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PKC Inhibitor (pkc + inhibitor)
Selected AbstractsGenistein prevents thyroid hormone-dependent tail regression of Rana catesbeiana tadpoles by targetting protein kinase C and thyroid hormone receptor ,DEVELOPMENTAL DYNAMICS, Issue 3 2007L. Ji Abstract Thyroid hormone (TH)-regulated gene expression is mainly mediated by TH binding to nuclear thyroid hormone receptors (TRs). Despite extensive studies in mammalian cell lines that show that phosphorylation signaling pathways are important in TH action, little is known about their roles on TH signaling in vivo during development. Anuran metamorphosis is a postembryonic process that is absolutely dependent upon TH and tadpole tail resorption can be precociously induced by exogenous administration of 3,5,3,-triiodothyronine (T3). We demonstrate that genistein (a major isoflavone in soy products and tyrosine kinase inhibitor) and the PKC inhibitor (H7) prevent T3 -induced regression of the Rana catesbeiana tadpole tail. T3 -induced protein kinase C tyrosine phosphorylation and kinase activity are inhibited by genistein while T3 -induced up-regulation of TR, mRNA, but not TR, mRNA, is significantly attenuated, most likely through inhibition of T3 -dependent phosphorylation of the TR, protein. This phosphorylation may be modulated through PKC. These data demonstrate that T3 signaling in the context of normal cells in vivo includes phosphorylation as an important factor in establishing T3 -dependent tail regression during development. Developmental Dynamics 236:777,790, 2007. © 2007 Wiley-Liss, Inc. [source] Econazole-induced Ca2+ fluxes and apoptosis in human oral cancer cellsDRUG DEVELOPMENT RESEARCH, Issue 4 2010Daih-Huang Kuo Abstract The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of >1,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+ -free medium, after treatment with 1,µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30,µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2,µM U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations of 5,70,µM econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50,µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,,N,-tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10,µM), also enhanced 20,µM econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10,70,µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC-regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240,248, 2010. © 2010 Wiley-Liss, Inc. [source] SHORT COMMUNICATION Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKCEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2002Yaron Seroussi Abstract We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl- sn -glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells. [source] Vasoactive intestinal peptide acts via multiple signal pathways to regulate hippocampal NMDA receptors and synaptic transmissionHIPPOCAMPUS, Issue 9 2009Kai Yang Abstract Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide, which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase-activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1-R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1-R only if it is used at relatively high concentrations (e.g., 100 nM); however, at lower concentrations (e.g., 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1-R activation signals through PKC/CAK,/Src pathway to regulate NMDA receptors; however, there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1 nM VIP enhanced NMDA currents by stimulating the VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac-Tyr1, D-Phe2]GRF (1,29). This enhancement of NMDA currents was blocked by both Rp-cAMPS and PKI14,22 (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP-induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g., 100 nM) was mediated by PAC1-R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways. © 2009 Wiley-Liss, Inc. [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source] Participation of protein kinase C , isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Youngshik Choe Abstract In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12- O -Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKC,-selective inhibitor), but not by rottlerinTM (a PKC,-selective inhibitor), indicating that PKC, may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and ,-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKC,, PKC,, and PKC, were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKC,-EGFP to growth cones and cell,cell adhesion sites, PKC,-EGFP to the nucleus, and PKC,-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKC, (PKC,-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKC, enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKC,-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKC, with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKC, isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway. [source] Fractalkine and fractalkine receptors in human neurons and glial cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2002Kozo Hatori Abstract Fractalkine has been identified as a novel chemokine that exhibits cell adhesion