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PKC Activation (pkc + activation)
Selected AbstractsSearching for Disease Modifiers,PKC Activation and HDAC Inhibition,A Dual Drug Approach to Alzheimer's Disease that Decreases A, Production while Blocking Oxidative StressCHEMMEDCHEM, Issue 7 2009Abstract A series of benzolactam compounds were synthesized, some of which caused a concentration-dependent increase in sAPP, and decrease in A, production in the concentration range of 0.1,10,,M. Moreover, some compounds showed neuroprotective effects in the 10,20,,M range in the HCA cortical neuron model of oxidative stress and no toxicity in measurements of neuron viability by MTT assay, even at the highest concentrations tested (20,,M). Alzheimer's disease (AD) is a well-studied neurodegenerative process characterized by the presence of amyloid plaques and neurofibrillary tangles. In this study, a series of protein kinase,C (PKC) activators were investigated, some of which also exhibit histone deacetylase (HDAC) inhibitory activity, under the hypothesis that such compounds might provide a new path forward in the discovery of drugs for the treatment of AD. The PKC-activating properties of these drugs were expected to enhance the ,-secretase pathway in the processing of amyloid precursor protein (APP), while their HDAC inhibition was anticipated to confer neuroprotective activity. We found that benzolactams 9 and 11,14 caused a concentration-dependent increase in sAPP, and decrease in ,-amyloid (A,) production in the concentration range of 0.1,10,,M, consistent with a shift of APP metabolism toward the ,-secretase-processing pathway. Moreover, compounds 9,14 showed neuroprotective effects in the 10,20,,M range in the homocysteate (HCA) cortical neuron model of oxidative stress. In parallel, we found that the most neuroprotective compounds caused increased levels of histone acetylation (H4), thus indicating their likely ability to inhibit HDAC activity. As the majority of the compounds studied also show nanomolar binding affinities for PKC, we conclude that it is possible to design, de,novo, agents that combine both PKC-activating properties along with HDAC inhibitory properties. Such agents would be capable of modulating amyloid processing while showing neuroprotection. These findings may offer a new approach to therapies that exhibit disease-modifying effects, as opposed to symptomatic relief, in the treatment of AD. [source] Protein kinase C and the development of diabetic vascular complicationsDIABETIC MEDICINE, Issue 12 2001K. J. Way Abstract Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKC, isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKC, isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia. Diabet. Med. 18, 945,959 (2001) [source] Reversible protein kinase C activation in PC12 cells: effect of NGF treatmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2000Jean-Luc Dupont Abstract Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+ -dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized. [source] Protein Kinase C Regulation of Rat Jejunal Transport Systems: Mechanisms Involved in Lactate MovementEXPERIMENTAL PHYSIOLOGY, Issue 6 2002Marisa Tosco We examined whether protein kinase C (PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+ -lactate symport, as well as on apical Na+ -glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4-,-PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+,K+ -ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis. [source] Spatiotemporal dynamics of lipid signaling: Protein kinase C as a paradigmIUBMB LIFE, Issue 12 2008Lisa L. Gallegos Abstract The lipid second messenger diacylglycerol (DAG) controls the rate, amplitude, duration, and location of protein kinase C (PKC) activity in the cell. There are three classes of PKC isozymes and, of these, the conventional and novel isozymes are acutely controlled by DAG. The kinetics of DAG production at various intracellular membranes, the intrinsic affinity of specific isoforms for DAG-containing membranes, the coordinated use of additional membrane-binding modules, the intramolecular regulation of DAG sensitivity, and the competition from other DAG-responsive proteins together result in a unique, context-dependent activation signature for each isoform. This review focuses on the spatiotemporal dynamics of PKC activation and how it is controlled by lipid second messengers. © 2008 IUBMB IUBMB Life, 60(12): 782,789, 2008 [source] Subcellular redistribution of protein kinase C isozymes