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  1. Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

    DEFF Research Database (Denmark)

    Marshall, W. S.; Ossum, Carlo Gunnar; Hoffmann, Else Kay

    2005-01-01

    at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 µmol l-1) inhibited Cl- secretion that was high, increased Cl- secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes......: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca2+ and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting...

  2. OSR1-sensitive small intestinal Na+ transport

    NARCIS (Netherlands)

    Pasham, V.; Pathare, G.T.; Fajol, A.; Rexhepaj, R.; Michael, D.; Pakladok, T.; Alesutan, I.; Rotte, A.; Foller, M.; Lang, F.

    2012-01-01

    The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explore

  3. Osr1 Interacts Synergistically with Wt1 to Regulate Kidney Organogenesis.

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    Jingyue Xu

    Full Text Available Renal hypoplasia is a common cause of pediatric renal failure and several adult-onset diseases. Recent studies have associated a variant of the OSR1 gene with reduction of newborn kidney size and function in heterozygotes and neonatal lethality with kidney defects in homozygotes. How OSR1 regulates kidney development and nephron endowment is not well understood, however. In this study, by using the recently developed CRISPR genome editing technology, we genetically labeled the endogenous Osr1 protein and show that Osr1 interacts with Wt1 in the developing kidney. Whereas mice heterozygous for either an Osr1 or Wt1 null allele have normal kidneys at birth, most mice heterozygous for both Osr1 and Wt1 exhibit defects in metanephric kidney development, including unilateral or bilateral kidney agenesis or hypoplasia. The developmental defects in the Osr1+/-Wt1+/- mouse embryos were detected as early as E10.5, during specification of the metanephric mesenchyme, with the Osr1+/-Wt1+/- mouse embryos exhibiting significantly reduced Pax2-positive and Six2-positive nephron progenitor cells. Moreover, expression of Gdnf, the major nephrogenic signal for inducing ureteric bud outgrowth, was significantly reduced in the metanephric mesenchyme in Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- littermates. By E11.5, as the ureteric buds invade the metanephric mesenchyme and initiate branching morphogenesis, kidney morphogenesis was significantly impaired in the Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- embryos. These results indicate that Osr1 and Wt1 act synergistically to regulate nephron endowment by controlling metanephric mesenchyme specification during early nephrogenesis.

  4. Regulation of ClC-2 Activity by SPAK and OSR1

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    Jamshed Warsi

    2014-10-01

    Full Text Available Background/Aims: SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1 are powerful regulators of diverse transport processes. Both kinases are activated by cell shrinkage and participate in stimulation of regulatory cell volume increase (RVI. Execution of RVI involves inhibition of Cl- channels. The present study explored whether SPAK and/or OSR1 regulate the activity of the Cl- channel ClC-2. Methods: To this end, ClC-2 was expressed in Xenopus laevis oocytes with or without additional expression of wild type SPAK, constitutively active SPAKT233E, WNK1 insensitive inactive SPAKT233A, catalytically inactive SPAKD212A, wild type OSR1, constitutively active OSR1T185E, WNK1 insensitive inactive OSR1T185A, and catalytically inactive OSR1D164A. Cl- channel activity was determined by dual electrode voltage clamp. Results: Expression of ClC-2 was followed by the appearance of a conductance (GCl, which was significantly decreased following coexpression of wild-type SPAK, SPAKT233E, wild type OSR1 or OSR1T185E, but not by coexpression of SPAKT233A, SPAKD212A, OSR1T185A, or OSR1D164A. Inhibition of ClC-2 insertion by brefeldin A (5 μM resulted in a decline of GCl which was similar in the absence and presence of SPAK or OSR1, suggesting that SPAK and OSR1 did not accelerate the retrieval of ClC-2 protein from the cell membrane. Conclusion: SPAK and OSR1 are powerful negative regulators of the cell volume regulatory Cl- channel ClC-2.

  5. The Pepper CaOSR1 Protein Regulates the Osmotic Stress Response via Abscisic Acid Signaling.

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    Park, Chanmi; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Plants are sessile organisms, and their growth and development is detrimentally affected by environmental stresses such as drought and high salinity. Defense mechanisms are tightly regulated and complex processes, which respond to changing environmental conditions; however, the precise mechanisms that function under adverse conditions remain unclear. Here, we report the identification and functional characterization of the CaOSR1 gene, which functions in the adaptive response to abiotic stress. We found that CaOSR1 gene expression in pepper leaves was up-regulated after exposure to abscisic acid (ABA), drought, and high salinity. In addition, we demonstrated that the fusion protein of CaOSR1 with green fluorescent protein (GFP) is localized in the nucleus. We used CaOSR1-silenced pepper plants and CaOSR1-OX-overexpressing (OX) transgenic Arabidopsis plants to show that the CaOSR1 protein regulates the osmotic stress response. CaOSR1-silenced pepper plants showed increased drought susceptibility, and this was accompanied by a high transpiration rate. CaOSR1-OX plants displayed phenotypes that were hypersensitive to ABA and hyposensitive to osmotic stress, during the seed germination and seedling growth stages; furthermore, these plants exhibited enhanced drought tolerance at the adult stage, and this was characterized by higher leaf temperatures and smaller stomatal apertures because of ABA hypersensitivity. Taken together, our data indicate that CaOSR1 positively regulates osmotic stress tolerance via ABA-mediated cell signaling. These findings suggest an involvement of a novel protein in ABA and osmotic stress signalings in plants.

  6. OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

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    Bernat Elvira

    2016-04-01

    Full Text Available Background/Aims: The oxidative stress-responsive kinase 1 (OSR1 and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase are under the control of WNK (with-no-K [Lys] kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels. An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

  7. WNK-OSR1/SPAK-NCC signal cascade has circadian rhythm dependent on aldosterone.

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    Susa, Koichiro; Sohara, Eisei; Isobe, Kiyoshi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2012-11-02

    Blood pressure and renal salt excretion show circadian rhythms. Recently, it has been clarified that clock genes regulate circadian rhythms of renal transporter expression in the kidney. Since we discovered the WNK-OSR1/SPAK-NaCl cotransporter (NCC) signal cascade, which is important for regulating salt balance and blood pressure, we have sought to determine whether NCC protein expression or phosphorylation shows diurnal rhythms in the mouse kidneys. Male C57BL/6J mice were sacrificed every 4h (at 20:00, 0:00, 4:00, 8:00, 12:00, and 16:00), and the expression and phosphorylation of WNK4, OSR1, SPAK, and NCC were determined by immunoblot. (Lights were turned on at 8:00, which was the start of the rest period, and turned off at 20:00, which was the start of the active period, since mice are nocturnal.) Although expression levels of each protein did not show diurnal rhythm, the phosphorylation levels of OSR1, SPAK, and NCC were increased around the start of the active period and decreased around the start of the rest period. Oral administration of eplerenone (10mg/day) attenuated the phosphorylation levels of these proteins and also diminished the diurnal rhythm of NCC phosphorylation. Thus, the activity of the WNK4-OSR1/SPAK-NCC cascade was shown to have a diurnal rhythm in the kidney that may be governed by aldosterone. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels

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    Bernat Elvira

    2015-11-01

    Full Text Available Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1, which are under control of WNK (with-no-K[Lys] kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1. Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

  9. SPAK and OSR1 Dependent Down-Regulation of Murine Renal Outer Medullary K+ Channel ROMK1

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    Bernat Elvira

    2014-09-01

    Full Text Available Background/Aims: The kinases SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1 participate in the regulation of the NaCl cotransporter NCC and the Na+,K+,2Cl- cotransporter NKCC2. The kinases are regulated by WNK (with-no-K[Lys] kinases. Mutations of genes encoding WNK kinases underly Gordon's syndrome, a monogenic disease leading to hypertension and hyperkalemia. WNK kinases further regulate the renal outer medullary K+ channel ROMK1. The present study explored, whether SPAK and/or OSR1 have similarly the potential to modify the activity of ROMK1. Methods: ROMK1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active T233ESPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1 and catalytically inactive D164AOSR1. Channel activity was determined utilizing dual electrode voltage clamp and ROMK1 protein abundance in the cell membrane utilizing chemiluminescence of ROMK1 containing an extracellular hemagglutinin epitope (ROMK1-HA. Results: ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type SPAK and T233ESPAK, but not by D212ASPAK. Similarly, ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type OSR1 and T185EOSR1, but not by D164AOSR1. Conclusion: ROMK1 protein abundance and activity are down-regulated by SPAK and OSR1.

