The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.
We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.
We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.
The cellular response to excessive endoplasmic reticulum (ER) stress includes the activation of signaling pathways, which lead to apoptotic cell death. Here we show that treatment of cultured cardiac myocytes with tunicamycin, an agent that induces ER stress, causes the rapid translocation of δPKC to the ER. We further demonstrate that inhibition of δPKC using the δPKC-specific antagonist peptide, δV1-1, reduces tunicamycin-induced apoptotic cell death, and inhibits expression of specific ER stress response markers such as CHOP, GRP78 and phosphorylation of JNK. The physiological importance of δPKC in this event is further supported by our findings that the ER stress response is also induced in hearts subjected to ischemia and reperfusion injury and that this response also involves δPKC translocation to the ER. We found that the levels of the ER chaperone, GRP78, the spliced XBP-1 and the phosphorylation of JNK are all increased following ischemia and reperfusion and that δPKC inhibition by δV1-1 blocks these events. Therefore, ischemia-reperfusion injury induces ER stress in the myocardium in a mechanism that requires δPKC activity. Taken together, our data show for the first time that δPKC activation plays a critical role in the ER stress-mediated response and the resultant cell death.
Cerebral ischemia causes blood flow derangements characterized by hyperemia (increased cerebral blood flow, CBF) and subsequent hypoperfusion (decreased CBF). We previously demonstrated that protein kinase C delta (δPKC) plays an important role in hippocampal neuronal death after ischemia. However, whether part of this protection is due to the role of δPKC on CBF following cerebral ischemia remains poorly understood. We hypothesized that δPKC exacerbates hyperemia and subsequent hypoperfusion resulting in CBF derangements following ischemia. Sprague-Dawley (SD) 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 2-vessel occlusion plus hypotension measured by 2-photon microscopy. In an asphyxial cardiac arrest model (ACA), SD rats treated with δV1-1 (pre- and post-ischemia) exhibited improved perfusion after 24 hrs and less hippocampal CA1 neuronal death 7 days after ACA. These results suggest possible therapeutic potential of δPKC in modulating CBF and neuronal damage after cerebral ischemia.
Protein Kinase C Delta; Asphyxial Cardiac Arrest; Neuroprotection; Two-vessel Occlusion; Two-photon Microscopy; Cerebral Ischemia
To improve myocardial flow during reperfusion after acute myocardial infarction and to elucidate the molecular and cellular basis that impedes it. According to the AHA/ACC recommendation, an ideal reperfusion treatment in patients with acute myocardial infarction (AMI) should not only focus on restoring flow in the occluded artery, but should aim to reduce microvascular damage to improve blood flow in the infarcted myocardium.
Transgenic mouse hearts expressing the δPKC (protein kinase C) inhibitor, δV1-1, in their myocytes only were treated with or without the δPKC inhibitor after ischemia in an ex vivo AMI model. δV1-1 or vehicle was also delivered at reperfusion in an in vivo porcine model of AMI. Microvascular dysfunction was assessed by physiological and histological measurements.
δPKC inhibition in the endothelial cells improved myocardial perfusion in the transgenic mice. In the porcine in vivo AMI model, coronary flow reserve (CFR), which is impaired for 6 days following infarction, was improved immediately following a one-minute treatment at the end of the ischemic period with the δPKC-selective inhibitor, δV1-1 (∼250 ng/Kg), and was completely corrected by 24 hrs. Myocardial contrast echocardiography, electron microscopy studies, and TUNEL staining demonstrated δPKC-mediated microvascular damage. δPKC-induced preconditioning, which also reduces infarct size by >60%, did not improve microvascular function.
These data suggest that δPKC activation in the microvasculature impairs blood flow in the infarcted tissue after restoring flow in the occluded artery and that AMI patients with no-reflow may therefore benefit from treatment with a δPKC inhibitor given in conjunction with removal of the coronary occlusion.
Previous studies demonstrate impairment of endothelial-dependent vasodilation after ischemia/reperfusion (I/R). Though we have demonstrated that inhibition of δ protein kinase C (δPKC) at reperfusion reduces myocyte damage and improves cardiac function in a porcine acute myocardial infarction (AMI) model, impact of the selective δPKC inhibitor on epicardial coronary endothelial function remains unknown.
