Activation Of Protein Kinase Cdelta Research Articles

Sepsis and Cerebral Dysfunction: BBB Damage, Neuroinflammation, Oxidative Stress, Apoptosis and Autophagy as Key Mediators and the Potential Therapeutic Approaches.

Sepsis-associated cerebral dysfunction is complex pathophysiology, generated from primary infections that are developed elsewhere in the body. The neonates, elderly population and chronically ill and long-term hospitalized patients are predominantly vulnerable to sepsis and related cerebral damage. Generally, electrophysiological recordings, severity and sedation scales, computerized imaging and spectroscopy techniques are used for its detection and diagnosis. About the underlying mechanisms, enhanced blood-brain barrier permeability and metalloprotease activity, tight junction protein loss and endothelial cell degeneration promote the influx of inflammatory and toxic mediators into the brain, triggering cerebrovascular damage. An altered neutrophil count and phenotype further dysregulate the normal neuroimmune responses and neuroendocrine stability via modulated activation of protein kinase C-delta, nuclear factor kappa-B and sphingolipid signaling. Glial activation, together with pro-inflammatory cytokines and chemokines and the Toll-like receptor, destabilize the immune system. Moreover, superoxides and hydroperoxides generate oxidative stress and perturb mitochondrial dynamics and ATP synthesis, propagating neuronal injury cycle. Activated mitochondrial apoptotic pathway, characterized by increased caspase-3 and caspase-9 cleavage and Bax/Bcl2 ratio in the hippocampal and cortical neurons, stimulate neurocognitive impairments. Additionally, altered LC3-II/I and P62/SQSTM1, p-mTOR, p-AMPK1 and p-ULK1 levels and dysregulated autophagosome-lysosome fusion decrease neuronal and glial energy homeostasis. The therapies and procedures for attenuating sepsis-induced brain damage include early resuscitation, cerebral blood flow autoregulation, implantable electric vagus nerve stimulation, antioxidants, statins, glucocorticoids, neuroimmune axis modulators and PKCδ inhibitors. The current review enumerates the pathophysiology of sepsis-induced brain damage, its diagnosis, the role of critical inducers and mediators and, ultimately, therapeutic measures attenuating cerebrovascular degeneration.

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

Paclitaxel Induces Apoptosis Through Activation of Nuclear Protein Kinase C-δ and Subsequent Activation of Golgi Associated Cdk1 in Human Hormone Refractory Prostate Cancer

Emerging evidence shows that the translocation of apoptosis related factors on cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus and nucleus, has a crucial role in the apoptotic process. We characterized the effect of paclitaxel (Sigma®) on Golgi involved apoptosis in human hormone refractory prostate cancer. FACScan™ flow cytometric analysis was used to determine cell cycle distribution and the subG1 (apoptosis) population. Protein expression and localization were detected by Western blot, confocal microscopic examination and the sucrose gradient separation technique. Paclitaxel induced Golgi apparatus disassembly and interaction between Golgi complexes and mitochondria. Discontinuous sucrose gradient fractionation was used to determine and collect Golgi containing fractions. Data revealed that paclitaxel induced an increase of Cdk1 activity and DR5 expression on the Golgi complex that was associated with increased cleavage of caspase-8, a DR5 downstream factor, and caspase-3 into catalytically active fragments. Data were validated by confocal immunofluorescence microscopy. Golgi associated effects were inhibited by the Cdk1 inhibitor roscovitine (Sigma), suggesting a critical role for Golgi-Cdk1. Also, paclitaxel caused an increase of nuclear but not of Golgi associated PKC-δ activity. The selective PKC-δ inhibitor rottlerin (Sigma) completely inhibited the increase of Golgi-Cdk1 activity, suggesting that nuclear PKC-δ served as an upstream regulator of Golgi-Cdk1. Data suggest that paclitaxel induces nuclear translocation and activation of PKC-δ, which in turn causes Golgi-Cdk1 activation, leading to Golgi associated DR5 up-regulation, and caspase-8 and 3 activation. Golgi mediated signaling cascades facilitate mitochondria involved apoptotic pathways and at least partly explain the anticancer activity of paclitaxel action.

