PKCδ Inhibitor Rottlerin Research Articles

Ginsenoside Re blocks Bay k-8644-induced neurotoxicity via attenuating mitochondrial dysfunction and PKCδ activation in the hippocampus of mice: Involvement of antioxidant potential.

Although the anticonvulsant effects of ginsenosides are recognized, little is known about their effects on the convulsive behaviors induced by the activation of L-type Ca2+ channels. Here, we investigated whether ginsenoside Re (GRe) modulates excitotoxicity induced by the L-type Ca2+ channel activator Bay k-8644. GRe significantly attenuated Bay k-8644-induced convulsive behaviors and hippocampal oxidative stress in mice. GRe-mediated antioxidant potential was more pronounced in the mitochondrial fraction than cytosolic fraction. As L-type Ca2+ channels are thought to be targets of protein kinase C (PKC), we investigated the role of PKC under excitotoxic conditions. GRe attenuated Bay k-8644-induced mitochondrial dysfunction, PKCδ activation, and neuronal loss. The PKCδ inhibition and neuroprotection mediated by GRe were comparable to those by the ROS inhibitor N-acetylcysteine, the mitochondrial protectant cyclosporin A, the microglial inhibitor minocycline, or the PKCδ inhibitor rottlerin. Consistently, the GRe-mediated PKCδ inhibition and neuroprotection were counteracted by the mitochondrial toxin 3-nitropropionic acid or the PKC activator bryostatin-1. GRe treatment did not have additional effects on PKCδ gene knockout-mediated neuroprotection, suggesting that PKCδ is a molecular target of GRe. Collectively, our results suggest that GRe-mediated anticonvulsive/neuroprotective effects require the attenuation of mitochondrial dysfunction and altered redox status and inactivation of PKCδ.

The role of reactive oxygen species and redoxsensitive protein kinases in the infarction-limiting effect of opioid peptide deltorphin II in cardiac reperfusion in rats

Background. Mortality from acute myocardial infarction with ST-segment elevation in cardiac hospitals ranges from 4.5 to 7 %, and these data has not decreased in recent years. The most common cause of death in patients is cardiogenic shock, the likelihood of which directly depends on infarct size. It is quite clear that there is an urgent need to create drugs to limit the size of infarction and prevent the occurrence of cardiogenic shock.The aim. To evaluate the role of reactive oxygen species and redox-sensitive protein kinases in the infarction-limiting effect of opioid peptide deltorphin II in cardiac reperfusion in rats.Materials and methods. Coronary occlusion (45 min) and reperfusion (120 min) were performed in rats anesthetized with α-chloralose. The selective δ2-opioid receptor agonist deltorphin II, a hydroxyl radical scavenger 2-mercaptoprpionyl glycine (2-MPG), a superoxide radical scavenger tempol, the protein kinase Cδ (PKCδ) inhibitor rottlerin, the PI3-kinase inhibitor wortmannin, the inhibitor of ERK1/2 kinase PD98059 were injected before of reperfusion of the heart.Results. Deltorphin II contributed to a two-fold decrease in infarction size. Injection of 2-MPG, tempol, rottlerin, wortmannin, PD98059 alone had no effect on infarction size in rats. 2-MPG and tempol did not affect the infarction-reducing effect of deltorphin II. Rottlerin, wortmannin, and PD98059 eliminated the cardioprotective effect of deltorphin II.Conclusion. The infarction-reducing effect of deltorphin II does not depend on the production of superoxide radical and hydroxyl radical. Superoxide radical and hydroxyl radical do not play a significant role in reperfusion injury of the heart after coronary occlusion (45 min). PKCδ, PI3-kinase, and ERK1/2 kinase are involved in the infarction-limiting effect of deltorphin II in myocardial reperfusion.

Interaction between adenosine diphosphate receptors and protein-kinase C isoforms in platelet adhesion under flow condition

Adenosine diphosphate (ADP) receptors and protein-kinase C (PKC) isoforms play different role in platelet activity. In the present study, whole blood platelet adhesion at 200 - 1800 s-1 shear rates was investigated by Impact-R system, measuring percent of surface coverage (SC) by platelets. Gradual heightened shear rate par-alleled increase of platelet adhesion. At relatively low shear (200 and 1000 s-1) blockade of neither P2Y1 receptor nor P2Y12 receptor (by A2P5P and 2MeSAMP, respectively) affected SC. At high shear rate (1800 s-1) reduction of SC was observed by 2MeSAMP. Treatment of blood with PKCδ inhibitor (rottlerin) but not PKCα,β inhibitor (Gö6976) diminished platelet adhe-sion. Among all the agents, only combination of 2MeSAMP and rottlerin used at subthreshold concentrations was able to inhibit platelet adhesion under high shear condition. We suggest that platelet agonist-induced P2Y12 and PKCδ signaling essentially stimulates platelet adhesion under flow condition, the important initiating step of thrombin formation.