Either δPKC inhibitor (δV1-1, n=5) or saline (n=5) was infused into the left anterior descending artery at the last 1 minute of the 30-minute ischemia by balloon occlusion. In vivo responses to bradykinin (endothelium-dependent vasodilator) or nitroglycerin (endothelium-independent vasodilator) were analyzed at 24 h after I/R using intravascular ultrasound. Vascular responses were calculated as the ratio of vessel area at each time point (30, 60, 90 and 120 seconds after the infusion), divided by values at baseline (before the infusion).
In control pigs, endothelial-dependent vasodilation following bradykinin infusion in infarct-related epicardial coronary artery was impaired, whereas in δPKC inhibitor treated-pigs the endothelial-dependent vasodilation was preserved. Nitroglycerin infusion caused similar vasodilatory responses in the both groups.
This is the first demonstration that a δPKC inhibitor preserves vasodilator capacity in epicardial coronary arteries in an in vivo porcine AMI model. Because endothelial dysfunction correlates with worse outcome in patients with AMI, this preserved endothelial function in epicardial coronary arteries might result in a better clinical outcome.
ultrasonography; angioplasty; myocardial infarction; protein kinases; endothelium
MEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2). Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA) leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126.
Methods and result
Rats were subjected to a 2-h middle cerebral artery occlusion (MCAO) followed by reperfusion for 48-h and the ischemic area was calculated. The expression of phosphorylated ERK1/2 and Elk-1, and of endothelin ETA and ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ETB receptor was analyzed by quantitative Western blot. We demonstrate that there is an increase in the number of contractile smooth muscle receptors in the MCA and in micro- vessels within the ischemic region. The enhanced expression occurs in the smooth muscle cells as verified by co-localization studies. This receptor upregulation is furthermore associated with enhanced expression of pERK1/2 and of transcription factor pElk-1 in the vascular smooth muscle cells. Blockade of transcription with the MEK1 inhibitor U0126, given at the onset of reperfusion or as late as 6 hours after the insult, reduced transcription (pERK1/2 and pElk-1), the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score.
Our results show that MCAO results in upregulation of cerebrovascular ETB, AT1 and 5-HT1B receptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced the enhanced vascular receptor expression and the associated cerebral infarction.
Hypertensive encephalopathy is a life-threatening condition due to elevation of cerebral perfusion pressure beyond the limits of autoregulation. Breakdown of the blood-brain barrier (BBB) leads to cerebral edema and reduced blood flow. In this issue of the JCI, Mochly-Rosen and colleagues demonstrate a novel molecular strategy for preserving the BBB in a model of hypertension-induced encephalopathy (see the related article beginning on page 173). Using a rationally designed peptide inhibitor of δPKC, they stabilized the BBB and improved mortality in hypertensive rats. This study highlights the therapeutic potential of δPKC inhibitors in hypertensive encephalopathy and provides incentive to elucidate δPKC signaling pathways that mediate BBB dysfunction in other disease states.
Although protein kinase C (PKC) plays a key role in ischemic preconditioning (IPC), the actual mechanism of that protection is unknown. We recently found that protection from IPC requires activation of adenosine receptors during early reperfusion. We, therefore, hypothesized PKC might act to increase the heart’s sensitivity to adenosine. IPC limited infarct size in isolated rabbit hearts subjected to 30-min regional ischemia/2-h reperfusion and IPC’s protection was blocked by the PKC inhibitor chelerythrine given during early reperfusion revealing involvement of PKC at reperfusion. Similarly chelerythrine infused in the early reperfusion period blocked the increased phosphorylation of the protective kinases Akt and ERK1/2 observed after IPC. Infusing phorbol 12-myristate 13-acetate (PMA), a PKC activator, during early reperfusion mimicked IPC’s protection. As expected, the protection triggered by PMA at reperfusion was blocked by chelerythrine, but surprisingly it was also blocked by MRS1754, an adenosine A2b receptor–selective antagonist, suggesting that PKC was somehow facilitating signaling from the A2b receptors. NECA [5′-(N-ethylcarboxamido) adenosine], a potent but not selective A2b receptor agonist, increased phosphorylation of Akt and ERK1/2 in a dose-dependent manner. Pretreating hearts with PMA or brief preconditioning ischemia had no effect on phosphorylation of Akt or ERK1/2 per se, but markedly lowered the threshold for NECA to induce their phosphorylation. BAY 60-6583, a highly selective A2b agonist, also caused phosphorylation of ERK 1/2 and Akt. MRS1754 prevented phosphorylation induced by BAY 60-6583. BAY 60-6583 limited infarct size when given to ischemic hearts at reperfusion. These results suggest that activation of cardiac A2b receptors at reperfusion is protective, but because of the very low affinity of the receptors endogenous cardiac adenosine is unable to trigger their signaling. We propose that the key protective event in IPC occurs when PKC increases the heart’s sensitivity to adenosine so that endogenous adenosine can activate A2b-dependent signaling.
adenosine A2b receptors; BAY 60-6583; NECA; preconditioning; protein kinase C
Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain.
