexpression of PKCε is tumorigenic resulting in metastatic disease. Perhaps most important in implicating the epsilon isoform in oncogenesis, PKCε has been found to be overexpressed in tumor-derived cell lines and histopathological tumor specimens from various organ sites. Combined, this body of work provides substantial evidence implicating PKCε as a transforming oncogene that plays a crucial role in establishing an aggressive metastatic phenotype. Reviewed here is the literature that has led to the current understanding of PKCε as an oncogene. Moreover, this review focuses on the PKCε-mediated signaling network for cell motility and explores the interaction of PKCε with three major PKCε signaling nodes: RhoA/C, Stat3 and Akt. Lastly, the emerging role of PKCε as a tumor biomarker is discussed.
A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCα, PKCδ, and PKCε), two isoforms (PKCδ and PKCε) have been implicated in profibrotic intracellular signaling. The role of PKCα-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCα was found mostly in the cytoplasm, whereas PKCδ and PKCε were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCα but not PKCδ or PKCε underwent rapid (within 5–10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCα and ERK2, but not PKCδ or PKCε, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCα Gö6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCα signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCα nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCα but not PKCδ or PKCε mediate the profibrotic effect of CCL18. PKCα may therefore become a viable target for future antifibrotic therapies.
chemokines; fibroblast; fibrosis; lung; signal transduction
Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus, and results in decreased protein kinase C epsilon (PKCε) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats.
The present study tested the hypothesis that fetal hypoxia-induced methylation of CpG dinucleotides at the PKCε promoter is repressive and contributes to PKCε gene repression in the heart of adult offspring.
Methods and Results
Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in significant decreases in PKCε protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCε promoter at SP1 binding sites, −346 and −268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O2 for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2’-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCε mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCε mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor α (ERα ) and β (ERβ ) isoforms in females than in males. Both ERα and ERβ interacted with the SP1 binding sites in the fetal heart, which may explain the gender differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCε restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring.
The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCε gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.
Fetal heart; PKCε; hypoxia; epigenetics; DNA methylation
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal (4-HNE) and the bioactivation of nitroglycerin (GTN) to nitric oxide (NO). In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our lab recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon (PKCε) and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator (Alda-1) which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2, and discuss potential clinical implications of these findings.
Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease.
Methods and Results
We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen.
Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.
Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin43 (Cx43) from gap junctions during subsequent ischemia.
To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCε), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCε (PKCε-KO). Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity.
The amount of Cx43 located in gap junctions, measured by confocal microscopy under basal conditions, was significantly greater in PKCε-KO hearts compared to wildtype but total Cx43 content measured by immunoblotting was not different. These unanticipated results indicate that PKCε regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented loss of Cx43 from gap junctions during ischemia in wildtype but not PKCε-KO hearts. Specific activation of PKCε, but not PKCδ, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wildtype but hastened uncoupling in PKCε-KO hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wildtype hearts and, surprisingly, by nearly 10-fold in PKCε-KO hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wildtype but not PKCε-KO hearts.
Taken together, these results indicate that PKCε plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.
preconditioning; gap junctions; connexin43; coupling; protein kinase C
The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin cancer. Here we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2, enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex, increases mitochondrial permeability and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associates with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.
We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε−/−) mice to examine this mechanistically. PKCε−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.
Our earlier study demonstrated the induction of PKC isoforms (beta II, PKC-alpha/beta, PKC-theta) by ionizing radiation induced bystander response in human cells. In this study, we extended our investigation to yet another important member of PKC family, PKC epsilon (PKCε). PKCε functions both as an anti-apoptotic and pro-apoptotic protein and it is the only PKC isozyme implicated in oncogenesis. Given the importance of PKCε in oncogenesis, we wished to determine whether or not PKCε is involved in bystander response. Gene expression array analysis demonstrated a 2-3 fold increase in PKCε expression in the bystander human primary fibroblast cells that were co-cultured in double sided Mylar dishes for 3 h with human primary fibroblast cells irradiated with 5 Gy of α-particles. The elevated PKCε expression in bystander cells was verified by quantitative real time PCR. Suppression of PKCε expression by small molecule inhibitor Bisindolylmaleimide IX (Ro 31-8220) considerably reduced the frequency of micronuclei (MN) induced both by 5 Gy of γ-rays (low LET) and α-particles (high LET) in bystander cells. Similar cytoprotective effects were observed in bystander cells after siRNA mediated silencing of PKCε suggestive of its critical role in mediating some of the bystander effects (BE). Our novel study suggests the possibility that PKC signaling pathway may be a critical molecular target for suppression of ionizing radiation induced biological effects in bystander cells.
bystander effects; protein kinase Cε; ionizing radiation; signal transduction pathway
Adenosine A1 receptor (A1R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β1-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β1-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A1R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β1-adrenergic stimulation on PKCε translocation elicited by A1R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A1R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β1-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A1R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A1R. This increase was attenuated by the A1R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A1R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A1R, it is apparent that the interactive effects of adenosine and β1-adrenergic agonists on function are complex in the heart.
adrenergic; antiadrenergic; heart; RACK2; PKA; PKCε
Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCε) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCε signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCε in the amygdala reduced ethanol consumption and preference in a limited access paradigm. Further, mice which are heterozygous for the PKCε allele consume less ethanol compared to wild type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCε in the amygdala compared with wild-type mice. We conclude that amygdala PKCε is important for ethanol consumption in mice.
Protein kinase C epsilon (PKCε), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCε-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCε signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCε. Stat3 has two conserved amino acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCε interacts with Stat3α isoform which has Ser727 and not with Stat3β isoform which lacks Ser727. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that: 1) PKCε physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231). 2) Inhibition of PKCε expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion. 3) PKCε mediates Stat3Ser727 phosphorylation via integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCε-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.
PKCε; Stat3; Human cancer
The activity of transient receptor potential vanilloid subtype-1 (TRPV1) receptors, key nociceptive transducers in dorsal root ganglion sensory neurons, is enhanced by protein kinase C ε (PKCε) activation. The intravenous anesthetic propofol has been shown to activate PKCε. Our objectives were to examine whether propofol modulates TRPV1 function in dorsal root ganglion neurons via activation of PKCε.
Lumbar dorsal root ganglion neurons from wild-type and PKCε-null mice were isolated and cultured for 24 h. Intracellular free Ca2+ concentration was measured in neurons by using fura-2 acetoxymethyl ester. The duration of pain-associated behaviors was also assessed. Phosphorylation of PKCε and TRPV1 and the cellular translocation of PKCε from cytosol to membrane compartments were assessed by immunoblot analysis.
In wild-type neurons, repeated stimulation with capsaicin (100 nM) progressively decreased the transient rise in intracellular free Ca2+ concentration. After desensitization, exposure to propofol rescued the Ca2+ response. The resensitizing effect of propofol was absent in neurons obtained from PKCε-null mice. Moreover, the capsaicin-induced desensitization of TRPV1 was markedly attenuated in the presence of propofol in neurons from wild-type mice but not in neurons from PKCε-null mice. Propofol also prolonged the duration of agonist-induced pain associated behaviors in wild-type mice. In addition, propofol increased phosphorylation of PKCε as well as TRPV1 and stimulated translocation of PKCε from cytosolic to membrane fraction.
Our results indicate that propofol modulates TRPV1 sensitivity to capsaicin and that this most likely occurs through a PKCε-mediated phosphorylation of TRPV1.