Ginsenoside Re attenuates 8-OH-DPAT-induced serotonergic behaviors in mice via interactive modulation between PKCδ gene and Nrf2

It has been recognized that serotonergic blocker showed serious side effects, and that ginsenoside modulated serotonergic system with the safety. However, the effects of ginsenoside on serotonergic impairments remain to be clarified. Thus, we investigated ginsenoside Re (GRe), a major bioactive component in the mountain-cultivated ginseng on (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT), a 5-HT1A receptor agonist. In the present study, we observed that the treatment with GRe resulted in significant inhibition of protein kinase C δ (PKCδ) phosphorylation induced by the 5-HT1A receptor agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) in the hypothalamus of the wild-type (WT) mice. The inhibition of GRe was comparable with that of the PKCδ inhibitor rottlerin or the 5-HT1A receptor antagonist WAY100635 (WAY). 8-OH-DPAT-induced significant reduction in nuclear factor erythroid-2-related factor 2 (Nrf2)-related system (i.e., Nrf2 DNA binding activity, γ-glutamylcysteine ligase modifier (GCLm) and γ-glutamylcysteine ligase catalytic (GCLc) mRNA expression, and glutathione (GSH)/oxidized glutathione (GSSG) ratio) was significantly attenuated by GRe, rottlerin, or WAY in WT mice. However, PKCδ gene knockout significantly protected the Nrf2-dependent system from 8-OH-DPAT insult in mice. Increases in 5-hydroxytryptophan (5-HT) turnover rate, overall serotonergic behavioral score, and hypothermia induced by 8-OH-DPAT were significantly attenuated by GRe, rottlerin, or WAY in WT mice. Consistently, PKCδ gene knockout significantly attenuated these parameters in mice. However, GRe or WAY did not provide any additional positive effects on the serotonergic protective potential mediated by PKCδ gene knockout in mice. Therefore, our results suggest that PKCδ is an important mediator for GRe-mediated protective activity against serotonergic impairments/oxidative burden caused by the 5-HT1A receptor.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Ouabain inhibitor rostafuroxin attenuates dextromethorphan-induced manic potential

Dextromethorphan (DM) abuse produces mania-like symptoms in humans. ERK/Akt signaling activation involved in manic potential can be attenuated by the inhibition of ouabain-like cardiac steroids. In this study, increased phosphorylations of ERK/Akt and hyperlocomotion induced by DM (30 mg/kg, i.p./day × 7) were significantly protected by the ouabain inhibitor rostafuroxin (ROSTA), suggesting that DM induces the manic potential. ROSTA significantly attenuated DM-induced protein kinase C δ (PKCδ) phosphorylation, GluN2B (i.e., MDA receptor subunit) expression, and phospho-PKCδ/GluN2B interaction. DM instantly upregulated the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent system. However, DM reduced Nrf2 nuclear translocation, Nrf2 DNA binding activity, γ-glutamylcysteine mRNA expression, and subsequent GSH/GSSG level and enhanced oxidative parameters following 1-h of administration. ROSTA, PKCδ inhibitor rottlerin, and GluN2B inhibitor traxoprodil significantly attenuated DM-induced alterations in Nrf2-related redox parameters and locomotor activity induced by DM in wild-type mice. Importantly, in PKCδ knockout mice, DM failed to alter the above parameters. Further, ROSTA and traxoprodil also failed to enhance PKCδ depletion effect, suggesting that PKCδ is a critical target for the anti-manic potential of ROSTA or GluN2B antagonism. Our results suggest that ROSTA inhibits DM-induced manic potential by attenuating ERK/Akt activation, GluN2B/PKCδ signalings, and Nrf2-dependent system.

Signalling pathway of U46619-induced vascular smooth muscle contraction in mouse coronary artery.

Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. A multi myograph system was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration-dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but was not blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1μmol/L nifedipine, then mostly inhibited by 100μmol/L 2-APB and 10μmol/L Y27632. We further found that the response to U46619 was inhibited, respectively, by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels did not contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signalling pathway in TP downstream proteins, while SOC channels did not participate in the vasoconstriction.

Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells.

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

PKCδ Mediates Mineralocorticoid Receptor Activation by Angiotensin II to Modulate Smooth Muscle Cell Function

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10μmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50μmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10μmol/L, and a selective PKCδ inhibitor rottlerin at 10μmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10μmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100μmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1μmol/L and 2-APB at 50 and 100μmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100μmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells.

Hypoxia-inducible factor 1 (HIF-1) plays important roles in cancer cell biology. HIF-1α is reportedly activated by several factors, including protein kinase C (PKC), in addition to hypoxia. We investigated the role of PKC isoforms and the effects of vitamin K2 (VK2) in the activation process of HIF-1α. Human hepatocellular carcinoma (HCC)-derived Huh7 cells were cultured under normoxic and hypoxic (1% O2) conditions with or without the PKC stimulator TPA. The expression, transcriptional activity and nuclear translocation of HIF-1α were examined under treatment with PKC inhibitors, siRNAs against each PKC isoform and VK2. Hypoxia increased the expression and activity of HIF-1α. TPA increased the HIF-1α activity several times under both normoxic and hypoxic conditions. PKC-δ siRNA-mediated knockdown, PKC-δ inhibitor (rottlerin) and pan-PKC inhibitor (Ro-31-8425) suppressed the expression and transcriptional activity of HIF-1α. VK2 significantly inhibited the TPA-induced HIF-1α transcriptional activity and suppressed the expression and nuclear translocation of HIF-1α induced by TPA without altering the HIF-1α mRNA levels. These data indicate that PKC-δ enhances the HIF-1α transcriptional activity by increasing the nuclear translocation, and that VK2 might suppress the HIF-1α activation through the inhibition of PKC in HCC cells.

Berberine alleviates hepatic lipid accumulation by increasing ABCA1 through the protein kinase C δ pathway

Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH). The ATP-binding cassette transporter A1 (ABCA1) protein mediates the transport of cholesterol and phospholipids from cells to apolipoprotein A-I (apoA-I) to generate nascent HDL particles. Previous studies revealed that the overexpression of ABCA1 alleviated hepatic lipid levels by modifying lipid transport. Here, we examined the effect of berberine (BBR) on ABCA1 in QSG-7701 hepatocytes and in mice. BBR decreased hepatic cholesterol and triglyceride levels. It also increased ABCA1 protein levels but not mRNA levels in a time- and dose-dependent manner. The PKCδ inhibitor rottlerin and PKCδ siRNA completely abolished the effect of BBR on ABCA1. BBR also decreased the phosphorylation of ABCA1 serine residues and PKCδ Tyr 311. The inhibition of ABCA1 with its siRNA abolished the reduction in cellular cholesterol levels by BBR. BBR administration to mice fed a methionine choline-deficient diet also significantly increased ABCA1 protein levels and reduced hepatic lipid levels. These results suggest that BBR can reduce steatosis by increasing ABCA1 protein levels through PKCδ to reduce the phosphorylation of serine residues in ABCA1.

Protein kinase C δ-dependent regulation of Ubiquitin-proteasome system function in breast cancer.

Besides the crucial role of hyperinsulinemia in the development of breast cancer with Type 2 diabetes mellitus (T2DM), it has been shown that hyperglycemia could contribute to promote cancer progression. A remarkable association within hyperglycemia, PKCδ and Ubiquitin-proteasome system (UPS) has been reported, suggesting that PKCδ may mediate high glucose-induced UPS activation in breast cancer cells. Although the independent effects of PKCδ or UPS on breast cancer and T2DM are increasingly supported by experimental evidence, the complex interactional link between PKCδ and UPS is still unclear. Hence, we focus on the relationship between PKCδ and UPS in breast cancer with T2DM. We hypothesize that PKCδ may have the function to regulate the activity of UPS. Further, we speculate that PKCδ combine with proteasome α2 promoter, that indicate PKCδ regulate the function of UPS by change the composition of proteasome. Therefore, we surmise that PKCδ mediated high glucose-induced UPS activation in breast cancer cells, and specific PKCδ inhibitor rottlerin significantly suppressed elevated glucose induced the activity of UPS. We hope that our paper will stimulate further studies the relationship between PKCδ and UPS, and a new targeted therapy and early medical intervention for PKCδ could be a useful option for breast cancer cases complicated with T2DM or hyperglycemia.

