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1.  Selective activation of PKC epsilon in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice 
Journal of neuroscience research  2013;91(6):799-807.
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.
doi:10.1002/jnr.23186
PMCID: PMC3905808  PMID: 23426889
mitochondria; astrocytes; acute stroke; cell culture; animal models
2.  Protein Kinase Cε Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria 
Circulation research  2003;92(8):873-880.
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.
doi:10.1161/01.RES.0000069215.36389.8D
PMCID: PMC3691672  PMID: 12663490
mitochondria; signal transduction; permeability transition pore; cardioprotection
3.  PROTEOMIC AND METABOLOMIC ANALYSIS OF CARDIOPROTECTION: INTERPLAY BETWEEN PROTEIN KINASE C EPSILON AND DELTA IN REGULATING GLUCOSE METABOLISM OF MURINE HEARTS 
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.
doi:10.1016/j.yjmcc.2008.10.008
PMCID: PMC3661410  PMID: 19027023
proteomics; metabolism; cardioprotection; protein kinase C
4.  PKCε Promotes Cardiac Mitochondrial and Metabolic Adaptation to Chronic Hypobaric Hypoxia by GSK3β Inhibition 
Journal of cellular physiology  2011;226(9):2457-2468.
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.
doi:10.1002/jcp.22592
PMCID: PMC3411281  PMID: 21660969
5.  Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCε) knockout mice 
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.
doi:10.1016/j.yjmcc.2009.10.030
PMCID: PMC2837767  PMID: 19913552
6.  Loss of ischaemic preconditioning in ovariectomized rat hearts: possible involvement of impaired protein kinase C ε phosphorylation 
Cardiovascular Research  2008;79(3):387-394.
Aims
The aims of this study were to determine whether chronic oestrogen withdrawal influences the development of ischaemic preconditioning (IPC) in female hearts, to investigate the mechanism whereby IPC is impaired, and to assess whether direct activation of protein kinase C (PKC) can mimic IPC in female hearts with chronic oestrogen depletion.
Methods and results
We performed Sham-operation (Sham) or bilateral ovariectomy on 16-week-old Sprague–Dawley female rats. Ovariectomized rats were randomized to subcutaneous implantation of 17β-estradiol (OxE) or placebo (OxP) pellets. Four weeks later, isolated, perfused hearts were subjected to 30 min of ischaemia followed by 120 min of reperfusion with or without three cycles of 5 min ischaemia/5 min reperfusion. The cardioprotective effect of IPC was completely lost in the OxP group. Western immunoblots revealed that in the OxP group, IPC failed to translocate PKCε to the membranous fraction and that phosphorylation of PKCε (Ser729) and phosphoinositide-dependent kinase (PDK) 1 (Ser241) was impaired. Oestrogen replacement restored the IPC effect, the translocation and phosphorylation of PKCε, and the phosphorylation of PDK1. In the OxP group, pre-treatment with a PKCε selective activator peptide (Ψ–εRACK) mimicked the IPC effect. Pre-treatment with a phosphatidylinositol-3 kinase inhibitor before IPC abrogated the translocation and phosphorylation of PKCε in the Sham group.
Conclusions
The cardioprotective effect of IPC is lost in female hearts with chronic oestrogen withdrawal and this is due, at least in part, to impaired translocation and phosphorylation of PKCε. Selective activation of PKCε-mediated signalling can fully restore the IPC effect in a manner analogous to oestrogen replacement.
doi:10.1093/cvr/cvn086
PMCID: PMC2492728  PMID: 18390563
Oestrogen; Gender; Myocardial infarction; Protein kinase C; Reperfusion injury
7.  PKCε Promotes HuD-Mediated Neprilysin mRNA Stability and Enhances Neprilysin-Induced Aβ Degradation in Brain Neurons 
PLoS ONE  2014;9(5):e97756.