  10. Gene network and familial analyses uncover a gene network involving Tbx5/Osr1/Pcsk6 interaction in the second heart field for atrial septation.

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    Zhang, Ke K; Xiang, Menglan; Zhou, Lun; Liu, Jielin; Curry, Nathan; Heine Suñer, Damian; Garcia-Pavia, Pablo; Zhang, Xiaohua; Wang, Qin; Xie, Linglin

    2016-03-15

    Atrial septal defects (ASDs) are a common human congenital heart disease (CHD) that can be induced by genetic abnormalities. Our previous studies have demonstrated a genetic interaction between Tbx5 and Osr1 in the second heart field (SHF) for atrial septation. We hypothesized that Osr1 and Tbx5 share a common signaling networking and downstream targets for atrial septation. To identify this molecular networks, we acquired the RNA-Seq transcriptome data from the posterior SHF of wild-type, Tbx5(+/) (-), Osr1(+/-), Osr1(-/-) and Tbx5(+/-)/Osr1(+/-) mutant embryos. Gene set analysis was used to identify the Kyoto Encyclopedia of Genes and Genomes pathways that were affected by the doses of Tbx5 and Osr1. A gene network module involving Tbx5 and Osr1 was identified using a non-parametric distance metric, distance correlation. A subset of 10 core genes and gene-gene interactions in the network module were validated by gene expression alterations in posterior second heart field (pSHF) of Tbx5 and Osr1 transgenic mouse embryos, a time-course gene expression change during P19CL6 cell differentiation. Pcsk6 was one of the network module genes that were linked to Tbx5. We validated the direct regulation of Tbx5 on Pcsk6 using immunohistochemical staining of pSHF, ChIP-quantitative polymerase chain reaction and luciferase reporter assay. Importantly, we identified Pcsk6 as a novel gene associated with ASD via a human genotyping study of an ASD family. In summary, our study implicated a gene network involving Tbx5, Osr1 and Pcsk6 interaction in SHF for atrial septation, providing a molecular framework for understanding the role of Tbx5 in CHD ontogeny.

  11. Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone.

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    Chiga, Motoko; Rai, Tatemitsu; Yang, Sung-Sen; Ohta, Akihito; Takizawa, Toichiro; Sasaki, Sei; Uchida, Shinichi

    2008-12-01

    Pseudohypoaldosteronism type II (PHAII) is caused by mutations in the WNK1 and WNK4 genes (WNK with-no-lysine kinase). In a mouse model of this disease where a mutant of Wnk4 D561A was knocked in, increased phosphorylation of the sodium chloride cotransporter (NCC) was found and the transporter was concentrated on the apical membrane of the distal tubules. In addition, we recently found that other kinases, such as the oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK), also showed increased phosphorylation in these mice. Here we determined whether this kinase cascade is regulated by dietary salt intake. We found that the phosphorylation states of NCC and OSR1/SPAK were increased by low-salt diets and decreased by high-salt diets; a regulation completely lost in the knock-in mice. Increased phosphorylation was reversed by spironolactone and this decreased phosphorylation was reversed by administration of exogenous aldosterone. These studies suggest that that the WNK-OSR1/SPAK-NCC cascade may be a novel effector system of aldosterone action in the kidney.

  12. Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

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    Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2012-10-01

    Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

  13. PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

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    Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2013-01-01

    Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt-sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently-identified WNK kinase-OSR1/SPAK kinases-NCC transporter phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the PI3K/Akt signaling cascade in the kidney in response to hyperinsulinemia. A PI3K inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific PI3K inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the PI3K/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions such as the metabolic syndrome. PMID:22949526

  14. Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney.

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    Talati, Gulibaha; Ohta, Akihito; Rai, Tatemitsu; Sohara, Eisei; Naito, Shotaro; Vandewalle, Alain; Sasaki, Sei; Uchida, Shinichi

    2010-03-19

    In our recent study using Wnk4(D561A/+) knockin mice, we determined that the WNK-OSR1/SPAK-NaCl cotransporter (NCC) phosphorylation cascade is important for regulating NCC function in vivo. Phosphorylation of NCC was necessary for its plasma membrane localization. Previously, angiotensin II infusion was shown to increase apical membrane expression of NCC in rats. Therefore, we investigated whether angiotensin II was an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in cultured cells and in vivo kidney. In mpkDCT cells, the phosphorylation of OSR1 and NCC was increased 30 min after the addition of angiotensin II (10(-9)-10(-7)M) but returned to baseline after 18 h. In mice, a 5-min infusion of angiotensin II (5 ng/g/min) increased NCC phosphorylation in the kidney at 30 min and 2h after the injection but returned to baseline 24h later. This increase was inhibited by angiotensin II receptor blocker (valsartan) but not by aldosterone receptor blocker (eplerenone). Ten-day infusions of angiotensin II (720 ng/day) also increased phosphorylation of OSR1 and NCC in the mouse kidney, and both valsartan and eplerenone inhibited the increased phosphorylation. Although angiotensin II is identified as an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in vivo, aldosterone appears to be the major regulator of this signal cascade in the long-term regulation by angiotensin II. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Rafoxanide and Closantel Inhibit SPAK and OSR1 Kinases by Binding to a Highly Conserved Allosteric Site on Their C-terminal Domains.

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    AlAmri, Mubarak A; Kadri, Hachemi; Alderwick, Luke J; Simpkins, Nigel S; Mehellou, Youcef

    2017-05-09

    SPAK and OSR1 are two protein kinases that have emerged as attractive targets in the discovery of novel antihypertensive agents due to their role in regulating electrolyte balance in vivo. Herein we report the identification of an allosteric pocket on the highly conserved C-terminal domains of these two kinases, which influences their activity. We also show that some known WNK signaling inhibitors bind to this allosteric site. Using in silico screening, we identified the antiparasitic agent rafoxanide as a novel allosteric inhibitor of SPAK and OSR1. Collectively, this work will facilitate the rational design of novel SPAK and OSR1 kinase inhibitors that could be useful antihypertensive agents. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Effect of heterozygous deletion of WNK1 on the WNK-OSR1/ SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels.

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    Susa, Koichiro; Kita, Satomi; Iwamoto, Takahiro; Yang, Sung-Sen; Lin, Shih-Hua; Ohta, Akihito; Sohara, Eisei; Rai, Tatemitsu; Sasaki, Sei; Alessi, Dario R; Uchida, Shinichi

    2012-08-01

    We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1(+/-)mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1(+/-) mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1(+/-) mice have not been performed. We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. Contrary to the previous report, the blood pressure of WNK1(+/-) mice was not decreased, even under a low-salt diet. Under a WNK4(D561A/+) background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1(+/-) mice or WNK1(+/-)WNK4(D561A/+) mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1(+/-) mice. The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.