Environmental neurotoxic pesticide dieldrin activates a non receptor tyrosine kinase to promote pkcδ-mediated dopaminergic apoptosis in a dopaminergic neuronal cell model

Oxidative stress and apoptosis are two key pathophysiological mechanisms underlying dopaminergic degeneration in Parkinson's disease (PD). Recently, we identified that proteolytic activation of protein kinase C-delta (PKCδ), a member of the novel PKC family, contributes to oxidative stress-induced dopaminergic degeneration and that phosphorylation of tyrosine residue 311 (tyr311) on PKCδ is a key event preceding the PKCδ proteolytic activation during oxidative damage. Herein, we report that a non-receptor tyrosine kinase Fyn is significantly expressed in a dopaminergic neuronal N27 cell model. Exposure of N27 cells to the dopaminergic toxicant dieldrin (60μM) rapidly activated Fyn kinase, PKCδ-tyr311 phosphorylation and proteolytic cleavage. Fyn kinase activation precedes the caspase-3-mediated proteolytic activation of PKCδ. Pre-treatment with p60-tyrosine-specific kinase inhibitor (TSKI) almost completely attenuated dieldrin-induced phosphorylation of PKCδ-tyr311 and its proteolytic activation. Additionally, TSKI almost completely blocked dieldrin-induced apoptotic cell death. To further confirm Fyn's role in the pro-apoptotic function of PKCδ, we adopted the RNAi approach. siRNA-mediated knockdown of Fyn kinase also effectively attenuated dieldrin-induced phosphorylation of PKCδ-tyr311, caspase-3-mediated PKCδ proteolytic cleavage, and DNA fragmentation, suggesting that Fyn kinase regulates the pro-apoptotic function of PKCδ. Collectively, these results demonstrate for the first time that Fyn kinase is a pro-apoptotic kinase that regulates upstream signaling of the PKCδ-mediated apoptotic cell death pathway in neurotoxicity models of pesticide exposure.

Inhibition of Protein Kinase C δ Attenuates Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

Vision loss in diabetic retinopathy is due to macular edema characterized by increased vascular permeability, which involves phosphorylation associated with activation of protein kinase C (PKC) isoforms. Herein, we demonstrated PKC delta inhibition could prevent blood-retinal barrier breakdown in diabetic retinopathy. Increased vascular permeability of diabetic retina was accompanied by a decrease of zonula occludens (ZO)-1 and ZO-2 expression. In diabetic retina and advanced glycation end product-treated human retinal microvascular endothelial cells, vascular leakage and loss of ZO-1 and ZO-2 on retinal vessels were effectively restored or prevented with treatment of rottlerin, transfection of PKC-delta-DN, or siRNA for PKC delta. Interestingly, PKC delta translocated from cytosol to membrane in advanced glycation end product-treated human retinal microvascular endothelial cells, which was blocked by PKC delta inhibition. Taken together, PKC delta activation, related to its subcellular translocation, is involved in vascular permeability in response to diabetes, and inhibition of PKC delta effectively restores loss of tight junction proteins in retinal vessels. Therefore, we suggest that inhibition of PKC delta could be an alternative treatment to blood-retinal barrier breakdown in diabetic retinopathy.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67 phox, one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67 phox membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.

Negative regulation of diacylglycerol kinase θ mediates adenosine-dependent hepatocyte preconditioning

In liver ischemic preconditioning (IP), stimulation of adenosine A2a receptors (A2aR) prevents ischemia/reperfusion injury by promoting diacylglycerol-mediated activation of protein kinase C (PKC). By concerting diacylglycerol to phosphatidic acid, diacylglycerol kinases (DGKs) act as terminator of diacylglycerol signalling. This study investigates the role of DGK in the development of hepatocyte IP. DGK activity and cell viability were evaluated in isolated rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by the treatment with the A2aR agonist, CGS21680, and subsequently exposed to prolonged hypoxia. We observed that after IP or A2aR activation, a decrease in DGK activity was associated with the onset of hepatocyte tolerance to hypoxia. CGS21680-induced stimulation of A2aR specifically inhibited DGK isoform theta by activating RhoA-GTPase. Consistently, both siRNA-mediated downregulation of DGK theta and hepatocyte pretreatment with the DGK inhibitor R59949 induced cell tolerance to hypoxia. The pharmacological inhibition of DGK was associated with the diacylglycerol-dependent activation of PKC delta and epsilon and of their downstream target p38 MAPK. In conclusion, we unveil a novel signalling pathway contributing to the onset of hepatocyte preconditioning, which through RhoA-GTPase, couples A2aR to the downregulation of DGK. Such an inhibition is essential for the sustained accumulation of diacylglycerol required for triggering PKC-mediated survival signals.