A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period ("time window" as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126.
The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.
bone cancer pain; hyperalgesia; spinal cord; extracellular signal-regulated protein kinase (ERK); cAMP response element-binding protein (CREB); rat
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
We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C2-ceramide but not C2-dihydroceramide induced translocation of δPKC-GFP to the Golgi complex, while αPKC- and ζPKC-GFP did not respond to ceramide. The Golgi-associated δPKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where δPKC was translocated by ceramide. Gamma interferon also induced the δPKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg2+-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of δPKC-GFP to the Golgi complex but induces the continuous association and dissociation of δPKC with the Golgi complex. Ceramide inhibited the kinase activity of δPKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of δPKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated δPKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of δPKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg2+-dependent sphingomyelinase induced δPKC-specific translocation to the Golgi complex and that translocation results in δPKC activation through tyrosine phosphorylation of the enzyme.
We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained α-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.
Actin; Adhesion; Atherosclerosis; Cytoskeleton; Metalloproteinase; Remodeling
A zinc-induced signaling pathway leading to ERK1/2 activation and subsequent neuronal death has been investigated. We find that an extracellular zinc application stimulates biphasic phosphorylation of ERK1/2 and p38 MAPK in rat cultured neurons. The activation of ERK1/2, but not p38, is responsible for zinc neurotoxicity as only U0126, a MEK inhibitor that blocks ERK1/2 phosphorylation, significantly protects cortical neurons from zinc exposure. Overexpression of a dominant negative Ras mutant blocks zinc-induced Elk1-dependent gene expression in neurons, indicating the involvement of Ras activation in the zinc pathway leading to ERK phosphorylation and Elk1 signaling. We also find that zinc treatment results in neuronal mitochondrial hyperpolarization. Importantly, both U0126 and bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase (ANT), effectively reduce zinc-triggered mitochondrial changes. As bongkrekic acid also prevents zinc-triggered neuronal death but not ERK1/2 phosphorylation, activation of MAPK signaling precedes and is required for mitochondrial dysfunction and cell death. These results provide new insight on the mechanism of extracellular zinc-induced toxicity in which the regulation of mitochondrial function by the Ras/MEK/ERK pathway is closely associated with neuronal viability.
zinc; neurotoxicity; Ras; extracellular signal-regulated kinase 1/2; cortical neuron; mitochondria
Intercellular adhesion molecule-1 (ICAM-1) in cerebral vascular endothelium induced by ischemic insult triggers leukocyte infiltration and inflammatory reaction. We investigated the mechanism of hypothermic suppression of ICAM-1 in a model of focal cerebral ischemia. Rats underwent 2 hours of middle cerebral artery occlusion and were kept at 37°C or 33°C during occlusion and rewarmed to normal temperature immediately after reperfusion. Under hypothermic condition, robust activation of extracellular signal-regulated kinase-1/2 (ERK1/2) was observed in vascular endothelium of ischemic brain. Hypothermic suppression of ICAM-1 was reversed by ERK1/2 inhibition. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) in ischemic vessel was attenuated by hypothermia. STAT3 inhibitor suppressed ICAM-1 production induced by stroke. ERK1/2 inhibition enhanced phosphorylation and DNA binding activity of STAT3 in hypothermic condition. In this study, we demonstrated that hypothermic suppression of ICAM-1 induction is mediated by enhanced ERK1/2 activation and subsequent attenuation of STAT3 action.