  17. The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps

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    Ryffel Gerhart U

    2011-01-01

    Full Text Available Abstract Background The three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis. Results We have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter. Conclusions The early

  18. Formation of Kv2.1-FAK Complex as a Mechanism of FAK Activation, Cell Polarization and Enhanced Motility

    OpenAIRE

    Wei, Jian-Feng; Wei, Ling; ZHOU, XIN; Lu, Zhong-yang; Francis, Kevin; Hu, Xin-yang; Liu, Yu; Xiong, Wen-Cheng; Zhang, Xiao; Banik, Naren L.; Zheng, Shu-Sen; Yu, Shan Ping

    2008-01-01

    Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK397 and FAK576/577. Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv...

  19. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

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    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  20. Endothelial FAK as a therapeutic target in disease.

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    Infusino, Giovanni A; Jacobson, Jeffrey R

    2012-01-01

    Focal adhesions (FA) are important mediators of endothelial cytoskeletal interactions with the extracellular matrix (ECM) via transmembrane receptors, integrins and integrin-associated intracellular proteins. This communication is essential for a variety of cell processes including EC barrier regulation and is mediated by the non-receptor protein tyrosine kinase, focal adhesion kinase (FAK). As FA mediate the basic response of EC to a variety of stimuli and FAK is essential to these responses, the idea of targeting EC FAK as a therapeutic strategy for an assortment of diseases is highly promising. In particular, inhibition of FAK could prove beneficial in a variety of cancers via effects on EC proliferation and angiogenesis, in acute lung injury (ALI) via the attenuation of lung vascular permeability, and in rheumatoid arthritis via reductions in synovial angiogenesis. In addition, there are potential therapeutic benefits of FAK inhibition in cardiovascular disease and diabetic nephropathy as well. Several drugs that target EC FAK are now in existence and include agents currently under investigation in preclinical models as well as drugs that are readily available such as the sphingolipid analog FTY720 and statins. As the role of EC FAK in the pathogenesis of a variety of diseases continues to be explored and new insights are revealed, drug targeting of FAK will continue to be an important area of investigation and may ultimately lead to highly novel and effective strategies to treat these diseases.

  1. Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles

    Directory of Open Access Journals (Sweden)

    Abdelkader Hamadi

    2010-01-01

    Full Text Available Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs. Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

  2. Altering FAK-paxillin interactions reduces adhesion, migration and invasion processes.

    Directory of Open Access Journals (Sweden)

    Thérèse B Deramaudt

    Full Text Available Focal adhesion kinase (FAK plays an important role in signal transduction pathways initiated at sites of integrin-mediated cell adhesion to the extracellular matrix. Thus, FAK is involved in many aspects of the metastatic process including adhesion, migration and invasion. Recently, several small molecule inhibitors which target FAK catalytic activity have been developed by pharmaceutical companies. The current study was aimed at addressing whether inhibiting FAK targeting to focal adhesions (FA represents an efficient alternative strategy to inhibit FAK downstream pathways. Using a mutagenesis approach to alter the targeting domain of FAK, we constructed a FAK mutant that fails to bind paxillin. Inhibiting FAK-paxillin interactions led to a complete loss of FAK localization at FAs together with reduced phosphorylation of FAK and FAK targets such as paxillin and p130Cas. This in turn resulted in altered FA dynamics and inhibition of cell adhesion, migration and invasion. Moreover, the migration properties of cells expressing the FAK mutant were reduced as compared to FAK-/- cells. This was correlated with a decrease in both phospho-Src and phospho-p130Cas levels at FAs. We conclude that targeting FAK-paxillin interactions is an efficient strategy to reduce FAK signalling and thus may represent a target for the development of new FAK inhibitors.

  3. Altering FAK-paxillin interactions reduces adhesion, migration and invasion processes.

    Science.gov (United States)

    Deramaudt, Thérèse B; Dujardin, Denis; Noulet, Fanny; Martin, Sophie; Vauchelles, Romain; Takeda, Ken; Rondé, Philippe

    2014-01-01

    Focal adhesion kinase (FAK) plays an important role in signal transduction pathways initiated at sites of integrin-mediated cell adhesion to the extracellular matrix. Thus, FAK is involved in many aspects of the metastatic process including adhesion, migration and invasion. Recently, several small molecule inhibitors which target FAK catalytic activity have been developed by pharmaceutical companies. The current study was aimed at addressing whether inhibiting FAK targeting to focal adhesions (FA) represents an efficient alternative strategy to inhibit FAK downstream pathways. Using a mutagenesis approach to alter the targeting domain of FAK, we constructed a FAK mutant that fails to bind paxillin. Inhibiting FAK-paxillin interactions led to a complete loss of FAK localization at FAs together with reduced phosphorylation of FAK and FAK targets such as paxillin and p130Cas. This in turn resulted in altered FA dynamics and inhibition of cell adhesion, migration and invasion. Moreover, the migration properties of cells expressing the FAK mutant were reduced as compared to FAK-/- cells. This was correlated with a decrease in both phospho-Src and phospho-p130Cas levels at FAs. We conclude that targeting FAK-paxillin interactions is an efficient strategy to reduce FAK signalling and thus may represent a target for the development of new FAK inhibitors.

  4. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

    OpenAIRE

    1996-01-01

    It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 ...

  5. FAK kinase activity is required for the progression of c-Met/β-catenin-driven HCC

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Zaidi, Ali; Cotler, Scott J.; Breslin, Peter; Ding, Xianzhong; Kuo, Paul; Nishimura, Michael; Zhang, Jiwang; Qiu, Wei

    2016-01-01

    Background & Aims There is an urgent need to develop new and more effective therapeutic strategies and agents to treat hepatocellular carcinoma (HCC). We have recently found that deletion of Fak in hepatocytes before tumors form inhibits tumor development and prolongs survival of animals in a c-Met (MET)/β-catenin (CAT)-driven HCC mouse model. However, it has yet to be determined whether FAK expression in hepatocytes promotes MET/CAT-induced HCC progression after tumor initiation. In addition, it remains unclear whether FAK promotes HCC development through its kinase activity. Methods We generated hepatocyte-specific inducible Fak-deficient mice (Alb-creERT2; Fakflox/flox) to examine the role of FAK in HCC progression. We re-expressed wild-type and mutant FAK in Fak-deficient mice to determine FAK’s kinase activity in HCC development. We also examined the efficacy of a FAK kinase inhibitor PF-562271 on HCC inhibition. Results We found that deletion of Fak after tumors form significantly repressed MET/CAT-induced tumor progression. Ectopic FAK expression restored HCC formation in hepatocyte-specific Fak-deficient mice. However, overexpression of a FAK kinase-dead mutant led to reduced tumor load compared to mice which express wild-type FAK. Furthermore, PF-562271 significantly suppressed progression of MET/CAT-induced HCC. Conclusion Fak kinase activity is important for MET/CAT-induced HCC progression. Inhibiting FAK kinase activity provides a potential therapeutic strategy to treat HCC. PMID:27142958

  6. Contraction stimulates muscle glucose uptake independent of atypical PKC.

    Science.gov (United States)

    Yu, Haiyan; Fujii, Nobuharu L; Toyoda, Taro; An, Ding; Farese, Robert V; Leitges, Michael; Hirshman, Michael F; Mul, Joram D; Goodyear, Laurie J

    2015-11-01

    Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC-λ/ζ) have been shown to be necessary for insulin-, AICAR-, and metformin-stimulated glucose uptake in skeletal muscle, but not for treadmill exercise-stimulated muscle glucose uptake. To investigate if PKC-λ/ζ activity is required for contraction-stimulated muscle glucose uptake, we used mice with tibialis anterior muscle-specific overexpression of an empty vector (WT), wild-type PKC-ζ (PKC-ζ(WT)), or an enzymatically inactive T410A-PKC-ζ mutant (PKC-ζ(T410A)). We also studied skeletal muscle-specific PKC-λ knockout (MλKO) mice. Basal glucose uptake was similar between WT, PKC-ζ(WT), and PKC-ζ(T410A) tibialis anterior muscles. In contrast, in situ contraction-stimulated glucose uptake was increased in PKC-ζ(T410A) tibialis anterior muscles compared to WT or PKC-ζ(WT) tibialis anterior muscles. Furthermore, in vitro contraction-stimulated glucose uptake was greater in soleus muscles of MλKO mice than WT controls. Thus, loss of PKC-λ/ζ activity increases contraction-stimulated muscle glucose uptake. These data clearly demonstrate that PKC-λζ activity is not necessary for contraction-stimulated glucose uptake.

