We have previously shown that domains involved in binding of protein kinase C (PKC1) isozymes to their respective anchoring proteins (RACKs2) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named ψRACK3 peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the ψRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the ψRACK sequence. We focused on εPKC ψRACK peptide, where the pseudo-εRACK sequence (ψεRACK; HDAPIGYD; corresponding to εPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to εRACK285-292) in the second amino acid. Here we show that changing the charge of the ψεRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.
PKC (protein kinase C); RACK (receptor for activated C-kinase); ψRACK (pseudo RACK); intramolecular interaction; carrier peptide
Pervious biochemical and hemodymanic studies have highlighted the important role of εPKC in cardioprotection during ischemic preconditioning. However, little is known about the electrophysiological consequences of εPKC modulation in ischemic hearts. Membrane permeable peptide εPKC selective activator and inhibitor were used to investigate the role of εPKC modulation in reperfusion arrhythmias.
Protein transduction domain from HIV- TAT was used as a carrier for peptide delivery into intact Langendorff perfused guinea pig hearts. Action potentials were imaged and mapped (124 sites) using optical techniques and surface ECG was continuously recorded. Hearts were exposed to 30 min stabilization period, 15 min of no-flow ischemia, followed by 20 min reperfusion. Peptides (0.5 μM) were infused as follows: a) control (vehicle-TAT peptide; TAT-scrambled ψεRACK peptide); b) εPKC agonist (TAT-ψεRACK); c) εPKC antagonist (TAT-εV1).
Hearts treated with εPKC agonist ψεRACK had reduced incidence of ventricular tachycardia (VT, 64%) and fibrillation (VF, 50%) compared to control (VT, 80%, p<0.05) and (VF, 70%, P<0.05). However, the highest incidence of VT (100%, P<0.05) and VF (80%) occurred in hearts treated with εPKC antagonist peptide εV1 compared to control and to εPKC agonist ψεRACK. Interestingly, at 20 min reperfusion, 100% of hearts treated with εPKC agonist ψεRACK exhibited complete recovery of action potentials compared to 40% (p<0.05) of hearts treated with εPKC antagonist peptide, εV1 and 65% (P<0.5) of hearts in control. At 20 min reperfusion, maps of action potential duration from εPKC agonist ψεRACK showed minimal dispersion (48.2±9 ms) compared to exacerbated dispersion (115.4±42 ms, P<0.05) in εPKC antagonist and control (67±20 ms, P<0.05). VT/VF and dispersion from hearts treated with scrambled agonist or antagonist peptides were similar to control.
the results demonstrate that εPKC activation by ψεRACK peptide protects intact hearts from reperfusion arrhythmias and affords better recovery. On the other hand, inhibition of εPKC increased the incidence of arrhythmias and worsened recovery compared to controls. The results carry significant therapeutic implications for the treatment of acute ischemic heart disease by preconditioning-mimicking agents.
cardiac electrophysiology; Protein Kinase C; reperfusion arrhythmia; optical mapping
Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial
KATP+ channel (
mtKATP+) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC targets
mtKATP+ channels, but its presence in brain mitochondria is controversial, we determined the presence of the
KATP+ channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether
mtKATP+ channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the
KATP+ channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or εPKC agonist-induced preconditioning. Blockade of
mtKATP+ channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic andεPKC agonist-mediated preconditioning resulted in phosphorylation of the
mtKATP+ channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC or its downstream effectors. Our results support the hypothesis that the brain
mtKATP+ channel is an important target of IPC and the signal transduction pathways initiated by εPKC.
ischemic tolerance; diazoxide; protein kinase C; organotypic slice culture; cell death; signal transduction
Activation of ε protein kinase C (εPKC) protects hearts from ischemic injury. However, some of the mechanism(s) of εPKC mediated cardioprotection are still unclear. Identification of εPKC targets may aid to elucidate εPKC–mediated cardioprotective mechanisms. Previous studies, using a combination of εPKC transgenic mice and difference in gel electrophoresis (DIGE), identified a number of proteins involved in glucose metabolism, whose expression was modified by εPKC. These studies, were accompanied by metabolomic analysis, and suggested that increased glucose oxidation may be responsible for the cardioprotective effect of εPKC. However, whether these εPKC-mediated alterations were due to differences in protein expression or phosphorylation was not determined.