Oxidative stress after cerebral ischemia and reperfusion activates extracellular signal-regulated kinases (ERK) in brain. However, the mechanism of this activation has not been elucidated. We have previously reported that in an in vitro model of oxidative stress in immature cortical neuronal cultures, the inhibition of ERK phosphatase activity contributes to ERK1/2 activation and subsequent neuronal toxicity. This study examined whether ERK activation was associated with altered activity of ERK phosphatases in a rat cardiac arrest model. Rats in experimental groups were subjected to asphyxial cardiac arrest for 8 min and then resuscitated for 30 min. Significant ERK activation was detected in both cortex and hippocampus following ischemia/reperfusion by immunoblotting. ERK phosphatase activity was reversibly inhibited in cerebral cortex but not affected in hippocampus following ischemia/reperfusion. MEK1/2 was activated in both cerebral cortex and hippocampus following ischemia/reperfusion. Using a specific inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), we have identified PP2A to be the major ERK phosphatase that is responsible for regulating ERK activation in ischemic brain tissues. Orthovanadate inhibited ERK phosphatase activity in brain tissues, suggesting that tyrosine phosphatases and dual specificity phosphatases may also contribute to the ERK phosphatase activity in brain tissues. Together, these data implicate ERK phosphatase in the regulation of ERK activation in distinct brain regions following global ischemia.
Ischemia/reperfusion; ERK; Phosphatase; Oxidative stress
Thiazolidinediones (TZDs) dramatically reduce the growth of human prostate cancer cells in vitro and in vivo. To determine whether the antitumor effects of TZDs were due in part to changes in the MEK/Erk signaling pathway, we examined the regulation of Erk phosphorylation by the TZD troglitazone within the PC-3 and C4-2 human prostate cancer cell lines. Western blot analysis revealed troglitazone-induced phosphorylation of Erk in both PC-3 and C4-2 cells. Troglitazone-induced increases in Erk phosphorylation were suppressed by the MEK inhibitor U0126 but not by the PPARγ antagonist GW9662. Pretreatment with U0126 did not alter the ability of troglitazone to regulate expression of two proteins that control cell cycle, p21, and c-Myc. Troglitazone was also still effective at reducing PC-3 proliferation in the presence of U0126. Therefore, our data suggest that troglitazone-induced Erk phosphorylation does not significantly contribute to the antiproliferative effect of troglitazone.
Hemoglobin breakdown produces an iron-dependent neuronal injury after experimental CNS hemorrhage that may be attenuated by heme oxygenase (HO) inhibitors. The HO enzymes are phosphoproteins that are activated by phosphorylation in vitro. While testing the effect of kinase inhibitors in cortical cell cultures, we observed that HO activity was consistently decreased by the MEK inhibitor U0126. The present study tested the hypothesis that MEK/ERK pathway inhibitors reduce HO activity and neuronal vulnerability to hemoglobin. The MEK inhibitors U0126 and SL327 and the ERK inhibitor FR180204 reduced baseline culture HO activity by 35–50%, without altering the activity of recombinant HO-1 or HO-2; negative control compounds U0124 and FR180289 had no effect. Hemoglobin exposure for 16 hours produced widespread neuronal injury, manifested by release of 59.2±7.8% of neuronal lactate dehydrogenase and a twelve-fold increase in malondialdehyde; kinase inhibitors were highly protective. HO-1 induction after hemoglobin treatment was also decreased by U0126, SL327, and FR180204. These results suggest that reduction in HO activity may contribute to the protective effect of MEK and ERK inhibitors against heme-mediated neuronal injury.
cell culture; free radical; hemoglobin toxicity; intracerebral hemorrhage; mouse; oxidative stress
Cerebral ischemia that develops after subarachnoid hemorrhage (SAH) carries high morbidity and mortality. Inflammatory mediators are involved in the development of cerebral ischemia through activation of the mitogen-activated protein kinase pathway. We hypothesized that blockade of the MAPkinase/ERK (MEK)/extracellular signal-regulated kinase (ERK) pathway upstream with a specific raf inhibitor would prevent SAH-induced activation of the cerebrovascular inflammatory response. The raf inhibitor SB-386023-b was injected intracisternally in our rat model at 0, 6, or 12 hours after the SAH. After 48 hours, cerebral arteries were harvested, and iNOS, interleukin (IL)-6, IL-1β, matrix metalloproteinase (MMP)-9, tissue inhibitors of metalloproteinase (TIMP)-1, and phosphorylated ERK1/2 were investigated by immunofluorescence, real-time polymerase chain reaction (PCR), and Western blot analysis. Cerebral blood flow (CBF) was measured using autoradiography. Protein levels of MMP-9, TIMP-1, iNOS, IL-6, and IL-1β were increased after SAH, as were mRNA levels of IL-6, MMP-9, and TIMP-1. After SAH, pERK1/2 was increased, but CBF was reduced. Treatment with SB-386023-b at 0 or 6 hours after SAH normalized CBF and prevented SAH-induced upregulation of MMPs, pro-inflammatory cytokines, and pERK1/2 proteins. These results suggested that inhibition of MEK/ERK signal transduction by a specific raf inhibitor administered up to 6 hours after SAH normalized the expression of pro-inflammatory mediators and extracellular matrix-related genes.