  7. Pilocarpine-induced status epilepticus alters hippocampal PKC expression in mice.

    Science.gov (United States)

    Liu, Jian Xin; Liu, Yong; Tang, Feng Ru

    2011-01-01

    We investigated the protein expression of different protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta1, PKC-beta2, PKC-gamma, PKC-delta, PKC-epsilon, PKC-eta and PKC-zeta) in the hippocampus of normal control mice and progressive changes in PKC isoforms expression during and after pilocarpine induced status epilepticus (PISE). We showed the reduced expression of PKC-delta, PKC-eta and PKC-zeta in interneurons in the CA1 area and in the hilus of the dentate gyrus during or after PISE. Increased expression of PKC-alpha and PKC-beta1 was demonstrated in the stratum pyramidale of CA3 area, and PKC-epsilon was up-regulated in the stratum lucidum of the CA3 area during or after PISE. Our results suggest that hippocampal PKC isoforms may play different roles in seizure generation, and be targets for development of anti-convulsive drugs.

  8. Cystine dimethyl ester induces apoptosis through regulation of PKC-δ and PKC-ε in prostate cancer cells.

    Science.gov (United States)

    Gurbuz, Nilgun; Park, Margaret A; Dent, Paul; Abdel Mageed, Asim B; Sikka, Suresh C; Baykal, Asli

    2015-01-01

    Protein kinase C-δ (PKC-δ) and PKC-ε are reported to be effective in cancer prevention via S-thiolation-mediated mechanisms. This may be through stimulation of the pro-apoptotic, tumor-suppressive isozyme PKC-δ and/or inactivation of the growth stimulatory, oncogenic isozyme PKC-ε. We investigated oxidative regulatory responses of PKC-δ and PKC-ε to cystine dimethyl ester (CDME), a metabolic precursor of cystine, which, by inducing release of cellular cystine stimulates apoptosis in different prostate cancer cells, PC3 and LNCaP, compared to normal RWPE1 cells. Treatment of CDME in doses of 0.5mM and 5mM significantly induces apoptosis due to regulation of concentration-dependent PKC-δ stimulation and PKC-ε reduction in these prostate cancer cells. This apoptotic regulation was confirmed by immunoblot analyses and specific PKC enzyme assays in immunoprecipitated samples. Additionally, inhibition of PKC-δ by small interfering RNA (siRNA) proved that CDME-induced cell death was dependent on PKC-δ activity in prostate cancer cells. These data demonstrated that CDME induces apoptosis by cysteinylation of both PKC-δ and PKC-ε in tumorigenic prostate epithelial cells compared to control nontumorigenic cells. Cellular cystine may play a critical role in treatment and/or prevention of prostate cancer by regulating PKC activity.

  9. FAK dimerization controls its kinase-dependent functions at focal adhesions

    KAUST Repository

    Brami-Cherrier, Karen

    2014-01-30

    Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

  10. PIAS1-FAK Interaction Promotes the Survival and Progression of Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jerfiz D. Constanzo

    2016-05-01

    Full Text Available The sequence of genomic alterations acquired by cancer cells during tumor progression and metastasis is poorly understood. Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that integrates cytoskeleton remodeling, mitogenic signaling and cell survival. FAK has previously been reported to undergo nuclear localization during cell migration, cell differentiation and apoptosis. However, the mechanism behind FAK nuclear accumulation and its contribution to tumor progression has remained elusive. We report that amplification of FAK and the SUMO E3 ligase PIAS1 gene loci frequently co-occur in non-small cell lung cancer (NSCLC cells, and that both gene products are enriched in a subset of primary NSCLCs. We demonstrate that endogenous FAK and PIAS1 proteins interact in the cytoplasm and the cell nucleus of NSCLC cells. Ectopic expression of PIAS1 promotes proteolytic cleavage of the FAK C-terminus, focal adhesion maturation and FAK nuclear localization. Silencing of PIAS1 deregulates focal adhesion turnover, increases susceptibility to apoptosis in vitro and impairs tumor xenograft formation in vivo. Nuclear FAK in turn stimulates gene transcription favoring DNA repair, cell metabolism and cytoskeleton regulation. Consistently, ablation of FAK by CRISPR/Cas9 editing, results in basal DNA damage, susceptibility to ionizing radiation and impaired oxidative phosphorylation. Our findings provide insight into a mechanism regulating FAK cytoplasm-nuclear distribution and demonstrate that FAK activity in the nucleus promotes NSCLC survival and progression by increasing cell-ECM interaction and DNA repair regulation.

  11. FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Chitsike, Lennox; Wang, Fang; Viswakarma, Navin; Breslin, Peter; Qiu, Wei

    2016-01-01

    Understanding the molecular mechanisms of liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. Focal adhesion kinase (FAK) regulates different cellular functions, including cell survival, proliferation and cell migration. The role of FAK in liver regeneration remains unknown. In this study, we found that Fak is activated and induced during liver regeneration after two-thirds partial hepatectomy (PHx). We used mice with liver-specific deletion of Fak and investigated the role of Fak in liver regeneration in 2/3 PHx model (removal of 2/3 of the liver). We found that specific deletion of Fak accelerates liver regeneration. Fak deletion enhances hepatocyte proliferation prior to day 3 post-PHx but attenuates hepatocyte proliferation 3 days after PHx. Moreover, we demonstrated that the deletion of Fak in liver transiently increases EGFR activation by regulating the TNFα/HB-EGF axis during liver regeneration. Furthermore, we found more apoptosis in Fak-deficient mouse livers compared to WT mouse livers after PHx. Conclusion: Our data suggest that Fak is involved in the process of liver regeneration, and inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients after liver transplantation. PMID:27677358

  12. FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing.

    Directory of Open Access Journals (Sweden)

    Katherine A Owen

    Full Text Available BACKGROUND: Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression. CONCLUSIONS: In the colon, FAK functions as a regulator of

  13. Targeting FAK Radiosensitizes 3-Dimensional Grown Human HNSCC Cells Through Reduced Akt1 and MEK1/2 Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Hehlgans, Stephanie [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt am Main (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Eke, Iris [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Cordes, Nils, E-mail: Nils.Cordes@OncoRay.de [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany)

    2012-08-01

    Purpose: Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures. Methods and Materials: Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation. Results: FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization. Conclusions: Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy.

  14. FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells.

    OpenAIRE

    2014-01-01

    Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also ...