Methods and Results
Here, we used an εPKC-specific activator peptide, ψεRACK, in combination with phosphoproteomics to identify εPKC targets, and identified proteins whose phosphorylation was altered by selective activation of εPKC most of the identified proteins were mitochondrial proteins and analysis of the mitochondrial phosphoproteome, led to the identification of 55 spots, corresponding to 37 individual proteins, which were exclusively phosphorylated, in the presence of ψεRACK. The majority of the proteins identified were proteins involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins.
In summary the protective effect of εPKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose, lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by εPKC phosphorylation may lead to εPKC-mediated cardioprotection induced by ψεRACK.
εPKC; ischemia; phosphorylation; mitochondria
Protein Kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that εPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of εPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of εPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that εPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of εPKC activation during ischemia on cerebral blood flow (CBF). We found that ψε-Receptors for activated C kinase (RACK), a εPKC-selective peptide activator, injected intravenously 30 minutes before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψεRACK compared to Tat peptide (vehicle). Our results suggest that εPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, εPKC may be one of the treatment modalities against ischemic injury.
Ischemia; epsilon Protein Kinase C; Cerebral Blood Flow; Neuroprotection
In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of ε protein kinase C (εPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of εPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal εPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of εPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific εPKC activating peptide, tat-ψεRACK (tat-ψε-receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that εPKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal εPKC. Activation of εPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, εPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the εPKC signaling pathway.
cerebral ischemia; phosphorylation; electron transport chain; neuroprotection; cell death; hippocampus
The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, δ and εPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.
Methods and results
Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished δPKC translocation by 3.8-fold and increased εPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of δPKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of εPKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of εPKC during IPC restored δPKC levels at the mitochondria while decreasing εPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.
Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, εPKC.
Cardioprotection; Ischaemia/reperfusion; Apoptosis; Proteasome; PKC; Ischaemic preconditioning
Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the ε-isoform of protein kinase C (εPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and εPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or εPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or ψεRACK, a selective εPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABAA receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, εPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABAA receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9 ± 6.1%. When applied during test ischemia, 100 nmol/L bicuculline abolished neuroprotection conferred by either IPC or εPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.
εPKC; inhibition; ischemia; ischemic tolerance; organotypic slice
In response to mild ischemic stress, the brain elicits endogenous survival mechanisms to protect cells against a subsequent lethal ischemic stress, referred to as ischemic tolerance. The molecular signals that mediate this protection are thought to involve the expression and activation of multiple kinases, including protein kinase C (PKC). Here we demonstrate that εPKC mediates cerebral ischemic tolerance in vivo. Systemic delivery of ψεRACK, an εPKC-selective peptide activator, confers neuroprotection against a subsequent cerebral ischemic event when delivered immediately prior to stroke. In addition, activation of εPKC by ψεRACK treatment decreases vascular tone in vivo, as demonstrated by a reduction in microvascular cerebral blood flow. Here we demonstrate the role of acute and transient εPKC in early cerebral tolerance in vivo and suggest that extra-parenchymal mechanisms, such as vasoconstriction, may contribute to the conferred protection.
Ischemia; preconditioning; protein kinase C; cerebral blood flow
The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
infarct size; remote; incision; protein kinase C; gamma; epsilon
Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen–glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.
Cerebral ischemia; Ischemic tolerance; Epsilon protein kinase C; Extracellular signal-regulated kinase (ERK1/2); Neuroprotection; Mixed cortical neuron/astrocyte cell cultures
The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (εPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of εPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 minutes prior to (but not 15 minutes prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of εPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and εPKC-mediated protection from cardiac ischemia, in vivo.