and chemoattractive properties in the central nervous system (CNS), and the fractalkine receptors, CX3CR1, are also expressed in the CNS. In the present study, the expression of fractalkine and fractalkine receptors was investigated in enriched populations of human CNS neurons, astrocytes, and microglia. In addition, the regulatory role played by protein kinase C (PKC) in fractalkine secretion in neurons was determined in A1 human hybrid neuronal cell line produced between a human cerebral neuron and a human neuroblastoma cell. Human neurons and astrocytes expressed fractalkine mRNA as determined by the revserse transcriptase-polymerase chain reaction (RT-PCR) analysis, while human microglia preparation did not express the fractalkine message. Human neurons and microglia expressed CX3CR1 mRNA, but astrocytes did not. These results suggest that fractalkine secreted by CNS neurons and astrocytes produce biological effects in neurons and microglia. Although phorbol ester did not change the expression of fractalkine mRNA level in A1 hybrid neurons, it did upregulate fractalkine secretion over unstimulated controls. This upregulation of fractalkine production was suppressed by the treatment with Ro32-0432, a PKC inhibitor. These results indicate that intracellular signals transduced by PKC play an important role in the regulation of soluble fractalkine at the post-transcriptional level in human neurons. As for the biological function of fractalkine, extracellularly applied fractalkine increased the number of bromodeoxyuridine-labeled microglia 3-fold over the untreated controls, indicating fractalkine induces proliferation of human microglia. These observations suggest that fractalkine released by injured neurons could induce proliferation, activation and/or migration of microglia at the injured brain sites. © 2002 Wiley-Liss, Inc. [source] Prohibitin regulates TGF-, induced apoptosis as a downstream effector of smad-dependent and -independent signalingTHE PROSTATE, Issue 1 2010Brian Zhu Abstract BACKGROUND Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-, (TGF-,) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-,. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-, via Smad-dependent mechanism. METHOD TGF-, activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-, induced apoptosis in prostate cancer cells. RESULTS These finding indicates that induction of TGF-, apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-, treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites. CONCLUSION Our results demonstrate that TGF-, led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-,, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-, via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival). Prostate 70: 17,26, 2010. © 2009 Wiley-Liss, Inc. [source] Molecular cloning and expression of protein kinase C from Bombyx moriARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2006Tomohide Uno Abstract Two partial cDNA clones (Protein kinase C alpha and Protein kinase C iota), each of which encoded a different member of PKC-protein family, were isolated using RT-PCR from mRNA of Bombyx mori. The full-length cDNAs were isolated using SMART-RACE. The cDNAs were expressed in HepG2 cells and the recombinant proteins were partially purified using an affinity chromatography. Protein kinase C alpha (BPKC alpha) showed a calcium-dependent kinase activity of histones. Whereas protein kinase C iota (BPKC iota) showed a calcium-independent activity. Bisindolyl maleimide I, a PKC inhibitor, inhibited these kinase activities. Furthermore, in vitro BPKC alpha interacted and phosphorylated two proteins expressed in the brain of Bombyx mori: Rab protein, which plays important roles in the vesicle transport in the brain, and bMBD2/3, which is a methyl DNA-binding protein and regulates transcription. These results suggest that these PKCs phosphorylate various substrate proteins and function in the brain of Bombyx mori. Arch. Insect Biochem. Physiol. 61:65,76, 2006. © 2006 Wiley-Liss, Inc. [source] Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteriesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2009R Rodríguez-Rodríguez Background and purpose:, Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries. Experimental approach:, The non-hydrolyzable selective P2Y1 agonist ADP,S (3 µM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca2+]i were obtained in the presence and absence of inhibitors of protein kinase C (PKC). Key results:, ADP,S evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 µM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 µM) or BIS-VIII (1 µM) tended to augment concentration-dependent dilatation to ADP,S (0.1,3 µM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 µM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca2+]i in pressurized arteries confirmed the P2Y1 receptor but not M3 muscarinic receptor desensitization. Conclusions and implications:, These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides. [source] Intra- and extrarenal arteries exhibit different profiles of contractile responses in high glucose conditionsBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2008K Nobe