  15. Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

    Science.gov (United States)

    Parsons, Sean A; Sharma, Ritu; Roccamatisi, Dawn L; Zhang, Hong; Petri, Björn; Kubes, Paul; Colarusso, Pina; Patel, Kamala D

    2012-02-01

    During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

  16. FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo12

    Science.gov (United States)

    Gillory, Lauren A; Stewart, Jerry E; Megison, Michael L; Nabers, Hugh C; Mroczek-Musulman, Elizabeth; Beierle, Elizabeth A

    2013-01-01

    Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas. PMID:23544173

  17. Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes

    Directory of Open Access Journals (Sweden)

    Rukmini B. Govekar

    2012-01-01

    Full Text Available The molecular mechanism mediating expression of senescent cell antigen-aggregated or cleaved band 3 and externalized phosphatidylserine (PS on the surface of aged erythrocytes and their premature expression in certain anemias is not completely elucidated. The erythrocytes with these surface modifications undergo macrophage-mediated phagocytosis. In this study, the role of protein kinase C (PKC isoforms in the expression of these surface modifications was investigated. Inhibition of PKC α by 30 μM rottlerin (R30 and 2.3 nM Gö 6976 caused expression of both the senescent cell marker-externalized PS measured by FACS analysis and aggregated band 3 detected by western blotting. In contrast to this observation, but in keeping with literature, PKC activation by phorbol-12-myristate-13-acetate (PMA also led to the expression of senescence markers. We explain this antithesis by demonstrating that PMA-treated cells show reduction in the activity of PKC α, thereby simulating inhibition. The reduction in PKC α activity may be attributed to the known downregulation of PMA-activated PKC α, caused by its membrane translocation and proteolysis. We demonstrate membrane translocation of PKC α in PMA-treated cells to substantiate this inference. Thus loss of PKC α activity either by inhibition or downregulation can cause surface modifications which can trigger erythrophagocytosis.

  18. [Study on FAK regulation of migration of vascular endothelial cells depending upon focal adhesion proteins].

    Science.gov (United States)

    Gao, Min; Liu, Xiaoheng; Sun, Heng; Ren, Hongyi; Wang, Lijuan; Shen, Yang

    2013-06-01

    Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.

  19. A Calcium- and Diacylglycerol-Stimulated Protein Kinase C (PKC), Caenorhabditis elegans PKC-2, Links Thermal Signals to Learned Behavior by Acting in Sensory Neurons and Intestinal Cells.

    Science.gov (United States)

    Land, Marianne; Rubin, Charles S

    2017-10-01

    Ca(2+)- and diacylglycerol (DAG)-activated protein kinase C (cPKC) promotes learning and behavioral plasticity. However, knowledge of in vivo regulation and exact functions of cPKCs that affect behavior is limited. We show that PKC-2, a Caenorhabditis elegans cPKC, is essential for a complex behavior, thermotaxis. C. elegans memorizes a nutrient-associated cultivation temperature (Tc ) and migrates along the Tc within a 17 to 25°C gradient. pkc-2 gene disruption abrogated thermotaxis; a PKC-2 transgene, driven by endogenous pkc-2 promoters, restored thermotaxis behavior in pkc-2(-/-) animals. Cell-specific manipulation of PKC-2 activity revealed that thermotaxis is controlled by cooperative PKC-2-mediated signaling in both AFD sensory neurons and intestinal cells. Cold-directed migration (cryophilic drive) precedes Tc tracking during thermotaxis. Analysis of temperature-directed behaviors elicited by persistent PKC-2 activation or inhibition in AFD (or intestine) disclosed that PKC-2 regulates initiation and duration of cryophilic drive. In AFD neurons, PKC-2 is a Ca(2+) sensor and signal amplifier that operates downstream from cyclic GMP-gated cation channels and distal guanylate cyclases. UNC-18, which regulates neurotransmitter and neuropeptide release from synaptic vesicles, is a critical PKC-2 effector in AFD. UNC-18 variants, created by mutating Ser(311) or Ser(322), disrupt thermotaxis and suppress PKC-2-dependent cryophilic migration. Copyright © 2017 American Society for Microbiology.

  20. CCK causes PKD1 activation in pancreatic acini by signaling through PKC-δ and PKC-independent pathways

    Science.gov (United States)

    Berna, Marc J.; Hoffmann, K. Martin; Tapia, Jose A.; Thill, Michelle; Pace, Andrea; Mantey, Samuel A.; Jensen, Robert T.

    2007-01-01

    Summary Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependant increase in serine phosphorylation by activation of high- and low-affinity CCKA receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-ζ pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-δ, but not PKC-ε, or treatment with PKC-δ translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCKA receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways. PMID:17306383

  1. Involvement of distinct PKC gene products in T cell functions

    Directory of Open Access Journals (Sweden)

    Gottfried eBaier

    2012-08-01

    Full Text Available It is well established that members of the Protein kinase C(PKC family seem to have important roles in T cells. Focusing on the physiological and non-redundant PKC functions established in primary mouse T cells via germline gene-targeting approaches, our current knowledge defines two particularly critical PKC gene products, PKCθ and PKCα, as the flavor of PKC in T cells that appear to have a positive role in signaling pathways that are necessary for full antigen receptor-mediated T cell activation ex vivo and T cell-mediated immunity in vivo. Consistently, in spite of the current dogma that PKCθ inhibition might be sufficient to achieve complete immunosuppressive effects, more recent results have indicated that the pharmacological inhibition of PKCθ, and additionally, at least PKCα, appears to be needed to provide a successful approach for the prevention of allograft rejection and treatment of autoimmune diseases.

  2. Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

    DEFF Research Database (Denmark)

    Horton, Edward R.; Humphries, Jonathan D.; Stutchbury, Ben

    2016-01-01

    of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAKY397 phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK...... inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data...... demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals....

  3. Pharmacological blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide

    OpenAIRE

    2012-01-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15–18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report we tested the FAK autophosphorylation inhibitor, Y15 in DBTRG and U87 gl...

  4. Functional Analysis of PKC Phosphorylation Sites on Myelin Protein Zero

    Institute of Scientific and Technical Information of China (English)

    GangXu; MichaelShy; JohnKamhoz; JanneBalsamo

    2003-01-01

    Objective To analyze the function of Protein kinase C(PKC) phosphorylation sites on mylelin protein zero (P0) at adhesion and myelination.Methods Mutations of p0 cyto-plasmic domain motif (RSTK) and adjacent sequence which are targeted by PKC were studied.Results The point mutations in this region or an adjacent serine residue could abolish P0 adhe-sion function. PKCα,along with the PKC binding protein RACK1,were associated with wild type P0.Inhibition of PKC activity abolished the P0 mediated adhesion.Point mutation in the RSTKtarget site that abolished adhesion did not alter the association of PKC with P0,but deletion of a 14 amino acid region,which included the PSTK motif,could abolish the association.Conclusion PKC mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is neces-sary for P0 mediated adhesion.The alteration of this phoporylation can cause demyelinating neu-ropathy in human.

  5. Soft matrices downregulate FAK activity to promote growth of tumor-repopulating cells.

    Science.gov (United States)

    Tan, Youhua; Wood, Adam Richard; Jia, Qiong; Zhou, Wenwen; Luo, Junyu; Yang, Fang; Chen, Junwei; Chen, Junjian; Sun, Jian; Seong, Jihye; Tajik, Arash; Singh, Rishi; Wang, Ning

    2017-01-29

    Tumor-repopulating cells (TRCs) are a tumorigenic sub-population of cancer cells that drives tumorigenesis. We have recently reported that soft fibrin matrices maintain TRC growth by promoting histone 3 lysine 9 (H3K9) demethylation and Sox2 expression and that Cdc42 expression influences H3K9 methylation. However, the underlying mechanisms of how soft matrices induce H3K9 demethylation remain elusive. Here we find that TRCs exhibit lower focal adhesion kinase (FAK) and H3K9 methylation levels in soft fibrin matrices than control melanoma cells on 2D rigid substrates. Silencing FAK in control melanoma cells decreases H3K9 methylation, whereas overexpressing FAK in tumor-repopulating cells enhances H3K9 methylation. Overexpressing Cdc42 or RhoA in the presence of FAK knockdown restores H3K9 methylation levels. Importantly, silencing FAK, Cdc42, or RhoA promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices, whereas overexpressing each gene suppresses Sox2 expression and reduces growth of TRCs in soft but not in stiff fibrin matrices. Our findings suggest that low FAK mediated by soft fibrin matrices downregulates H3K9 methylation through reduction of Cdc42 and RhoA and promotes TRC growth.