Protection of Neurons from Apoptosis by Apolipoprotein E-containing Lipoproteins Does Not Require Lipoprotein Uptake and Involves Activation of Phospholipase Cγ1 and Inhibition of Calcineurin

Apolipoprotein E-containing lipoproteins (LpE) are generated in the central nervous system by glial cells, primarily astrocytes, and are recognized as key players in lipid metabolism and transport in the brain. We previously reported that LpE protect retinal ganglion neurons from apoptosis induced by withdrawal of trophic additives (Hayashi, H., Campenot, R. B., Vance, D. E., and Vance, J. E. (2007) J. Neurosci. 27, 1933-1941). LpE bind to low density lipoprotein receptor-related protein-1 and initiate a signaling pathway that involves activation of protein kinase Cdelta and inhibition of the pro-apoptotic glycogen synthase kinase-3beta. We now show that uptake of LpE is not required for the neuroprotection. Experiments with inhibitors of phospholipase Cgamma1 and RNAi knockdown studies demonstrate that activation of phospholipase Cgamma1 is required for the anti-apoptotic signaling pathway induced by LpE. In addition, the protein phosphatase-2B, calcineurin, is involved in a neuronal death pathway induced by removal of trophic additives, and LpE inhibit calcineurin activation. LpE also attenuate neuronal death caused by oxidative stress. Moreover, physiologically relevant apoE3-containing lipoproteins generated by apoE3 knock-in mouse astrocytes more effectively protect neurons from apoptosis than do apoE4-containing lipoproteins. Because inheritance of the apoE4 allele is the strongest known genetic risk factor for Alzheimer disease, the reduced neuroprotection afforded by apoE4-containing LpE might contribute to the neurodegeneration characteristic of this disease.

Wnt5a Increases Cardiac Gene Expressions of Cultured Human Circulating Progenitor Cells via a PKC Delta Activation

BackgroundWnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC.Methodology/Principal FindingsImmunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca2+-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca2+-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC.Conclusions/SignificanceThese data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta.

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Endothelial PKCδ activation attenuates neutrophil transendothelial migration

The process of neutrophil extravasation is involved in acute and chronic inflammatory diseases; however our understanding of the role of endothelial second messengers in the regulation of leukocyte emigration is still incomplete. We investigated this using an in vitro model of neutrophil migration across human endothelial cells. Activation of endothelial protein kinase C (PKC) by either phorbol myristate acetate (PMA) or bryostatin-1 (a potent PKC delta and epsilon activator) completely abolished neutrophil migration mediated by either endothelial TNF-alpha stimulation or LTB4. Pretreatment with Gö-6983 (PKC alpha, beta, delta, inhibitor) prior to addition of bryostatin-1 restored LTB4 induced PMN migration, while pretreatment with Gö-6976 (PKC alpha and beta inhibitor) did not. PKC delta specific siRNA knockdown of PKC delta expression in endothelial cells also restored LTB4 induced PMN migration. In addition, PMA and bryostatin-1 both increased endothelial adhesion to the substratum that was also reversed using PKC delta siRNA knockdown of PKC delta expression. PMA and bryostatin-1 additionally altered the staining pattern of FAK[pY397], paxillin, and vinculin from a dot-like pattern to a dash-like pattern around the cell perimeter. While PMA reduced transendothelial resistance (TER), bryostatin-1 had no effect on TER. These observations show that endothelial PKC delta activation eliminates neutrophil transendothelial migration through a mechanism unrelated to endothelial barrier integrity. These data are consistent with PKC delta mediated increased cell substrate adhesion as a limiting factor for neutrophil transendothelial migration towards a chemoattractant.

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.

Protein kinase C promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha, delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation. PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC alpha-dependent positive feedback mechanism.