Background and purpose: The renal artery (RA) has been extensively investigated for the assessment of renal vascular function/dysfunction; however, few studies have focused on the intrarenal vasculature. Experimental approach: We devised a microvascular force measurement system, which allowed us to measure contractions of interlobar arteries (ILA), isolated from within the mouse kidney and prepared without endothelium. Key results: KCl (50 mM) induced similar force development in the aorta and RA but responses in the ILA were about 50% lower. Treatment of RA with 10 ,M phenylephrine (PE), 10 nM U46619 (thromboxane A2 analogue) or 10 ,M prostaglandin F2, elicited a response greater than 150% of that induced by KCl. In ILA, 10 nM U46619 elicited a response that was 130% of the KCl-induced response; however, other agonists induced levels similar to that induced by KCl. High glucose conditions (22.2 mM glucose) significantly enhanced responses in RA and ILA to PE or U46619 stimulation. This enhancement was suppressed by rottlerin, a calcium-independent PKC inhibitor, indicating that glucose-dependent, enhanced small vessel contractility in the kidney was linked to the activation of calcium-independent PKC. Conclusion and implications: Extra- and intrarenal arteries exhibit different profiles of agonist-induced contractions. In ILA, only U46619 enhanced small vessel contractility in the kidney, which might lead to renal dysfunction and nephropathy through reduced intrarenal blood flow rate. A model has been established, which will allow the assessment of contractile responses of intrarenal arteries from murine models of renal disease, including type 2 diabetes. British Journal of Pharmacology (2008) 155, 1204,1213; doi:10.1038/bjp.2008.365; published online 22 September 2008 [source] Protein kinase C, mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neuronsBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2005Hui-Fang Li 1The present studies were designed to test the hypothesis that neuronal-specific protein kinase C, (PKC,) plays a critical role in acute ethanol withdrawal hyper-responsiveness in spinal cord. 2Patch-clamp studies were carried out in motor neurons in neonatal rat spinal cord slices. Postsynaptic currents were evoked by brief pulses of 2 mMN -methyl- D -aspartic acid (NMDA) in the presence of bicuculline methiodide 10 ,M; strychnine 5 ,M and tetrodotoxin 0.5 ,M. 3Both ethanol depression and withdrawal hyper-responsiveness of NMDA-evoked currents are dependent on increases in intracellular Ca2+. Blocking intracellular increase in Ca2+ by 30 mM 1,2-bis(2-aminophenoxy)-ethane- N,N,N,,N,-tetraacetic acid (BAPTA) not only decreased the ethanol-induced depression of NMDA-evoked currents (33±5% in control vs 20±3% in BAPTA, P<0.05) but also eliminated acute ethanol withdrawal hyper-responsiveness. 4Immunohistochemistry studies revealed that neonatal spinal cord motor neurons contain an abundance of nuclear PKC,. 5Exposure to ethanol (100 mM) induced PKC, translocation from the nucleus to cytoplasm in motor neurons. Pretreatment with the , -isozyme-specific peptide PKC inhibitor, ,V5-3, blocked ethanol-induced translocation and also blocked withdrawal hyper-responsiveness. 6The results show that PKC, mediates ethanol withdrawal hyper-responsiveness in spinal motor neurons; the results may be relevant to some symptoms of ethanol withdrawal in vivo. British Journal of Pharmacology (2005) 144, 301,307. doi:10.1038/sj.bjp.0706033 [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] NEW INSIGHT INTO THE SIGNALLING PATHWAYS OF HEAT STRESS-INDUCED MYOCARDIAL PRECONDITIONING: PROTEIN KINASE C, TRANSLOCATION AND HEAT SHOCK PROTEIN 27 PHOSPHORYLATIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2004Claire Arnaud SUMMARY 1.,Heat stress (HS) is known to induce delayed preconditioning against myocardial infarction 24 h later, but the exact signalling pathway of this response remains to be elucidated. In previous studies, we have shown evidence for the implication of protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK) in the HS-induced reduction in infarct size. Furthermore, in their phosphorylated state, small heat shock proteins (Hsp27) seem to confer cytoskeletal protection. In the present study, we sought to determine the effect of HS on the subcellular distribution of PKC isoforms and on Hsp27 phosphorylation. 2.,Rats were subjected to either HS (42°C for 15 min; HS group) or sham anaesthesia (sham group) before their hearts were excised. Myocardial tissue extracts obtained 20 min or 24 h after HS were processed for western blot analysis. 3.,In the HS group, PKC, translocated from the cytosolic to the particulate fraction (4426 ± 128 vs 6258 ± 316 arbitrary units; P = 0.002). Chelerythrine (5 mg/kg, i.p.), a PKC inhibitor, abolished this translocation. Western blot analysis of Hsp27 24 h after HS showed a marked increase in protein expression and phosphorylation in the particulate fraction. 