We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP.
cAMP; intracellular targeting; phosphodiesterase (PDE); protein kinase C (PKC); receptor for activated C-kinase 1 (RACK1); β2-AR, β2-adrenoreceptor; CREB, cAMP-response-element-binding protein; DMEM, Dulbecco's modified Eagle's medium; DOTAP, dioleoyltrimethylammonium propane; ECL, enhanced chemiluminescence; ERK, extracellular-signal-regulated kinase; GFX, GF109203X; GST, glutathione transferase; HEK, human embryonic kidney; HRP, horseradish peroxidase; IGF, insulin-like growth factor; MEK, MAPK (mitogen-activated protein kinase)/ERK kinase; PDE, phosphodiesterase; PKA, protein kinase A; PKC, protein kinase C; PP2A, protein phosphatase 2A; RACK1, receptor for activated C-kinase 1; TBST, Tris-buffered saline containing 0.1% Tween 20; VSV, vesicular stomatitis virus
Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of εPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether εPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered εV1-2 an εPKC-selective inhibitor peptide (3 mg/Kg/day), δV1-1, a δPKC-selective inhibitor peptide (3 mg/Kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/Kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3mg/Kg/day) between 11 weeks and 17 weeks. Treatment with εV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or δV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to εV1-2. Finally, εV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that εPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.
Mast cell degranulation; protein kinase C; PKC-selective inhibitor peptide; cardiac remodeling; heart failure
The signaling pathway of cyclooxygenase-2 (COX-2) induction following ischemic preconditioning (IPC) in brain remains undefined. To determine role of COX-2 in ischemic preconditioning, we used two in vitro models: mixed cortical neuron/astrocyte cell cultures and organotypic hippocampal slice cultures. We simulated IPC by exposing cell or slice cultures to 1 h or 15 min of oxygen/glucose deprivation (OGD), respectively, 48 h prior to ischemia. To mimic ischemia in vitro, we exposed cell or slice cultures to OGD of 4 h or 45 min, respectively. In cell cultures, these experiments revealed that COX-2 induction peaked at 24 h following IPC in cell culture. Inhibition of COX-2 activation with 50 μM NS-398 (a COX-2 selective inhibitor) abolished IPC-mediated neuroprotection in both in vitro models. Next, we tested whether εPKC and ERK1/2 activation were involved in IPC-mediated neuroprotection and COX-2 expression in cell culture. Cell cultures were treated with an εPKC-specific activating peptide (ψεRACK, 100 nM) for 1 h, and 48 h later were exposed to OGD. εPKC activation increased ERK1/2 phosphorylation and COX-2 induction and conferred neuroprotection similar to IPC. Additionally, inhibition of either εPKC or ERK1/2 activation abolished COX-2 expression and neuroprotection due to ischemic preconditioning. These results demonstrate a crucial role for the εPKC → ERK1/2 → COX-2 pathway in the induction of neuroprotection via ischemic preconditioning.
Neuroprotection; ERK1/2; mixed cortical neuron/astrocyte cell cultures; organotypic hippocampal slice cultures; ischemia; oxygen/glucose deprivation
Protein kinase C-ε (εPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the εPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP2) binding of εPKC and its correlation with the neurite extension. We found that full length εPKC bound to PIP2 in a 12-ο-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of εPKC (εRD) bound to PIP2 without any stimulation. To identify the PIP2 binding region, we made mutants lacking several regions from εRD, and examined their PIP2 binding activity. The mutants lacking variable region 1 (V1) bound to PIP2 stronger than intact εRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP2 binding ability of the V3-deleted mutant was weakened. Those PIP2 bindings of εPKC, εRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cδ and the V3 region of εPKC revealed that PIP2 binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, εPKC directly binds to PIP2 mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.
