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
Protein kinase C (PKC) is a family of kinases that are critical in many cellular events. These enzymes are activated by lipid-derived second messengers, are dependent on binding to negatively charged phospholipids and some members also require calcium to attain full activation. The interaction with lipids and calcium activators is mediated by binding to the regulatory domains C1 and C2. In addition, many protein-protein interactions between PKC and other proteins have been described. These include interactions with adaptor proteins, substrates and cytoskeletal elements. Regulation of the interactions between PKC, small molecules and other proteins is essential for signal transduction to occur. Finally, a number of auto-inhibitory intramolecular protein-protein interactions have also been identified in PKC. This chapter focuses on mapping the sites for many of these inter and intramolecular interactions and how this information may be used to generate selective inhibitors and activators of PKC signaling.
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.
Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Whereas the earlier inhibitory peptides are highly specific, we believe that peptides targeting additional interactions between PKC and selective substrates will generate even more selective tools that regulate different functions of individual isozymes. Here, we discuss the methodologies and applications for identifying selective regulators of PKC.
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 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
Two pathways that have been shown to mediate cerebral ischemic damage are the MEK/ERK cascade and the pro-apoptotic δPKC pathway. We investigated the relationship between these pathways in a rat model of focal ischemia by observing and modifying the activation state of each pathway. The ERK1/2 inhibitor, U0126, injected at ischemia onset, attenuated the increase in phosphorylated ERK1/2 (P-ERK1/2) after reperfusion. The δPKC inhibitor, δV1-1, delivered at reperfusion, did not significantly change P-ERK1/2 levels. In contrast, the δPKC activator, ψδRACK, injected at reperfusion, reduced ERK1/2 phosphorylation measured 4 h after reperfusion. Additionally, U0126 pretreatment at ischemia onset reduced infarct size compared with vehicle, but U0126 injected at the onset of reperfusion had no protection. Finally, combination of U0126 injection at ischemia onset plus δV1-1 injection at reperfusion further reduced infarct size, while combination of U0126 delivered at ischemia onset with ψδRACK injected at reperfusion increased infarct size compared with U0126 alone. In conclusion, we find that inhibiting both the MEK/ERK and the δPKC pathways offers greater protection than either alone, indicating they likely act independently.
Cerebral ischemia; MEK/ERK cascade; δPKC; ERK1/2
There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here an unbiased proteomic search identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats prior to an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wildtype or mutant ALDH2 subjected to cardiac ischemia, such as during coronary bypass surgery. (140/140 words)
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
Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCβII was activated during increased angiogenesis and that PKCβII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCβII-selective inhibitor, βIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCβII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCβII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be upregulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is upregulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCβII inhibitor such as βIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.
Previously we found that neural responses to ethanol and the dopamine D2 receptor (D2) agonist NPA involve both epsilon protein kinase C (εPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of εPKC and the relationship to PKA activation. In the present study, we used a new εPKC antibody, 14E6, that selectively recognizes active εPKC when not bound to its anchoring protein εRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators, to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated εPKC and also induced translocation of both εPKC and its anchoring protein, εRACK to a new cytosolic site. The selective εPKC agonist, pseudo-εRACK, activated εPKC but did not cause translocation of the εPKC/εRACK complex to the cytosol. These data suggest a step-wise activation and translocation of εPKC following NPA or ethanol treatment where εPKC first translocates and binds to its RACK and subsequently the εPKC/εRACK complex translocates to a new subcellular site. Direct activation of PKA by Sp-cAMPS, PGE1 or the adenosine A2A receptor is sufficient to cause εPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between εPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.
Heart failure (HF) afflicts about 5 million people and causes 300 000 deaths a year in the United States alone. An integral part of the pathogenesis of HF is cardiac remodelling, and the signalling events that regulate it are a subject of intense research. Cardiac remodelling is the sum of responses of the heart to causes of HF, such as ischaemia, myocardial infarction, volume and pressure overload, infection, inflammation, and mechanical injury. These responses, including cardiomyocyte hypertrophy, myocardial fibrosis, and inflammation, involve numerous cellular and structural changes and ultimately result in a progressive decline in cardiac performance. Pharmacological and genetic manipulation of cultured heart cells and animal models of HF and the analysis of cardiac samples from patients with HF are all used to identify the molecular and cellular mechanisms leading to the disease. Protein kinase C (PKC) isozymes, a family of serine–threonine protein kinase enzymes, were found to regulate a number of cardiac responses, including those associated with HF. In this review, we describe the PKC isozymes that play critical roles in specific aspects of cardiac remodelling and dysfunction in HF.
Protein kinase C; Heart failure; Cardiac remodeling; Hypertrophy; Fibrosis and inflammation
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.
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
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.
Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.
Src; RACK1; PKC; tyrosine kinases; G1/S transition; cell cycle regulation
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.
Protein kinase C (PKC) family members have been implicated in numerous cellular processes. However, identifying the substrates of each PKC isozyme remains a challenge. Here, we describe a method using two dimensional (2-D) isoelectric focusing gel electrophoresis to identify substrates of delta PKC (δPKC) in MCF-7 breast carcinoma cells. We show that M2 pyruvate kinase is a substrate of δPKC, and further characterize the interaction between M2 pyruvate kinase and δPKC in MCF-7 cells by immunoprecipitation. δPKC activation in vitro or in cells did not appear to alter the enzyme activity or polymerization of M2 pyruvate kinase.
Protein Kinase C; Signal transduction; M2-type pyruvate kinase; Heat shock protein 27
The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D1 agonist, fenoldopam, but not with the dopaminergic D2 agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.