  6. Oral administration of FAK inhibitor TAE226 inhibits the progression of peritoneal dissemination of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Hui-fang [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Takaoka, Munenori [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Bao, Xiao-hong [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Wang, Zhi-gang [College of Life Science, Inner Mongolia University, The Key Laboratory of Mammal Reproductive Biology and Biotechnology, Ministry of Education, Hohhot 010021 (China); Tomono, Yasuko [Division of Molecular and Cell Biology, Shigei Medical Research Institute, 2117 Yamada, Okayama 700-0202 (Japan); Sakurama, Kazufumi; Ohara, Toshiaki [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Fukazawa, Takuya; Yamatsuji, Tomoki [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Fujiwara, Toshiyoshi [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Naomoto, Yoshio, E-mail: ynaomoto@med.kawasaki-m.ac.jp [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer A novel FAK inhibitor TAE226 suppressed FAK activity in HCT116 colon cancer cells. Black-Right-Pointing-Pointer TAE226 suppressed proliferation and migration, with a modest effect on adhesion. Black-Right-Pointing-Pointer Silencing of FAK by siRNA made no obvious difference on cancer cell attachment. Black-Right-Pointing-Pointer TAE226 treatment suppressed the progression of peritoneal dissemination. Black-Right-Pointing-Pointer Oral administration of TAE226 prolonged the survival of tumor-bearing mice. -- Abstract: Peritoneal dissemination is one of the most terrible types of colorectal cancer progression. Focal adhesion kinase (FAK) plays a crucial role in the biological processes of cancer, such as cell attachment, migration, proliferation and survival, all of which are essential for the progression of peritoneal dissemination. Since we and other groups have reported that the inhibition of FAK activity exhibited a potent anticancer effect in several cancer models, we hypothesized that TAE226, a novel ATP-competitive tyrosine kinase inhibitor designed to target FAK, can prevent the occurrence and progression of peritoneal dissemination. In vitro, TAE226 greatly inhibited the proliferation and migration of HCT116 colon cancer cells, while their adhesion on the matrix surface was minimally inhibited when FAK activity and expression was suppressed by TAE226 and siRNA. In vivo, when HCT116 cells were intraperitoneally inoculated in mice, the cells could attach to the peritoneum and begin to grow within 24 h regardless of the pretreatment of cells with TAE226 or FAK-siRNA, suggesting that FAK is not essential, at least for the initial integrin-matrix contact. Interestingly, the treatment of mice before and after inoculation significantly suppressed cell attachment to the peritoneum. Furthermore, oral administration of TAE226 greatly reduced the size of disseminated tumors and prolonged survival in tumor-bearing mice. Taken

  7. pFAK-Y397 overexpression as both a prognostic and a predictive biomarker for patients with metastatic osteosarcoma

    Science.gov (United States)

    Thanapprapasr, Kamolrat; Nartthanarung, Adisak; Thanapprapasr, Duangmani

    2017-01-01

    Focal adhesion kinase (FAK) is important for tumor cell survival and metastasis in various cancers. However, its expression and prognostic value in patients with metastatic osteosarcoma remain unknown. We investigated the expression of FAK and its phosphorylated form (pFAK-Y397) in osteosarcoma tissues from 53 patients by immunohistochemistry and evaluated their correlations with clinicopathologic characteristics and outcomes. The prognostic values were assessed using Kaplan-Meier survival and Cox regression analyses. Total FAK and pFAK-Y397 were overexpressed in 48 (90.6%) and 33 (62.3%) cases, respectively. pFAK-Y397 overexpression was correlated with poor histologic response after neoadjuvant chemotherapy in patients with osteosarcoma regardless of the presence of metastasis or not. Kaplan-Meier curve showed that patients with metastatic osteosarcoma with pFAK-Y397 overexpression had significantly worse overall survival (OS) than those with non-overexpression (P = 0.044). Multivariate Cox regression analysis confirmed pFAK-Y397 overexpression as an independent prognostic predictor for OS and post metastases OS (PMOS) (P = 0.017, P = 0.006, respectively). Age at diagnosis was also an independent indicator for PMOS (P = 0.003). However, total FAK expression was not correlated with any clinicopathologic characteristics or OS in patients with metastatic osteosarcoma. In conclusion, our findings identified FAK as a common aberrant protein overexpression in various subtypes of osteosarcoma. pFAK-Y397 overexpression can be used as a prognostic biomarker predicting poor OS for patients with metastatic osteosarcoma, and the expression of pFAK-Y397 differentiated good and poor responders to neoadjuvant chemotherapy. PMID:28846700

  8. Inhibition of Focal Adhesion Kinase (FAK) Leads to Abrogation of the Malignant Phenotype in Aggressive Pediatric Renal Malignancies

    Science.gov (United States)

    Megison, Michael L.; Gillory, Lauren A.; Stewart, Jerry E.; Nabers, Hugh C.; Mrozcek-Musulman, Elizabeth; Beierle, Elizabeth A.

    2014-01-01

    Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and non-osseous renal Ewing sarcoma. Children with advanced, metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading us to hypothesize that FAK would be present in pediatric kidney tumors and would impact their cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric kidney tumor specimens. We also examined the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines utilizing a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse SK-NEP-1 xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in rare pediatric renal tumors, and may provide desperately needed novel therapeutic strategies and targets for these rare, but difficult to treat, malignancies. PMID:24464916

  9. Selective Function of PKC-θ in T cells

    Institute of Scientific and Technical Information of China (English)

    Santhakumar Manicassamy; Sonal Gupta; Zuoming Sun

    2006-01-01

    T cell activation is a critical process in initiating adaptive immune response since only through this process the na(i)ve antigen specific T cells differentiate into armed effector T cells that mediate the actual immune response.During T cell activation, na(i)ve T cells undergo clonal expansion and acquire the capability to kill target cells infected with pathogens or produce cytokines essential for regulating immune response. Inappropriate activation or inactivation of T cells leads to autoimmunity or severe immunodeficiencies. PKC-θ is selectively expressed in T cells and required for mediating T cell activation process. Mice deficient in PKC-θ exhibit defects in T cell activation, survival and activation-inducedcell death. PKC-θ selectively translocates to immunological synapse and mediates the signals required for activation of NF-κB, AP1 and NFAT that are essential for T cell activation.Furthermore, PKC-θ-/- mice displayed multiple defects in the development of T cell-mediated immune responses in vivo. PKC-θ is thus a critical molecule that regulates T cell function at multiple stages in T cell-mediated immune responses in vivo. Cellular & Molecular Immunology. 2006;3(4):263-270.