Amyloid-beta (Aβ) peptide accumulation in the brain is a pathological hallmark of all forms of Alzheimer’s disease. An imbalance between Aβ production and clearance from the brain may contribute to accumulation of neurotoxic Aβ and subsequent synaptic loss, which is the strongest correlate of the extent of memory loss in AD. The activity of neprilysin (NEP), a potent Aβ-degrading enzyme, is decreased in the AD brain. Expression of HuD, an mRNA-binding protein important for synaptogenesis and neuronal plasticity, is also decreased in the AD brain. HuD is regulated by protein kinase Cε (PKCε), and we previously demonstrated that PKCε activation decreases Aβ levels. We hypothesized that PKCε acts through HuD to stabilize NEP mRNA, modulate its localization, and support NEP activity. Conversely, loss of PKCε-activated HuD in AD leads to decreased NEP activity and accumulation of Aβ. Here we show that HuD is associated with NEP mRNA in cultures of human SK-N-SH cells. Treatment with bryostatin, a PKCε-selective activator, enhanced NEP association with HuD and increased NEP mRNA stability. Activation of PKCε also increased NEP protein levels, increased NEP phosphorylation, and induced cell surface expression. In addition, specific PKCε activation directly stimulated NEP activity, leading to degradation of a monomeric form of Aβ peptide and decreased Aβ neuronal toxicity, as measured by cell viability. Bryostatin treatment also rescued Aβ-mediated inhibition of HuD-NEP mRNA binding, NEP protein expression, and NEP cell membrane translocation. These results suggest that PKCε activation reduces Aβ by up-regulating, via the mRNA-binding protein HuD, Aβ-degrading enzymes such as NEP. Thus, PKCε activation may have therapeutic efficacy for AD by reducing neurotoxic Aβ accumulation as well as having direct anti-apoptotic and synaptogenic effects.
doi:10.1371/journal.pone.0097756
PMCID: PMC4029802  PMID: 24848988
8.  Foetal nicotine exposure causes PKCε gene repression by promoter methylation in rat hearts 
Cardiovascular Research  2010;89(1):89-97.
Aims
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.
Conclusion
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.
doi:10.1093/cvr/cvq270
PMCID: PMC3002869  PMID: 20733009
Nicotine; Heart; Norepinephrine; Protein kinase C; DNA methylation
9.  PKCε Increases Phosphorylation of the Cardiac Myosin Binding Protein C at Serine 302 both in Vitro and in Vivo† 
Biochemistry  2007;46(23):7054-7061.
Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCε (PKCε TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCε TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCε treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCε showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCε consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCε and determined phosphorylation by mass spectrometry and ELISA assay. PKCε induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCε-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCε phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCε activity.
doi:10.1021/bi700467k
PMCID: PMC3969456  PMID: 17503784
10.  Propofol Modulates Agonist-induced Transient Receptor Potential Vanilloid Subtype-1 Receptor Desensitization via a Protein Kinase Cε-dependent Pathway in Mouse Dorsal Root Ganglion Sensory Neurons 
Anesthesiology  2010;113(4):833-844.
Background
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.
Methods
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.
Results
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.
Discussion
Our results indicate that propofol modulates TRPV1 sensitivity to capsaicin and that this most likely occurs through a PKCε-mediated phosphorylation of TRPV1.
doi:10.1097/ALN.0b013e3181eaa9a0
PMCID: PMC3049262  PMID: 20808213
11.  Protein Kinase C as a Stress Sensor 
Cellular signalling  2007;19(9):1820-1829.
While there are many reviews which examine the group of proteins known as protein kinase C (PKC), the focus of this article is to examine the cellular roles of two PKCs that are important for stress responses in neurological tissues (PKCγ and ε) and in cardiac tissues (PKCε). These two kinases, in particular, seem to have overlapping functions and interact with an identical target, connexin 43 (Cx43), a gap junction protein which is central to proper control of signals in both tissues. While PKCγ and PKCε both help protect neural tissue from ischemia, PKCε is the primary PKC isoform responsible for responding to decreased oxygen, or ischemia, in the heart. Both do this through Cx43.
It is clear that both PKCγ and PKCε are necessary for protection from ischemia. However, the importance of these kinases has been inferred from preconditioning experiments which demonstrate that brief periods of hypoxia protect neurological and cardiac tissues from future insults, and that this depends on the activation, translocation, or ability for PKCγ and/or PKCε to interact with distinct cellular targets, especially Cx43.