is associated with rat liver cirrhotic changes induced by carbon tetrachloride or thioacetamideJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2001Da-Hee Jeong Abstract Background and Aims: Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA). Methods and Results: Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear-cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment. Discussion: When the subcellular redistribution of PKC isozymes (PKC,, -,1, -,, and -,) was examined, all the PKC isozymes in CCl4 -treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment. Conclusions: From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis. [source] Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogenJOURNAL OF NEUROCHEMISTRY, Issue 1 2006Myriam Cordey Abstract Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from ,-amyloid peptide (A,) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (,, ,I, ,II), novel (,, ,, ,) and atypical (,, ,/,) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against A, toxicity. In addition, peptide inhibitors of conventional, ,I, or ,II PKC isoforms significantly reduce protection afforded by PMA or 17,-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by A, toxicity. [source] Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptorJOURNAL OF NEUROCHEMISTRY, Issue 3 2001David R. Grosshans The N -methyl- d -aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nm phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 ± 11% above control) and NR2B (41 ± 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B. [source] Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2000B. Sorin Abstract There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx. [source] Prostaglandin F2, upregulates interleukin-6 production in human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2001Kazuyuki Noguchi Prostaglandin F2,(PGF2,) is a bioactive lipid mediator which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2, in periodontal lesions are poorly understood. In the present study, we investigated the effect of PGF2, on interleukin (IL)-6 production in human gingival fibroblasts (HGF). PGF2,stimulated IL-6 production in a time- and concentration-dependent fashion. IL-1, and tumor necrosis factor ,(TNF,), proinflammatory cytokines, induced IL-6 production in a time-dependent manner, and PGF2,synergistically enhanced IL-6 production induced by IL-1, and TNF,. IL-6 mRNA was expressed in PGF2, -stimulated HGF, and PGF2, increased IL-6 mRNA levels induced by IL-1, and TNF,. Fluprostenol, a selective FP receptor agonist, could mimic PGF2, -induced IL-6 production. Since FP receptors are coupled to elevation of intracellular calcium and activation of protein kinase C (PKC), the mechanism of IL-6 production by PGF2, was investigated using TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores, and calphostin C, an inhibitor of PKC. TMB-8 significantly suppressed PGF2, -induced IL-6 production, whereas calphostin C showed a stimulatory effect on PGF2, -induced IL-6 production. From these data, we suggest that PGF2, upregulates IL-6 production through FP receptors in HGF, that PGF2, synergistically enhances IL-6 production in IL-1,- and TNF,-stimulated HGF, and that PGF2, -induced IL-6 production may be dependent on intracellular Ca2+ mobilization and be downregulated by PKC activation. PGF2, may be involved in the pathogenesis of periodontal disease by enhancing IL-6 levels in periodontal lesions. [source] Critical role of ADP interaction with P2Y12 receptor in the maintenance of ,IIb,3 activation: association with Rap1B activationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2006T. KAMAE Summary.,Objective:,Platelet integrin ,IIb,3 plays a crucial role in platelet aggregation, and the affinity of ,IIb,3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which ,IIb,3 maintains its high-affinity state. Methods and results:,Washed platelets adjusted to 50 × 103 ,L,1 were stimulated with 0.2 U mL,1 thrombin or 5 ,m U46619 under static conditions. After the completion of ,IIb,3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated ,IIb,3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 ,m AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained ,IIb,3 activation by ,92% and ,38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 ,L,1 also disrupted the sustained ,IIb,3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5,-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL,1 thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions:,Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of ,IIb,3 activation. [source] The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cellsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 5 2010Jun Liu Abstract Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically applied. Protein kinase C (PKC) family members have been implicated in bone metabolism, which prompted us to use a pharmaceutical approach to manipulate PKC signalling in hMSCs. Inhibition of PKC resulted in a dose-dependent inhibition of dexamethasone-induced osteogenic differentiation. Surprisingly, PKC activation using phorbol 12-myristate 13-acetate (PMA) also resulted in inhibition of osteogenesis, although we observed that inhibition was more pronounced at low than at high concentrations of PMA. Furthermore, we observed that inhibition of PKC, blocked alkaline phosphatase (ALP, an early marker of osteogenic differentiation) expression, whereas inhibition of the conventional PKC subfamily and PKCµ using Gö6976 resulted in an induction of ALP activity, collagen (I) expression and mineralization. In conclusion, inhibition of the conventional PKCs/PKCµ and activation of PKC, could further benefit osteogenic differentiation of hMSCs in vitro and in vivo, which is currently under investigation. Copyright © 2009 John Wiley & Sons, Ltd. [source] A cyclic adenosine 3,,5,-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Hiroshi Harayama Abstract An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5°C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa. Mol. Reprod. Dev. 1169,1178, 2006. © 2006 Wiley-Liss, Inc. [source] Antiproliferative effect of Scutellaria barbata D. Don. on cultured human uterine leiomyoma cells by down-regulation of the expression of Bcl-2 proteinPHYTOTHERAPY RESEARCH, Issue 5 2008Kyung-Woon Kim Abstract Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of leiomyomal cells (LM). A time-dependent antiproliferative effect was noted when 10,5m buserelin, gonadotrophin-releasing hormone (GnRH) agonist or 20,40 µg/mL SB was added. The inhibition of cell growth decreased with the addition of the PKC activator (12-O-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of SB, and the decreases in the viable cells caused by the addition of SB were reversed completely by pretreatment with a protein kinase C (PKC) inhibitor (calphostin C). The findings suggest that SB inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied by PKC activation. Next, the study investigated the effect of SB on fetal development for toxicity. Pregnant Sprague-Dawley rats, from gestation day 6,15, were administered 20 g/L or 50 g/L SB in the drinking water and then killed on day 20. No maternal toxicity was observed, however, embryonic loss in the treatment groups was double that of the controls (p < 0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to SB were found to be significantly heavier than the controls, an effect that was greater in female fetuses and was not correlated with increased placental size. The results suggest that the SB had no toxicity and that in utero exposure to SB resulted in increased early embryo loss with increased growth in surviving fetuses. On the other hand, Western blot analyses revealed that Bcl-2 protein of a 26 kDa was abundant in leiomyomal cells, but not in normal myometrial cells. The addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells. However, the addition of SB (20 µg/mL) resulted in a significant reduction in Bcl-2 protein expression in the cells. The results indicated that human uterine leiomyomal cells express Bcl-2 protein and progesterone enhances its expression, however, SB reduces the expression of Bcl-2 protein in human uterine leiomyoma cells. Copyright © 2008 John Wiley & Sons, Ltd. [source] Toward the development of new medicinal leads with selectivity for protein kinase C isozymesTHE CHEMICAL RECORD, Issue 4 2005Kazuhiro Irie Abstract Tumor promoters such as phorbol esters bind strongly to protein kinase C (PKC) isozymes to induce their activation. Since each PKC isozyme is involved in diverse biological events in addition to tumor promotion, the isozymes serve as promising therapeutic targets. Tumor promoters bind to the C1A and/or C1B domain of conventional (,, ,I, ,II, and ,) and novel PKC isozymes (,, ,, ,, and ,). As these C1 domains play differential roles in PKC activation and their translocation in cells, the development of agents with binding selectivity for individual C1 domains is a pressing need. For this purpose, we established a synthetic