Relaxin Inhibits Cardiac Fibrosis in Diabetic Rats: Roles of Protein Kinase Cδ.

Relaxin (Rlx) is known to antagonize diabetic cardiac fibrosis. However, its mechanism is poorly understood. Protein kinase Cδ (PKCδ) plays a crucial role in diabetic cardiomyopathy (DCM). This study explored the involvement of PKCδ in Rlx's capacity of suppressing cardiac fibrosis in a rat model of type 2 diabetes mellitus (DM). Type 2 DM of 8-week-old male Sprague-Dawley (SD) rats was induced by a high-fat diet and the injection of streptozocin (STZ, 40 mg/kg). Fourteen-week-old rats with DM and rats in control group which were pre-treated for 1 week with human recombinant relaxin (rhRlx, 30 μg/kg.d), were assessed to detect cardiac fibrosis and PKCδ expression with Western blot. Cardiac fibroblasts of neonatal rats were treated for 72 h with rhRlx (100 ng/ml) under high glucose (HG). Western blot was utilized for detecting the membranous and cytoplasmic protein expressions of PKCδ. The effects of rhRlx and PKCδ inhibitor (rottlerin) were assessed either alone or in combination on the HG-induced proliferation and differentiation of cardiac fibroblasts and the release of collagen I.Rlx treatment inhibited the differentiation of cardiac fibroblasts and the expression of collagen I. The expression of PKCδ was regulated by Rlx in diabetic rats and cardiac fibroblasts under HG condition. The effects of Rlx upon the proliferation and differentiation of cardiac fibroblasts and the excretion of collagen I under HG were blunted by rottlerin. Rlx suppressed cardiac fibrosis in type 2 diabetic rats. This beneficial effect was associated with its ability of modulating the expression of PKCδ.

Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells.

Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression.

Duodenal GLP-1 signaling regulates hepatic glucose production through a PKC-\u03b4-dependent neurocircuitry

Intestinal glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion and acts as a neuropeptide to control glucose homeostasis, but little is known whether intestinal GLP-1 has any effect in the control of hepatic glucose production (HGP). Here we found that intraduodenal infusion of GLP-1 activated duodenal PKC-δ, lowered HGP and was accompanied by a decrease in hepatic expression of gluconeogenic enzymes and an increase in hepatic insulin signaling in rats. However, gut co-infusion of either the GLP-1 receptor antagonist Ex-9, or the PKC-δ inhibitor rottlerin with GLP-1, negated the ability of gut GLP-1 to lower HGP and to increase hepatic insulin signaling during clamps. The metabolic and molecular signal effects of duodenal GLP-1 were also negated by co-infusion with tetracaine, pharmacologic inhibition of N-methyl-d-aspartate receptors within the dorsalvagal complex, or hepatic vagotomy in rats. In summary, we identified a neural glucoregulatory function of gut GLP-1 signaling.

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity.

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.

Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay.

An assay for protein kinase C delta (PKCδ) activity based on the quantification of a synthetic substrate using capillary electrophoresis with laser-induced fluorescence detection was developed. The peptides labeled with fluorescein isothiocyanate F-ERK (where ERK is extracellular signal-regulated kinase) and the phosphorylated form, P-F-ERK, were utilized for the method development and validation. The migration time of F-ERK and P-F-ERK were 6.3 ± 0.1 and 8.7 ± 0.2 min, respectively. LOD and LOQ values of F-ERK were 2 and 6 ng/mL and those of P-F-ERK were 4 and 12 ng/mL. The correlation coefficients obtained from two standard curves were approximately 0.99. The reproducibility and accuracy of the method for F-ERK ranged 1.5-4.7 and 86-109%, respectively, and those for P-F-ERK were 1.6-6.1 and 93-109%, respectively. The activity of PKCδ was studied in vitro using the human gastric cancer cell line MKN-1. The use of PKCδ inhibitor candidates, including Gӧ6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC50 values of 50 nM, 15 nM, 795 nM, and 4 μM, respectively. Comparison of our assay with a commercial PKC kit revealed that our assay is more adaptable to differing enzyme isoforms. This method has potential for high throughput screening for kinase inhibitors as part of a drug discovery program.