extracellular signal-regulated kinase 1/2 (ERK1/2); matrix metalloproteinase; pro-inflammatory cytokines; subarachnoid hemorrhage (SAH); tissue inhibitor of metalloproteinase 1 (TIMP-1)
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 extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.
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
The extracellular signal-regulated kinase (ERK) pathway, a critical mediator of cell proliferation, is activated in cerebral ischemia/reperfusion (I/R) injury and is therefore a key target in the treatment of ischemic stroke. Acupuncture has long been used in China to clinically treat stroke. However, the precise mechanism of its neuroprotective activities remains largely unknown. In the present study, a focal cerebral I/R-injured rat model was used to evaluate the in vivo therapeutic efficacy of electroacupuncture (EA) and investigate the underlying molecular mechanisms. EA significantly ameliorated neurological deficits and cerebral infarction in cerebral I/R-injured rats. Moreover, EA significantly increased the phosphorylation levels of ERK, as well as the protein expression levels of Ras, cyclin D1 and cyclin-dependent kinase (CDK)4. Consequently, EA-mediated activation of the ERK pathway resulted in the stimulation of cerebral cell proliferation. The present data suggest that EA at the Quchi and Zusanli acupoints exerts a neuroprotective effect in ischemic stroke via the activation of ERK signaling.
electroacupuncture; Quchi (LI11) and Zusanli (ST36) acupoints; cerebral ischemia/reperfusion; extracellular signal-regulated kinase pathway; cell proliferation
During infection, viruses hijack various host cell components and programs for their amplification, among which is the canonical ERK signaling pathway, mainly consisting of three tiered serine/threonine kinases, Raf, MEK and ERK. MEK1 and MEK2 are two isoforms of the kinase operating immediately upstream of ERK, and connecting Raf and ERK by phosphorylating ERK. Previous studies have suggested that different isoforms of MEK have distinct biological functions, although their in vitro kinase function may be redundant. However, little is known about the isoform-specific effects of these kinases on viral propagation. In this study, we showed that herpes simplex virus type 2 (HSV-2) infection of human embryonic kidney (HEK) 293 cells induced a sustained activation of ERK1/2. Inhibition of this ERK activation by U0126, a specific inhibitor of MEK1/2, severely impaired virus production. A similar reduction of virus production was also seen following transfection of cells with siRNAs for MEK1/2. Interestingly, a specific knockdown of MEK1 with siRNAs caused a marked inhibition of viral titers, viral proteins and virus-induced cytopathic effect (CPE), whereas silencing MEK2 had little effect. Therefore, our results demonstrate that MEK1 and MEK2 act differently and that HSV-2 hijacks host MEK1 for its own amplification. To our knowledge, this is the first report showing inhibition of HSV-2 replication by targeting human MEK1. This study also suggests that MEK1 could be a potential target for anti-HSV-2 therapy, which may minimize damage to the host cells engendered by targeting both MEK1 and MEK2.
HSV-2; Viral replication; MEK1; MEK2; ERK; siRNA
Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of δPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using δV1-1 — a selective peptide inhibitor of δPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or δV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the δV1-1–treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with δV1-1 was also associated with decreased δPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with δV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that δPKC may specifically act to dysregulate tight junction components. Together, these results suggest that δPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.
Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1).
Here, we found an infarct volume of 24.8 ± 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume significantly (11.8 ± 2% and 14.6 ± 3%, respectively; P < 0.05), improved neurological function, normalized expression of phosphorylated ERK1/2, and reduced expression of MMP-9 and TIMP-1 in the vessel walls. Administration of U0126 12 hours after MCAO did not alter the expression of MMP-9. Immunocytochemistry showed no overlap in expression between MMP-9/TIMP-1 and the astrocyte/glial cell marker GFAP in the vessel walls.
These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia, are transcriptionally regulated via the MEK/ERK pathway.