C1 peptide library of all PKC isozymes. The library enabled us to identify indolactam-V (1) as a promising lead compound. Our diverse structure,activity studies on 1 indicated that the position of the hydrophobic substituent on the indole ring dominates the PKC isozyme- and C1 domain-selective binding rather than conformation of the nine-membered lactam. Moreover, we suggested that the indole ring of 1 could be involved in the CH/, interaction with Pro-11 of the C1B domain of PKC,. This invaluable information will lead to the structural optimization of the PKC, ligand as exemplified by the design and synthesis of naphtholactam-V8 (21). © 2005 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 5: 185,195; 2005: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20044 [source] Anti-DNA antibody induction of protein kinase C phosphorylation and fibronectin synthesis in human and murine lupus and the effect of mycophenolic acidARTHRITIS & RHEUMATISM, Issue 7 2009Susan Yung Objective To examine fibronectin (FN) expression in human lupus nephritis and the effect of anti-DNA antibodies on transforming growth factor ,1 (TGF,1) and FN synthesis in cultured human mesangial cells. The effects of mycophenolic acid (MPA) on this pathway, and the effects of mycophenolate mofetil (MMF) treatment in (NZB × NZW)F1/J mice were also studied. Methods Immunohistochemical analyses of renal biopsy samples from patients with active diffuse proliferative lupus nephritis were performed. Cultured human mesangial cells were incubated with human polyclonal anti-DNA antibodies, with or without MPA. (NZB × NZW)F1/J mice with active nephritis were randomized to receive either MMF (100 mg/kg/day) or vehicle treatment for 12 weeks. Results Glomerular FN expression was increased in patients with lupus nephritis, and it colocalized with IgG deposition. Anti-DNA antibodies induced protein kinase C, (PKC,), PKC,I, and PKC,II activation, increased levels of bioactive TGF,1, and increased FN synthesis in human mesangial cells (P < 0.001 for each comparison versus control conditions). Pretreatment of anti-DNA antibodies with exogenous DNA reduced their cellular binding and abrogated their induction of TGF,1 and FN synthesis. Inhibition of PKC activation in human mesangial cells prior to anti-DNA antibody stimulation had no effect on cell proliferation, but resulted in significantly reduced antibody-mediated TGF,1 secretion and FN synthesis. MPA treatment down-regulated PKC,, PKC,I, and PKC,II phosphorylation, reduced levels of TGF,1 bioactivation, and decreased FN synthesis and deposition into the extracellular matrix. MMF treatment in (NZB × NZW)F1/J mice resulted in a reduction in glomerular IgG deposition, PKC activation, and FN expression, as well as an amelioration of proteinuria. Conclusion Human polyclonal anti-DNA antibodies induce TGF,1 and FN synthesis in human mesangial cells through PKC activation, which is inhibited by MPA. [source] Formalin-Induced Short- and Long-Term Modulation of Cav Currents Expressed in Xenopus Oocytes: An In Vitro Cellular Model for Formalin-Induced PainBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2010Senthilkumar Rajagopal Cav channels were expressed with ,1,1b and ,2, sub-units and the currents (IBa) were studied by voltage clamp. None of the oocytes was dead during the exposure to formalin. Oocyte death was significant between day 1 and day 5 after the exposure to formalin and was uniform among the oocytes expressing various Cav channels. Peak IBa of all Cav and A1, the inactivating current component was decreased whereas the non-inactivated R current was not affected by 5 min. exposure to formalin. On day 1 after the exposure to formalin, Cav1.2c currents were increased, 2.1 and 2.2 currents were decreased and 2.3 currents were unaltered. On day 5, both peak IBa and A1 currents were increased. Cav1.2c, 2.2 and 2.3 currents were increased and Cav2.1 was unaltered on day 10 after the exposure to formalin. Protein kinase C (PKC) may be involved in formalin-induced increase in Cav currents due to the (i) requirement for Cav,1b sub-units; (ii) decreased phorbol-12-myristate,13-acetate potentiation of Cav2.3 currents; (iii) absence of potentiation of Cav2.3 currents following down-regulation of PKC; and (iv) absence of potentiation of Cav2.2 or 2.3 currents with Ser,Ala mutation of Cav,12.2 or 2.3 sub-units. Increased Cav currents and PKC activation may coincide with changes observed in in vivo pain investigations, and oocytes incubated with formalin may serve as an in vitro model for