4.,In the present study, we have shown that HS induces the translocation of PKC, from the cytosolic to the particulate fraction. Along with our previous observation that PKC is a trigger of HS-induced myocardial preconditioning, the results of the present study suggest an important role of the , isoform of PKC in this cardioprotective mechanism. Furthermore, we have also demonstrated that the cytoprotective protein Hsp27 is phosphorylated following HS. Therefore, we can conclude that PKC and MAPK/Hsp27 are involved in the signalling pathway of HS-induced cardioprotection. [source] Gangliosides activate microglia via protein kinase C and NADPH oxidaseGLIA, Issue 3 2004Kyoung-Jin Min Abstract Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)-1,, tumor necrosis factor-, (TNF-,), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL-1,, TNF-,, and iNOS in ganglioside-treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Gö6976, Ro31-8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC-,, ,II, and , and NADPH oxidase p67phox translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside-induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside-induced activation of NF-,B, an essential transcription factor that mediates the expression of IL-1,, TNF-,, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF-,B. © 2004 Wiley-Liss, Inc. [source] Transforming growth factor- , stimulates Interleukin-11 production by human periodontal ligament and gingival fibroblastsJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 3 2006R. Yashiro Abstract Background: Transforming growth factor (TGF)- , is a potent multifunctional polypeptide, abundant in the bone matrix. Interleukin (IL)-11 is a pleiotropic cytokine with effects on multiple cell types. The present study was performed to evaluate the regulatory effects of TGF- , on IL-11 production by human periodontal ligament cells (PDL) and human gingival fibroblasts (HGF). Material and Methods: The expression of TGF- , receptor in PDL and HGF were observed using flow cytometry. PDL and HGF were stimulated with TGF- , with or without protein kinase C (PKC) inhibitors and activator. IL-11, bone morphogenetic protein-2 (BMP-2) and TGF- , mRNA expression was quantified by real-time polymerase chain reaction (PCR). IL-11 production was measured using enzyme-linked immunosorbent assay. Results: PDL and HGF expressed both TGF- , receptor I and TGF- , receptor II on the cell surfaces. IL-11 mRNA expression and IL-11 production were augmented by TGF- , in both PDL and HGF, with higher values in PDL. PKC inhibitors partially suppressed TGF- , -induced IL-11 production in PDL and HGF, whereas activator enhanced it. TGF- , mRNA and BMP-2 mRNA expression were up-regulated by TGF- , in PDL. Conclusion: These results suggest that PDL produce IL-11 in response to TGF- ,. [source] Histamine-induced Ca2+ entry in human astrocytoma U373 MG cells: Evidence for involvement of store-operated channelsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2008Margarita Barajas Abstract Glial and glia-derived cells express a variety of receptors for neurotransmitters and hormones, the majority of which evoke both Ca2+ release from intracellular stores and Ca2+ entry across the plasma membrane. We investigated the links between histamine H1 receptor activation, Ca2+ release from intracellular stores and Ca2+ influx in human astrocytoma U373 MG cells. Histamine, through a H1 receptor-mediated effect, evoked an increase in cytoplasmic free calcium concentration ([Ca2+]i) that occurred in two phases: an initial, transient, increase owing to Ca2+ mobilization from intracellular pools, and a second, sustained increase dependent on both Ca2+ influx and continuous receptor occupancy. The characteristics of histamine-induced increases in [Ca2+]i were similar to the capacitative entry evoked by emptying of the Ca2+ stores with thapsigargine, and different from that observed when Ca2+ influx was activated with OAG (1-oleoyl-2-acetyl- sn -glycerol), a diacylglycerol (DAG) analog. OAG application or increased endogenous DAG, resulting from DAG kinase inhibition, reduced the histamine-induced response. Furthermore, activation of the DAG target, protein kinase C (PKC), by TPA (12-O-tetradecanoyl 4,-phorbol 13,-acetate) resulted in inhibition of the histamine-induced Ca2+ response, an action prevented by PKC inhibitors. By using reverse transcriptase,polymerase chain reaction analysis, mRNAs for transient receptor potential channels (TRPCs) 1, 4, and 6 as well as for STIM1 (stromal-interacting molecule) and Orai1 were found to be expressed in the U373 MG cells, and confocal microscopy using specific antibodies revealed the presence of the corresponding proteins. Therefore, TRPCs may be candidate proteins forming store-operated channels in the U373 MG cell line. Further, our results confirm the involvement of PKC in the regulation of H1 receptor-induced responses and point out to the existence of a feedback mechanism acting via PKC to limit the increase in [Ca2+]i. © 2008 Wiley-Liss, Inc. [source] Ethanol Uses cAMP-Independent Signal Transduction Mechanisms to Activate Proenkephalin Promoter Activity in Rat C6 Glioma CellsALCOHOLISM, Issue 7 2000Xiaoju Yang Background: Previous in vivo studies show that acute ethanol exposure sequentially increases protein kinase A (PKA) activity, the phosphorylation of the adenosine 3,:5,-cyclic monophosphate (cAMP) dependent