  10. Somatic mutational analysis of FAK in breast cancer: A novel gain-of-function mutation due to deletion of exon 33

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xu-Qian [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Liu, Xiang-Fan [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Yao, Ling [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Chen, Chang-Qiang; Gu, Zhi-Dong [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Ni, Pei-Hua [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Zheng, Xin-Min [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY (United States); Fan, Qi-Shi, E-mail: qishifan@126.com [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China)

    2014-01-10

    Highlights: •A novel FAK splicing mutation identified in breast tumor. •FAK-Del33 mutation promotes cell migration and invasion. •FAK-Del33 mutation regulates FAK/Src signal pathway. -- Abstract: Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

  11. Mast cell leukemia with prolonged survival on PKC412/midostaurin.

    Science.gov (United States)

    Xu, Xiangdong; Kreisel, Friederike H; Frater, John L; Hassan, Anjum

    2014-01-01

    Mast cell leukemia (MCL) is a rare and aggressive form of systemic mastocytosis. There are approximately 50 reported cases since 1950s. MCL is refractory to cytoreduction chemotherapy and the average survival is only six months. We report a MCL case in a 71 year-old woman with high tumor load at the initial presentation in 2005, who did not respond to either interleukin-2 or dasatinib therapy. After enrolled in a clinical trial of PKC412 (or Midostaurin) with a daily dose of 100 mg, the patient responded well to PKC412 and became transfusion independent in three months. Since then, her disease had been stably controlled. This is the first report of a high-tumor-load MCL case which achieved prolonged survival (101 months) by PKC 412. The 101-month overall survival is the longest among reported MCL cases in the English literature.

  12. Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

    Directory of Open Access Journals (Sweden)

    Teclise Ng

    Full Text Available Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397 and serine phosphorylation (Ser 732 of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

  13. Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

    Science.gov (United States)

    Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

    2017-07-04

    Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

  14. Improving nutrient values of palm kernel cake (PKC by reducing shell contamination and enzymes supplementation

    Directory of Open Access Journals (Sweden)

    Sinurat AP

    2013-03-01

    Full Text Available Inclusion of palm kernel cake (PKC in poultry feed is limited due to shell contamination and its low nutritive values, despite the increase of PKC production. Therefore, a series of experiment was conducted in order to improve nutritive values of palm kernel cake (PKC by sieving and enzyme supplementation. First experiment was designed to reduce shell content using shiever with different diameters (1, 2 and 4 mm. Shell content was measured manually to determine the effect of the sieving. The second experiment was carried out by blowing the after sieving at 2 mm shieve PKC, to produced heavy, medium and light fractions. The shell content and nutrient contents of the medium and light fractions were compared to those of unsieved PKC. In the third experiment, the sieved PKC was supplemented with 2 enzymes with different concentrations, i.e., BS4 at 10, 15 and 20 ml/kg PKC and a commercial multi enzymes at 0.5, 1.0 and 2.0 g/kg PKC. Digestibility of nutrients (dry matter, crude protein and TME were measured by force feeding method with six replications for each sample. Results of the study showed that sieving with 2 mm diameter siever without blowing was effective in reducing about 50% of PKC shell and improved crude protein, ether extract and amino acids, contents and reduced the crude fiber content of the PKC. Supplementation of enzymes improved the digestibility of dry matter, crude protein and the true metabolisable energy (TME of the PKC. Optimum improvement was obtained when PKC was supplemented with 20 ml BS4 enzymes/kg PKC. Similar improvement was obtained by supplementation of commercial multi enzymes at 2 g/kg PKC. Therefore, in order to improve the nutritive values of PKC, it is suggested to sieve the PKC followed by supplementation of enzyme prior to feeding.

  15. Nuclear FAK and Runx1 Cooperate to Regulate IGFBP3, Cell-Cycle Progression, and Tumor Growth.

    Science.gov (United States)

    Canel, Marta; Byron, Adam; Sims, Andrew H; Cartier, Jessy; Patel, Hitesh; Frame, Margaret C; Brunton, Valerie G; Serrels, Bryan; Serrels, Alan

    2017-08-14

    Nuclear focal adhesion kinase (FAK) is a potentially important regulator of gene expression in cancer, impacting both cellular function and the composition of the surrounding tumor microenvironment. Here, we report in a murine model of skin squamous cell carcinoma (SCC) that nuclear FAK regulates Runx1-dependent transcription of insulin-like growth factor binding protein 3 (IGFBP3), and that this regulates SCC cell-cycle progression and tumor growth in vivo Furthermore, we identified a novel molecular complex between FAK and Runx1 in the nucleus of SCC cells and showed that FAK interacted with a number of Runx1-regulatory proteins, including Sin3a and other epigenetic modifiers known to alter Runx1 transcriptional function through posttranslational modification. These findings provide important new insights into the role of FAK as a scaffolding protein in molecular complexes that regulate gene transcription. Cancer Res; 77(19); 1-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. PKC in Regenerative Therapy: New Insights for Old Targets

    Directory of Open Access Journals (Sweden)

    Marta Rui

    2017-05-01

    Full Text Available Effective therapies for chronic or non-healing wounds are still lacking. These tissue insults often result in severe clinical complications (i.e., infections and/or amputation and sometimes lead to patient death. Accordingly, several research groups have focused their efforts in finding innovative and powerful therapeutic strategies to overcome these issues. On the basis of these considerations, the comprehension of the molecular cascades behind these pathological conditions could allow the identification of molecules against chronic wounds. In this context, the regulation of the Protein Kinase C (PKC cascade has gained relevance in the prevention and/or reparation of tissue damages. This class of phosphorylating enzymes has already been considered for different physiological and pathological pathways and modulation of such enzymes may be useful in reparative processes. Herein, the recent developments in this field will be disclosed, highlighting the pivotal role of PKC α and δ in regenerative medicine. Moreover, an overview of well-established PKC ligands, acting via the modulation of these isoenzymes, will be deeply investigated. This study is aimed at re-evaluating widely known PKC modulators, currently utilized for treating other diseases, as fruitful molecules in wound-healing.

  17. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  18. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

    DEFF Research Database (Denmark)

    Lim, Ssang-Taek; Longley, Robert L; Couchman, John R

    2003-01-01

    Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC...... alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC...... alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating...

  19. Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility

    Science.gov (United States)

    Subauste, M. Cecilia; Pertz, Olivier; Adamson, Eileen D.; Turner, Christopher E.; Junger, Sachiko; Hahn, Klaus M.

    2004-01-01

    Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction. PMID:15138291

  20. Pharmacological blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide

    Science.gov (United States)

    Golubovskaya, Vita M.; Huang, Grace; Ho, Baotran; Yemma, Michael; Morrison, Carl D.; Lee, Jisook; Eliceiri, Brian P.; Cance, William G.

    2012-01-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15–18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report we tested the FAK autophosphorylation inhibitor, Y15 in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose and time-dependent manner, caused apoptosis and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide. PMID:23243059

  1. Pharmacologic blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide.

    Science.gov (United States)

    Golubovskaya, Vita M; Huang, Grace; Ho, Baotran; Yemma, Michael; Morrison, Carl D; Lee, Jisook; Eliceiri, Brian P; Cance, William G

    2013-02-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.