This review summarizes the recent findings which define the roles of PKCγ and PKCε in cardiac and neurological functions and their relationships to ischemia/reperfusion injury. In addition, a biochemical comparison of PKC γ and PKC ε and a proposed argument for why both forms are present in neurological tissue while only PKC ε is present in heart, are discussed. Finally, the biochemistry of PKCs and future directions for the field are discussed, in light of this new information.
doi:10.1016/j.cellsig.2007.05.014
PMCID: PMC1986756  PMID: 17629453
protein kinase C epsilon; protein kinase C gamma; ischemia; heart; neural tissues
12.  Formation of protein kinase Cε-Lck signaling modules confers cardioprotection 
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.
doi:10.1172/JCI13200
PMCID: PMC150872  PMID: 11854322
13.  Chronic prenatal hypoxia induces epigenetic programming of PKCε gene repression in rat hearts 
Circulation research  2010;107(3):365-373.
Rationale
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.
Objectives
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.
Conclusion
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.
doi:10.1161/CIRCRESAHA.110.221259
PMCID: PMC2919213  PMID: 20538683
Fetal heart; PKCε; hypoxia; epigenetics; DNA methylation
14.  Pre-clinical development of a bi-functional cancer cell homing, PKCε inhibitory peptide for the treatment of head and neck cancer 
Cancer research  2009;69(14):5829-5834.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide, comprising almost 50% of all malignancies in some developing nations. Our recent work identified protein kinase Cε (PKCε) as a critical and causative player in establishing an aggressive phenotype in HNSCC. In this study, we investigated the specificity and efficacy of HN1-PKCε, a novel bi-functional cancer cell homing, PKCε inhibitory peptide, as a treatment for HNSCC. HN1-PKCε peptide was designed by merging two separate technologies and synthesized as a capped peptide with two functional modules, HN1 (cancer cell homing) and PKCε (specific PKCε inhibitory), connected by a novel linker module. HN1-PKCε preferentially internalized into UMSCC1 and UMSCC36 cells, two HNSCC cell lines, in comparison to oral epithelial cells; 82.1% positive for UMSCC1 and 86.5% positive for UMSCC36 compared to 1.2% positive for oral epithelial cells. In addition, HN1-PKCε penetrated HNSCC cells in a dose-and time-dependent manner. Consistent with these in vitro observations, systemic injection of HN1-PKCε resulted in selective delivery of HN1-PKCε into UMSCC1 xenografts in nude mice. HN1-PKCε blocked the translocation of active PKCε in UMSCC1 cells confirming HN1-PKCε as a PKCε inhibitor. HN1-PKCε inhibited cell invasion by 72 ± 2% (p<0.001, n=12) and cell motility by 56 ± 2% (p<0.001, n=5) in UMSCC1 cells. Moreover, in vivo bioluminescence imaging demonstrated that HN1-PKCε significantly (83 ± 1% inhibition, p<0.02) retards the growth of UMSCC1 xenografts in nude mice. Our work indicates that the bi-functional HN1-PKCε inhibitory peptide represents a promising novel therapeutic strategy for HNSCC.
doi:10.1158/0008-5472.CAN-08-3465
PMCID: PMC2757013  PMID: 19567682
Experimental therapeutics; Head and Neck Cancer; Protein Kinase; and Oncogene
15.  Direct effect of cocaine on epigenetic regulation of PKCε gene repression in the fetal rat heart 
Maternal cocaine administration during gestation caused a down-regulation of PKCε expression in the heart of adult offspring resulting in an increased sensitivity to ischemia and reperfusion injury. The present study investigated the direct effect of cocaine in epigenetic modification of PKCε gene repression in the fetal heart. Hearts were isolated from gestational day 17 fetal rats and treated with cocaine in an ex vivo organ culture system. Cocaine treatment for 48 h resulted in significant decreases in PKCε protein and mRNA abundance and increases in CpG methylation at two SP1 binding sites in the PKCε promoter region (−346 and −268). Electrophoretic mobility shift assays demonstrated that CpG methylation of both SP1 sites inhibited SP1 binding. Consistently, chromatin immunoprecipitation assays showed that cocaine treatment significantly decreased binding of SP1 to the SP1 sites in the intact fetal heart. Reporter gene assays revealed that site-directed mutations of CpG methylation at both SP1 sites significantly reduced the PKCε promoter activity while methylation of a single site at either −346 or −268 did not have a significant effect. The causal effect of increased methylation in the cocaine-induced down-regulation of PKCε was demonstrated with the use of DNA methylation inhibitors. The presence of either 5-aza-2’-deoxycytodine or procainamide blocked the cocaine-induced increase in SP1 sites methylation and decrease in PKCε mRNA. The results demonstrate a direct effect of cocaine in epigenetic modification of DNA methylation and programming of cardiac PKCε gene repression linking prenatal cocaine exposure and pathophysiological consequences in the heart of adult offspring.
doi:10.1016/j.yjmcc.2009.06.004
PMCID: PMC2739252  PMID: 19538969
SP1; fetal programming; epigenetic; DNA methylation; gene regulation
16.  Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts 
PLoS ONE  2013;8(3):e58046.