Cardiopulmonary Bypass with Cardioplegic Arrest Activates Protein Kinase C in the Human Myocardium

The protein kinase C (PKC) family consists of 12 isoforms, 6 of which have been found in human myocardium (PKC alpha, beta I/beta II, delta, epsilon, eta, and lambda/iota). These kinases function in regulation of contractility, ion channels, and in cellular protection or damage during ischemia-reperfusion injury. This study investigated the effects of controlled ischemia-reperfusion injury through cardioplegic arrest on PKC activity in patients undergoing cardiac surgery. Myocardium and skeletal muscle were harvested from patients before and after cardiopulmonary bypass and cardioplegic arrest. Total PKC (n=12) was isolated and specifically, the PKC delta (n=8) and PKC epsilon (n=8) isoforms were immunoprecipitated for use in functional kinase assays. Cellular fractions (n=4) were separated by differential ultracentrifugation and analyzed by Western blotting for membrane translocation (an indirect indicator of increased activity). Immunofluorescent staining for PKC delta and PKC epsilon was performed on myocardial sections. An in vitro assay of hypoxic cardioplegic arrest followed by reoxygenation was performed using isolated cardiomyocytes, and apoptosis was assessed. Cardioplegic arrest was associated with a 24.4% +/- 4.6% increase (p < 0.05) in total PKC activity, a 26.7% +/- 4.9% increase (p < 0.05) in PKC delta activity, and a 35.3% +/- 14% increase (p < 0.05) in PKC epsilon activity in myocardium. Cardioplegic arrest induced migration of PKC delta and epsilon to the z-line of the cardiomyocyte. Inhibition of PKC delta in the in vitro studies demonstrated a considerable reduction in apoptotic cells. PKC delta and epsilon have previously been shown to mediate and protect, respectively, from ischemia-reperfusion injury after myocardial ischemia. Demonstration of increases in their activity after cardioplegic arrest provides support for their possible role in myocardial function after cardiac surgery. Isoform-specific modulators may be of potential therapeutic value in treating postoperative myocardial dysfunction.

Subcellular Proteome Analysis of Camptothecin Analogue NSC606985-Treated Acute Myeloid Leukemic Cells

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase Cdelta. Here, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula, and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells by two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. In total, 90 unique deregulated proteins, including 16 compartment-compartment translocated ones, were identified. They contributed to multiple functional activities such as DNA damage repairing, chromosome assembly, mRNA processing, biosynthesis, modification, and degradation of proteins. More interestingly, several increased oxidative stress-related proteins mainly presented in mitochondria, while upregulated glycolysis proteins mainly occurred in the nuclei. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were discussed. Collectively, these discoveries would shed new insights for systematically understanding the mechanisms of the camptothecin-induced apoptosis.

Arsenic trioxide and proteasome inhibitor bortezomib synergistically induce apoptosis in leukemic cells: the role of protein kinase Cδ

Arsenic trioxide (ATO) and proteasome inhibitor bortezomib have been successfully applied to treat acute promyelocytic leukemia (APL) and multiple myeloma (MM), respectively. Their synergistic effects with other anticancer drugs have been widely studied. Here, we investigated the potential synergy of bortezomib and ATO on Bcr-Abl(+) leukemic K562 cells. The results showed that cotreatment of bortezomib at 32 nM, a half concentration for growth arrest, and ATO at 1 microM, a dose with no significant cytotoxic effect, synergistically induced apoptosis in the cell line, followed by enhanced mitochondrial dysfunction, release of cytochrome c and apoptosis-inducing factor, caspase-3 cleavage and degradation of poly-adenosine diphosphate-ribose polymerase together with the decreased Bcr-Abl protein. These two drugs synergistically induced proteolytic activation of protein kinase Cdelta (PKCdelta) with enhanced activation of two mitogen-activated protein kinases phospho-c-Jun NH(2)-terminal kinase and p38. The specific PKCdelta inhibitor rottlerin markedly decreased bortezomib plus ATO-induced apoptosis, suggesting that PKCdelta plays an important role in bortezomib plus ATO-induced apoptosis. Moreover, apoptosis synergy of bortezomib and ATO could also be seen in some kinds of acute leukemic cell lines and primary cells. Totally, our results indicate that combined regimen of bortezomib and ATO might be a potential therapeutic remedy for the treatment of leukemia.