some cellular mechanisms of pain. [source] Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2004Cuiyan Xin Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGF,2, have no significant effect on S1P-induced MAPK activation. S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC- , inhibitor CGP41251, the PKC- , inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation. British Journal of Pharmacology (2004) 143, 581,589. doi:10.1038/sj.bjp.0705980 [source] In vivo evidence for a role of protein kinase C in peripheral nociceptive processingBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2002Adriano L S Souza The present study was designed to characterize the nociceptive response induced by protein kinase C (PKC) peripheral activation and to investigate if this biochemical event is important for the nociceptive response induced by formaldehyde, and bradykinin (BK). Intraplantar injection of phorbol-12,13-didecanoate (PDD; 0.01, 0.1 or 1 ,g), a PKC activator, but not of 4,-PDD (inactive analogue), dose-dependently induced thermal hyperalgesia in rats. This response was not observed at the contralateral hindpaw. Intraplantar injection of PDD (0.01, 0.1 or 1 ,g) also induced mechanical allodynia. In mice, injection of PDD (0.1 or 1 ,g) into the dorsum of the hindpaw induced a spontaneous licking behaviour. Intraplantar co-injection of chelerythrine (10 or 50 ,g), a PKC inhibitor, attenuated the thermal hyperalgesia induced by PDD (0.1 ,g) in rats. The second phase of the nociceptive response induced by the injection of formaldehyde (0.92%, 20 ,l) into the dorsum of mice hindpaws was inhibited by ipsi-, but not contralateral, pre-treatment with chelerythrine (1 ,g). Intraplantar injection of BK (10 ,g) induced mechanical allodynia in rats. Ipsi- but not contralateral injection of bisindolylmaleimide I (10 ,g), a PKC inhibitor, inhibited BK-induced mechanical allodynia. In conclusion, this study demonstrates that PKC activation at peripheral tissues leads to the development of spontaneous nociceptive response, thermal hyperalgesia and mechanical allodynia. Most importantly, it also gives in vivo evidence that peripheral PKC activation is essential for the full establishment of the nociceptive response induced by two different inflammatory stimuli. British Journal of Pharmacology (2002) 135, 239,247; doi:10.1038/sj.bjp.0704434 [source] Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C, activationCELL PROLIFERATION, Issue 6 2001P. W. Sylvester Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0,250 µm) of ,-, ,-, or ,-tocopherol or ,-, ,-, or ,-tocotrienol. Treatment with growth inhibitory doses of ,-tocopherol (100 µm), ,-tocotrienol (50 µm), or ,- or ,-tocotrienol (10 µm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC,. However, these treatments were found to inhibit EGF-induced PKC, activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 µm,- or ,-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC, activation. [source] Role of novel protein kinase C isoforms in Lyme arthritisCELLULAR MICROBIOLOGY, Issue 8 2007Ok S. Shin Summary Inflammation caused by Borrelia burgdorferi infection occurs as a result of induction of pro-inflammatory cytokines from activation of multiple signalling pathways. It has previously been shown that mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducer and activator of transcription signalling pathways are activated by B. burgdorferi in cultured human chondrocytes. Protein kinase C (PKC) signalling pathways are potential candidates that may control these downstream signalling pathways. Here we show that B. burgdorferi infection leads to phosphorylation and activation of novel PKC isoforms (PKC ,, ,, , and ,) in a time-dependent manner. A specific inhibitor of novel PKC isoforms blocked the induction of pro-inflammatory molecules in response to B. burgdorferi infection as did transient transfection of novel PKC dominant-negative plasmids into chondrocytes. B. burgdorferi -induced p38 MAPK phosphorylation was also significantly inhibited by an inhibitor of novel PKC isoforms, suggesting that PKC activation occurs upstream of p38 activation. In vivo, administration of an inhibitor of classical and novel PKC isoforms to C3H/HeN mice infected with B. burgdorferi resulted in significantly reduced ankle inflammation and swelling. In conclusion, these data suggest that novel PKC isoforms are specifically activated by B. burgdorferi infection and this can contribute to the regulation of inflammation in vitro and in vivo. [source] | ||||||||||||||||