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.
doi:10.1371/journal.pone.0058046
PMCID: PMC3585804  PMID: 23469261
17.  Protein Kinase Cε Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.hrthm.2007.05.030
PMCID: PMC2711555  PMID: 17765619
preconditioning; gap junctions; connexin43; coupling; protein kinase C
18.  Protein Kinase C epsilon is Important in Modulating Organic Dust-Induced Airway Inflammation 
Experimental lung research  2012;38(8):383-395.
Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased by greater than two-fold in PKCε KO mice following both a one-time challenge and three weeks of daily challenges with ODE as compared to WT mice. Likewise, lung pathology revealed that bronchiolar and alveolar inflammation and lymphoid aggregates were significantly increased in ODE-treated PKCε KO mice. Airway hyper-responsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT animals but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure, and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.
doi:10.3109/01902148.2012.714841
PMCID: PMC4066446  PMID: 22897707
protein kinase C epsilon; airway inflammation; airway hyper-responsiveness; nitric oxide; neutrophil; lung
19.  Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts 
Cardiovascular research  2006;73(3):488-496.
Objective
Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex.
Methods and Results
In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 minutes of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP).
Conclusion
Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε.
doi:10.1016/j.cardiores.2006.11.003
PMCID: PMC1852501  PMID: 17157283
20.  PKCε phosphorylation of the sodium channel NaV1.8 increases channel function and produces mechanical hyperalgesia in mice  
The Journal of Clinical Investigation  2012;122(4):1306-1315.
Mechanical hyperalgesia is a common and potentially disabling complication of many inflammatory and neuropathic conditions. Activation of the enzyme PKCε in primary afferent nociceptors is a major mechanism that underlies mechanical hyperalgesia, but the PKCε substrates involved downstream are not known. Here, we report that in a proteomic screen we identified the NaV1.8 sodium channel, which is selectively expressed in nociceptors, as a PKCε substrate. PKCε-mediated phosphorylation increased NaV1.8 currents, lowered the threshold voltage for activation, and produced a depolarizing shift in inactivation in wild-type — but not in PKCε-null — sensory neurons. PKCε phosphorylated NaV1.8 at S1452, and alanine substitution at this site blocked PKCε modulation of channel properties. Moreover, a specific PKCε activator peptide, ψεRACK, produced mechanical hyperalgesia in wild-type mice but not in Scn10a–/– mice, which lack NaV1.8 channels. These studies demonstrate that NaV1.8 is an important, direct substrate of PKCε that mediates PKCε-dependent mechanical hyperalgesia.
doi:10.1172/JCI61934
PMCID: PMC3315445  PMID: 22426212
21.  Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation 
Molecular Biology of the Cell  2002;13(1):12-24.
We have previously shown that protein kinase Cε (PKCε) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCε-mediated neurite induction. We show an increased association of PKCε to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCε is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCε actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCε overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCε independent of both serum and the actin-binding site, and PKCε colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCε in a pathway leading to neurite outgrowth. Localization of PKCε to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.
doi:10.1091/mbc.01-04-0210
PMCID: PMC65069  PMID: 11809819
22.  Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons 
PLoS ONE  2014;9(5):e97129.
Background
The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction.
Methods
In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot.
Results
The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε.
Conclusions
Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge.
doi:10.1371/journal.pone.0097129
PMCID: PMC4020800  PMID: 24828410
23.  Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms 
PLoS ONE  2014;9(4):e93473.
Background
Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin.
Methods and Results
In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity.
Conclusions
Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure.
doi:10.1371/journal.pone.0093473
PMCID: PMC3973555  PMID: 24695532
24.  The substrates and binding partners of protein kinase Cε 
The Biochemical journal  2010;427(2):189-196.
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.
doi:10.1042/BJ20091302
PMCID: PMC2966297  PMID: 20350291
25.  Protein kinase Cε: an oncogene and emerging tumor biomarker 
Molecular Cancer  2009;8:9.
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.
doi:10.1186/1476-4598-8-9
PMCID: PMC2647895  PMID: 19228372

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