Protein kinase Cδ‐mediated CREB activation regulates ghrelin‐induced cyclooxygenase‐2 expression and prostaglandin E2 production in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone release and appetite. Our recent study showed that ghrelin could stimulate protein kinase C-mediated activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 secretion in human colonic epithelial cells transfected with a functional ghrelin receptor. In the present study, the effect of ghrelin stimulation on cyclooxygenese-2 expression and prostaglandin E2 production was examined. The data indicate that ghrelin significantly increased the levels of cyclooxygenase-2 (COX-2) protein as well as its promoter activity, which leaded to profound increase in prostaglandin E2 secretion. In order to examine the involvement of NF-kappaB and cAMP responsive element-binding protein (CREB) in this response, the NF-kappaB inhibitory protein IkappaBalpha or a dominant negative mutant of CREB was co-transfected into cells and the data show that transfection of either IkappaBalpha or DN-CREB significantly attenuated ghrelin-induced COX-2 expression. Moreover ghrelin stimulated phosphorylation of CREB, which was mediated primarily via protein kinase Cdelta activation. Furthermore, inhibition of PKCdelta function significantly attenuated ghrelin-induced COX-2 expression. In addition, ghrelin stimulates phosphorylation of PKCdelta. Together, these results indicate that in addition to NF-kappaB, protein kinase Cdelta-mediated CREB activation plays an important role in the cellular responses of ghrelin.

Potent stimulation of large‐conductance Ca2+‐activated K+ channels by rottlerin, an inhibitor of protein kinase C‐δ, in pituitary tumor (GH3) cells and in cortical neuronal (HCN‐1A) cells

The effects of rottlerin, a known inhibitor of protein kinase C-delta activation, on ion currents were investigated in pituitary tumor (GH3) cells. Rottlerin (0.3-100 microM) increased the amplitude of Ca2+-activated K+ current (I K(Ca)) in a concentration-dependent manner with an EC50 value of 1.7 microM. In intracellular perfusion with rottlerin (1 microM) or staurosporine (10 microM), phorbol 12-myristate 13-acetate-induced inhibition of I K(Ca) in these cells was abolished. In cell-attached mode, rottlerin applied on the extracellular side of the membrane caused activation of large-conductance Ca2+-activated K+ (BK(Ca)) channels, and a further application of BAPTA-AM (10 microM) to the bath had no effect on rottlerin-stimulated channel activity. When cells were exposed to rottlerin, the activation curve of these channels was shifted to less positive potential with no change in the slope factor. Rottlerin increased BK(Ca)-channel activity in outside-out patches. Its change in kinetic behavior of BK(Ca) channels is primarily due to an increase in mean open time. With the aid of minimal kinetic scheme, a quantitative description of rottlerin stimulation on BK(Ca) channels in GH3 cells was also provided. Under current-clamp configuration, rottlerin (1 microM) decreased the firing of action potentials. I K(Ca) elicited by simulated action potential waveforms was enhanced by this compound. In human cortical HCN-1A cells, rottlerin (1 microM) could also interact with the BK(Ca) channel to stimulate I K(Ca). Therefore, rottlerin may directly activate BK(Ca) channels in neurons or endocrine cells.

Chronic-alcohol exposure alters IGF1 signaling in H9c2 cells via changes in PKC delta

Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western blot analyses of H9c2 protein preparations demonstrate that there are significant increases in the total protein levels of PKC delta and epsilon after 4 days exposure to alcohol, and similar increases were found after 2 and 6 days exposure. In addition, there was a significant increase in PKC delta and epsilon in the membranal fractions and a decrease in the cytosolic fractions. No change was found in the expression or activity levels for PKC alpha. Chronic-alcohol exposure (100 mM, 4 days) increased the basal tyrosine kinase activity of the IGF1 receptor (IGF1R), and altered its rate of activation. Chronic-alcohol exposure also reduced the rate of Erk1/Erk2 activation by IGF1. Chronic alcohol blocked the proliferative effects of IGF1 on cell growth and reduced cell viability both in the presence and absence of IGF1, and this alcohol-induced reduction in cell viability was blocked using siRNA to inhibit PKC delta. In addition, a reduction in the amount of myosin light chain 2 was found in the alcohol-exposed cells. In conclusion, chronic alcohol alters PKC delta and epsilon expression and activity, and suppresses the IGF1 signaling pathway in embryonic heart cell culture. Blockage of PKC delta expression using siRNA inhibits the suppressive effects of alcohol on cell viability.

IL-6 expression induced by adenosine A 2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A 1, A 2a, A 2b and A 3 adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA ( N6-( R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A 2b receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A 2b dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.