transcription factor, CREB, and finally proenkephalin gene expression. The present study was conducted to determine if ethanol could activate directly the adenylyl cyclase pathway and thus enhance proenkephalin promoter activity. Methods: Cultured rat C6 glioma cells stably transfected with a segment of the five prime flanking region of rat proenkephalin promoter (nucleotide -2700 + 53) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were employed to study the effects of ethanol on proenkephalin promoter activity. This region of proenkephalin promoter contains two cAMP response elements (CRE-1 and CRE-2) and one AP2 site located in the region upstream of the TATA box. Cultures were exposed to ethanol, isoproterenol, and phorbol-12, myristate 13-acetate (PMA) alone and in combination, in the presence and absence of PKA and protein kinase C (PKC) inhibitors. Results: Ethanol and isoproterenol increased proenkephalin promoter activity in a dose-dependent manner. Ethanol had an additive effect on maximal isoproterenol-stimulated proenkephalin promoter activity, which suggested that ethanol used a cAMP-independent signai transduction pathway to increase proenkephalin promoter activation. In contrast with isoproterenol, ethanol exposure did not increase cAMP accumulation, PKA activity, or the phosphorylated form of CREB. However, ethanol exposure modestly increased PKC activity. The PKA-specific inhibitor, Rp-cAMP, dampened isoproterenol-induced activation of CAT activity but did not alter ethanol's ability to increase CAT activity. However, the PKC inhibitors, chelerthyrine and G07874, abrogated ethanol's effect of CAT activity but did not alter isoproterenol's effects. Conclusions: Ethanol enhanced proenkephalin promoter activity and potentiated isoproterenol stimulated promoter activity through a cAMP-independent pathway. [source] Protein kinase C-, mediates von Willebrand factor secretion from endothelial cells in response to vascular endothelial growth factor (VEGF) but not histamineJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2008O. LORENZI Summary.,Background:,Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. Objectives:,We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF-induced and histamine-induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Results:,Several PKC inhibitors (staurosporine, Ro31-8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF-induced but not histamine-induced VWF secretion. PKC-, and novel PKCs (PKC-,, PKC-,, and PKC-,), but not PKC-,, are expressed in HUVECs. Both VEGF and histamine activate PKC-,. However, gene inactivation experiments using small interfering RNA indicate that PKC-, (but not PKC-,) is involved in the regulation of VEGF-induced but not histamine-induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF-induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA-AM. Conclusions:,Our study identifies striking agonist specificities in signal,secretion coupling. Histamine-induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF-induced secretion is largely dependent on PKC-, and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC-, in VEGF-induced VWF release, but suggest that a third, VEGF-specific, signaling intermediate is required as a PKC-, coactivator. [source] Enhancement of anchorage-independent growth of human pancreatic carcinoma MIA PaCa-2 cells by signaling from protein kinase C to mitogen-activated protein kinaseMOLECULAR CARCINOGENESIS, Issue 4 2002Keiko Ishino Abstract We found that 12- O -tetradecanoylphorbol-13-acetate (TPA) promoted anchorage-independent growth but did not affect anchorage-dependent growth of MIA PaCa-2 human pancreatic carcinoma cells. TPA markedly activated mitogen-activated protein kinase (MAPK)/extracellular signal,regulated kinase in an anchorage-independent manner. Two protein kinase C (PKC) isoforms, conventional PKC (cPKC) and novel PKC (nPKC), but not apical PKC, translocated from the cytosolic to the particulate fraction upon TPA treatment. To identify the PKC isoforms involved in the regulation of anchorage-independent growth, four PKC isoforms (,, ,, ,, and ,) were forced to be expressed in MIA PaCa-2 cells with an adenovirus vector. Overexpression of nPKC, or nPKC, activated MAPK and promoted anchorage-independent growth. Overexpression of cPKC, alone did not influence anchorage-independent growth but lowered the concentration of TPA that was required to enhance such growth. Expression of constitutively active MAPK kinase-1 (MEK1) also promoted anchorage-independent growth. Furthermore, PKC inhibitors or an MEK inhibitor completely suppressed both TPA-induced activation of MAPK and promotion of anchorage-independent growth, but a cPKC-selective inhibitor partially suppressed TPA-induced promotion of the growth. Based on these results, we suggest that MAPK activation, mediated by certain isoforms of PKC, plays a part in oncogenic growth of MIA PaCa-2 cells. In summary, our data indicated that specific inhibitors of the cPKC and nPKC signaling pathway might be selective anti-oncogenic growth agents for some types of human pancreatic cancer. © 2002 Wiley-Liss, Inc. [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] Characterization