Synergistic protective effect of paeoniflorin and β-ecdysterone against rotenone-induced neurotoxicity in PC12 cells.

There are several factors, like oxidative stress and neurons loss, involving neurodegenerative diseases such as Parkinson's disease (PD). The combination of antioxidant and anti-apoptotic agent is becoming a promising approach to fight against PD. This study evaluates the hypothesis that paeoniflorin (PF) and β-ecdysterone (β-Ecd) synergize to protect PC12 cells against toxicity induced by PD-related neurotoxin rotenone. The combination of PF and β-Ecd, hereafter referred to as the PF/β-Ecd, at suboptimal concentrations increased the viability of rotenone-exposed PC12 cells in a synergistic manner. PF and β-Ecd cooperate to attenuate the rotenone-induced apoptosis by decrease in Bax expression, caspase-9 activity, and caspase-3 activity. PF or PF/β-Ecd, but not β-Ecd, inhibited rotenone-triggered protein kinase C-δkinase C-δ (PKCδ) upregulation and nuclear factor κB (NF-κB) activation. β-Ecd or PF/β-Ecd, but not PF, enhanced serine/threonine protein kinase (Akt) activation, promoted nuclear factor E2-related factor 2 (Nrf2) nuclear accumulation, suppressed reactive oxygen species (ROS) production. Neuroprotection of PF/β-Ecd could be completely blocked by PKCδ inhibitor rottlerin plus Akt specific inhibitor LY294002. Dual blockade of the PKCδ/NF-κB pathway by PF and activation of Akt/Nrf2 pathway by β-Ecd results in a synergistic neuroprotective effect against rotenone-induced neurotoxicity in vitro. These findings provide the rationale for determining the in vivo activity of combined therapy with PF and β-Ecd against PD.

Abstract 1868: Glycodelin-producing endometrial adenocarcinoma cells are resistant to TGFβ

Abstract Glycodelin is a secreted glycoprotein belonging to lipocalin protein family. It is expressed mainly in reproductive and glandular tissues. Glycodlin is involved in the regulation of reproductive and immune systems and it drives epithelial differentiation. In endometrium, the expression of glycodelin is abundant in secretory phase of the menstrual cycle, while in proliferative phase it is not expressed. Most studies agree that glycodelin is not expressed in endometrial cancer. To study the effects of glycodelin in endometrial cancer cells, we have made stable glycodelin-producing HEC-1B endometrial adenocarcinoma cells. Previously, we have shown that glycodelin differentiated HEC-1B cells towards less malignant phenotype and the cells formed significantly smaller tumors in preclinical xenograft mice. In addition, glycodelin-producing cells were resistant to phorbol ester-stimulated migration and phenotypic differentiation, which was mediated by repressed activation of PKCδ. Here we studied the effect of TGFβ on glycodelin-induced differentiation of the HEC-1B cells. Glycodelin-producing and control cells were plated on Matrigel and grown with or without TGFβ. The media was changed every second day and the cells were monitored up to one week. For Western blot analysis the cells were grown on Matrigel and TGFβ was added for 0-2h, after which the cells were detached from Matrigel and lysed. TGFβ induced control cells to form net-like structures, similar to those found after phorbol ester exposure. However, the glycodelin-producing cells remained unresponsive. PKCδ siRNA and PKCδ inhibitor Rottlerin inhibited the effects of TGFβ. Western blot analysis showed equal phosphorylation levels of SMAD2 in glycodelin-transfected and control cells. In contrast, PKCδ and ERK1/2 were differently phosphorylated, indicating the involvement of MAPK-pathway in the TGFβ-induced changes of the control cells. Endometrial cancer is the most common cancer of the female reproductive tract in developed countries. However, the molecular mechanisms affecting the pathogenesis of the disease are still poorly understood. It has been suggested that TGFβ is involved in carcinogenesis of endometrial cancer. Therefore, our results showing that glycodelin-producing HEC-1B endometrial adenocarcinoma cells are resistant to the effects of TGFβ, due to the repressed PKCδ phosphorylation, may be important to understand the pathogenic mechanisms of endometrial cancer. Citation Format: Laura Hautala, Hannu Koistinen. Glycodelin-producing endometrial adenocarcinoma cells are resistant to TGFβ. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1868.