actin; neurite outgrowth; neuroblastoma; phosphatidylinositol 4,5-bisphosphate; protein kinase C
Cerebral ischemia causes cerebral blood flow (CBF) derangements resulting in neuronal damage by enhanced protein kinase C delta (δPKC) levels leading to hippocampal and cortical neuronal death after ischemia. Contrarily, activation of εPKC mediates ischemic tolerance by decreasing vascular tone providing neuroprotection. However, whether part of this protection is due to the role of differential isozymes of PKCs on CBF following cerebral ischemia remains poorly understood. Rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after two-vessel occlusion plus hypotension. In an asphyxial cardiac arrest (ACA) model, rats treated with δ V1-1 (pre- and postischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 and cortical neuronal death 7 days after ACA. On the contrary, εPKC-selective peptide activator, conferred neuroprotection in the CA1 region of the rat hippocampus 30 min before induction of global cerebral ischemia and decreased regional CBF during the reperfusion phase. These opposing effects of δ v. εPKC suggest a possible therapeutic potential by modulating CBF preventing neuronal damage after cerebral ischemia.
The signal transducers and activators of transcription (STATs) were found to be essential for cardioprotection. However, their role in preconditioning (PC) neuroprotection remains undefined. Previously, our studies showed that PC mediated a signaling cascade that involves activation of epsilon protein kinase C (εPKC), extracellular signal-regulated kinase (ERK1/2), and cyclooxygenase-2 (COX-2) pathways. However, the intermediate pathway by which ERK1/2 activates COX-2 was not defined. In this study, we investigated whether the PC-induced signaling pathway requires phosphorylation of STAT isoforms for COX-2 expression. To mimic PC or lethal ischemia, mixed cortical neuron/astrocyte cell cultures were subjected to 1 and/or 4 h of oxygen–glucose deprivation (OGD), respectively. The results indicated serine phosphorylation of STAT3 after PC or εPKC activation. Inhibition of either εPKC or ERK1/2 activation abolished PC-induced serine phosphorylation of STAT3. Additionally, inhibition of STAT3 prevented PC-induced COX-2 expression and neuroprotection against OGD. Therefore, our findings suggest that PC signaling cascade involves STAT3 activation after εPKC and ERK1/2 activation. Finally, we show that STAT3 activation mediates COX-2 expression and ischemic tolerance.
cerebral ischemia; extracellular signal-regulated kinase (ERK1/2); ischemic tolerance; neuroprotection; phosphorylation; protein kinase C
During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 minutes of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel, prophylactic agents to induce ischemic tolerance in patients at risk of stroke or cardiac arrest. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared to rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na+/K+-ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1–2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1–2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS.
brain ischemia; heart arrest; tolerance; neuroprotection
β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca2+-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways.
Methods and Results
DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p<0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p<0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca2+ release by 75% and peak [Ca2+]i by 20% respectively in isolated cardiomyocytes; the Ca2+ channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%.
β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, thus predisposing the mitochondria to opening of the MPT.
Adrenergic receptors; cardiomyopathy; mitochondria; signal transduction; protein kinases
Dopamine cellular signaling, via the D1 receptor (D1R), involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D1R-mediated inhibition of NADPH oxidase activity involves cross-talk between PKA and specific PKC isoform(s). In HEK-293 cells heterologously expressing human D1R (HEK-hD1), fenoldopam, a D1R agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D1R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two different PKA inhibitors, H89 (10 µmol/L) (88±8.1%) and Rp-cAMP (10 µmol/L) (97.7±6.7%), and two different PKC inhibitors, bisindolylmaleimide I (1 µmol/L) (94±6%) and staurosporine (10 nmol/L) (93±8%), which by themselves, had no effect (n=4–8/group). The inhibitory effect of PMA (1 µmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%) (n=5–6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθS676, an effect that was also blocked by H89. Fenoldopam (1 µmol/L) also increased PKCθS676 in HEK-hD1 and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross-talk between PKA and PKCθ plays an important role in the D1R-mediated negative regulation of NADPH oxidase activity in human kidney cells.
NADPH oxidase; protein kinase A; protein kinase C; D1 dopamin receptor
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31- 8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp- cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.
Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.