of apoptosis induced by protein kinase C inhibitors and its modulation by the caspase pathway in acute promyelocytic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2000Hesham M. Amin Acute promyelocytic leukaemia (APL;M3) is a unique form of acute myelogenous leukaemia characterized by t(15;17) translocation. The induction of apoptosis via inhibiting protein kinase C (PKC) has been recently viewed as a promising tool for the eradication of several malignant disorders. In the present study, we investigated the effect of two different protein kinase C inhibitors, Gö6976 and safingol, on the induction of apoptosis in the APL cell line NB4 and its all trans retinoic acid (ATRA)-resistant variant NB4.306. The effect of the PKC inhibitors on leukaemic cells obtained from three APL patients was also studied. We also evaluated the possible involvement of the caspases in apoptosis induced by PKC inhibitors. Significant time- and concentration-dependent apoptotic changes were demonstrated using Gö6976 and safingol. In addition, our results demonstrated that the caspases were involved in the apoptosis induced by the PKC inhibitors. In conclusion, our study illustrates that the PKC inhibitors Gö6976 and safingol induce apoptosis in APL and hence could be potential therapeutic agents for the treatment of this disease. [source] YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Ming-Shyan Chang YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-,, -,, -,, -, and -, isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-,, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-,. The MEK inhibitor, PD 98059 (10,50 ,M), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-, activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression. British Journal of Pharmacology (2002) 136, 558,567; doi:10.1038/sj.bjp.0704777 [source] Non surgical approach in diabetic macular edema : the future ?ACTA OPHTHALMOLOGICA, Issue 2009C CHIQUET Purpose To present the different non surgical therapeutical options of diabetic macular edema Methods The pathogenesis of diabetic macular edema is multifactorial. Hyperglycemia and poor systemic factor balance are major risk factors. Laser treatemnts and antiagiogenic treatments represent the main non surgical options to treat macular edema. Results Focal macular edema remains the best indication of laser treatment. Laser remains also the standard of care of diffuse macular edema but some edemas remain resistant. Several therapeutic options have been proposed : Steroid intravitreal injection and antiVEGF therapy (either PKC inhibitors, VEGF aptamers or VEGF antibodies) represent the future alternative treatments as well as their potential combination. Conclusion Laser remains the main treatment of diabetic macular edema. However, steroids and antiangiogenic agents either isolated or combined represent the main alternative treatment for non responding diffuse macular edema. [source] HIGH GLUCOSE-INDUCED HUMAN UMBILICAL VEIN ENDOTHELIAL CELL HYPERPERMEABILITY IS DEPENDENT ON PROTEIN KINASE C ACTIVATION AND INDEPENDENT OF THE Ca2+,NITRIC OXIDE SIGNALLING PATHWAYCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 9 2005Lei Dang SUMMARY 1.,Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. The precise molecular mechanisms by which hyperglycaemia causes the increased permeability in endothelial cells are not yet well understood. In the present study, we investigated whether high concentrations of glucose induce endothelial permeability through the activation of protein kinase C (PKC) and/or the calcium,nitric oxide (NO) signalling pathway in human umbilical vein endothelial cells (HUVEC). 2.,Endothelial permeability was measured by albumin diffusion across endothelial monolayers under the stimuli of high glucose (HG; 20 mmol/L), 100 nmol/L phorbol-myristate-acetate (PMA) or 100 nmol/L histamine. The intracellular calcium concentration ([Ca2+]i) was detected in HUVEC using the fluorescent probe fura-2 AM. The effects of PKC inhibitors (LY379196 and hypocrellin A) and the NO synthase (NOS) inhibitor NG -monomethyl- l -arginine (l -NMMA) on endothelial permeability and [Ca2+]i were determined. 3.,High glucose and PMA increased endothelial permeability associated with decreased [Ca2+]i, whereas histamine triggered significant increases in endothelial permeability, accompanied by increases in [Ca2+]i in HUVEC. Hypocrellin A (HA) and LY379196 reversed both HG- and histamine-induced endothelial permeability. The NOS inhibitor l -NMMA only abolished histamine- and not HG-induced endothelial permeability. Neither LY379196, HA nor l -NMMA had any significant effects on alterations in [Ca2+]i caused by HG and histamine. 4.,These results indicate that increased endothelial permeability in HUVEC induced by HG is dependent on PKC activity and is independent of the [Ca2+]i,NO pathway. Increased endothelial permeability due to other inflammatory factors, such as histamine, may also be mediated by the PKC pathway. Thus, PKC inhibitors would be a potential therapeutic approach to endothelial dysfunction induced by hyperglycaemia, as well as other inflammatory factors, in diabetes. [source] | |||||||||||||