  2. Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

    Science.gov (United States)

    Ding, Lei; Wang, Liyong; Sui, Leiming; Zhao, Huanying; Xu, Xiaoxue; Li, Tengyan; Wang, Xiaonan; Li, Wenjing; Zhou, Ping; Kong, Lu

    2016-08-01

    The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

  3. Correlation between Gli2, FAK expression in colonic adenocarcinoma tissue with different clinical pathological characteristics and cancer cell proliferation, invasion

    Institute of Scientific and Technical Information of China (English)

    Zhe Su

    2017-01-01

    Objective:To study the correlation between glioma-associated oncogene homologue 2 (Gli2), focal adhesion kinase (FAK) expression in colonic adenocarcinoma tissue with different clinical pathological characteristics and cancer cell proliferation, invasion.Methods: 56 patients with colonic adenocarcinoma who received surgical resection in our hospital between May 2012 and December 2015 were selected, cancer tissue and para-carcinoma tissue were collected respectively, immunohistochemical staining was used to detect the Gli2 and FAK protein-positive rate, and fluorescence quantitative PCR was used to determine the mRNA expression of Gli2 and FAK as well as the proliferation and invasionn genes.Results:Gli2 and FAK mRNA expression and protein-positive rate in colonic adenocarcinoma tissues were significantly higher than those in para-carcinoma tissues (P<0.05); Gli2 and FAK mRNA expression and protein-positive rate in colonic adenocarcinoma tissues with low differentiation, no differentiation, extraserosal infiltration and Dukes stage D were significantly higher than those in colonic adenocarcinoma tissues with high differentiation, medium differentiation, intraserosal infiltration, Dukes stage B-C (P<0.05); CyclinD1, CDK4, c-myc, N-cadherin and vimentin mRNA expression in Gli2- and FAK-positive colonic adenocarcinoma tissues were significantly higher than those in Gli2- and FAK-negative colonic adenocarcinoma tissues (P<0.05).Conclusions:Gli2 and FAK expression are high in colonic adenocarcinoma tissues and associated with the clinical pathological staging of tumor, and highly expressed Gli2 and FAK can promote cell proliferation and invasion.

  4. αν and β1 Integrins mediate Aβ-induced neurotoxicity in hippocampal neurons via the FAK signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hai-Yan Han

    Full Text Available αν and β1 integrins mediate Aβ-induced neurotoxicity in primary hippocampal neurons. We treated hippocampal neurons with 2.5 µg/mL 17E6 and 5 µg/mL ab58524, which are specific αν and β1 integrin antagonists, respectively, for 42 h prior to 10 µM Aβ treatment. Next, we employed small interfering RNA (siRNA to silence focal adhesion kinase (FAK, a downstream target gene of integrins. The siRNAs were designed with a target sequence, an MOI of 10 and the addition of 5 µg/mL polybrene. Under these conditions, the neurons were transfected and the apoptosis of different cell types was detected. Moreover, we used real-time PCR and Western blotting analyses to detect the expression of FAK and ρFAK genes in different cell types and investigated the underlying mechanism and signal pathway by which αν and β1 integrins mediate Aβ-induced neurotoxicity in hippocampal neurons. An MTT assay showed that both 17E6 and ab58524 significantly increased cell viability compared with the Aβ-treated neurons (P<0.01 and P<0.05, respectively. However, this protective effect was markedly attenuated after transfection with silencing FAK (siFAK. Moreover, TUNEL immunostaining and flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ (P<0.05 compared with the Aβ-treated cells. However, this protective effect was reversed with siFAK treatment. Both the gene and protein expression of FAK increased after Aβ treatment. Interestingly, as the gene and protein levels of FAK decreased, the ρFAK protein expression markedly increased. Furthermore, both the gene and protein expression of FAK and ρFAK were significantly diminished. Thus, we concluded that both αν and β1 integrins interfered with Aβ-induced neurotoxicity in hippocampal neurons and that this mechanism partially contributes to the activation of the Integrin-FAK signaling pathway.

  5. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    Science.gov (United States)

    2013-09-01

    tyrosine-kinase and G-protein-coupled receptor ( GPCR ) activation [3]. Despite our extensive knowledge on PKC regulation and function, the specific roles for...the receptor. LNCaP cells were stimulate with TNFα (25 ng/ml) for 5 min and subjected to IP with an anti-TNFR-I antibody . Western blots were probed...with phosphoamino acid antibodies . It was observed that TNFα treatment mediates the phosphorylation on serine and threonine residues but not on

  6. Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy.

    Directory of Open Access Journals (Sweden)

    Shaon Naskar

    Full Text Available A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy or detrimental (pathological hypertrophy. This study was undertaken to establish the role of different protein kinase-C (PKC isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

  7. Differential and Conditional Activation of PKC-Isoforms Dictates Cardiac Adaptation during Physiological to Pathological Hypertrophy

    Science.gov (United States)

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation. PMID:25116170

  8. Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy.

    Science.gov (United States)

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

  9. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    Science.gov (United States)

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-05-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

  10. PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity

    Science.gov (United States)

    Matsuda, Shinya; Kawamoto, Kohei; Miyamoto, Kenji; Tsuji, Akihiko; Yuasa, Keizo

    2017-01-01

    PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway. PMID:28361970

  11. The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

    Science.gov (United States)

    Kanno, Takeshi; Yamamoto, Hideyuki; Yaguchi, Takahiro; Hi, Rika; Mukasa, Takeshi; Fujikawa, Hirokazu; Nagata, Tetsu; Yamamoto, Satoshi; Tanaka, Akito; Nishizaki, Tomoyuki

    2006-06-01

    This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

  12. Role of Peg10 in FAK-mediated CAM-DR in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    ZHANG Hai

    2015-02-01

    Full Text Available ObjectiveTo preliminarily investigate the relationship of Peg10 with focal adhesion kinase (FAK-mediated cell adhesion-mediated drug resistance (CAM-DR in hepatocellular carcinoma (HCC, and to explore the underlying molecular mechanism. MethodsThe effects of 5-fluorouracil (5-Fu and adriamycin (ADR on the proliferation of LO2 cells, ADR-resistant human HCC cells BEL-7404/ADR (7404/ADR, and Peg10-silenced cells siRNA-BEL-7404/ADR (siRNA-7404/ADR were examined by MTT assay. To build a tumor-bearing nude mouse model, 48 rats were randomly divided into six groups (n=8 each: groups A, C, and D were injected with 7404/ADR cells; groups B, E, and F were injected with siRNA-7404/ADR cells. For experimental treatments, groups A and B revived saline by intravenous injection through the tail vein; groups C and E were given 5-Fu; groups D and F received ADR by intravenous injection through the tail vein; tumor mass volume was measured after injection. Peg10 mRNA expression was assayed by RT-PCR, and PEG10, p-FAK, p-JNK, p-ERK, and p-P38 protein expression was assayed by Western blotting. ResultsThe 24-h IC50 values of ADR and 5-Fu on SiRNA-7404/ADR were both significantly reduced compared with those on 7404/ADR (t=7.641 and 7.560, respectively; both P<0.01. Significantly smaller tumor mass volume was observed in groups B, C, and D than in group A (P<0.05, and in groups E and F than in group B (P<0.01. Additionally, tumor mass volume was significantly different between groups E and C as well as between groups F and D (P<0.05. PEG10 mRNA was rarely expressed in groups B, E, and F. Compared with that in group A, PEG10 mRNA expression in groups C and D was relatively reduced. PEG10 protein was rarely expressed in group B and its expression level significantly differed from that in group A (P<0.01. Additionally, there were statistically significant differences in p-FAK, p-JNK, p-ERK, and p38MAPK protein expression between groups B and A (P<0.05. In

  13. Abnormal cell properties and down-regulated FAK-Src complex signaling in B lymphoblasts of autistic subjects.

    Science.gov (United States)

    Wei, Hongen; Malik, Mazhar; Sheikh, Ashfaq M; Merz, George; Ted Brown, W; Li, Xiaohong

    2011-07-01

    Recent studies suggest that one of the major pathways to the pathogenesis of autism is reduced cell migration. Focal adhesion kinase (FAK) has an important role in neural migration, dendritic morphological characteristics, axonal branching, and synapse formation. The FAK-Src complex, activated by upstream reelin and integrin β1, can initiate a cascade of phosphorylation events to trigger multiple intracellular pathways, including mitogen-activated protein kinase-extracellular signal-regulated kinase and phosphatidylinositol 3-kinase-Akt signaling. In this study, by using B lymphoblasts as a model, we tested whether integrin β1 and FAK-Src signaling are abnormally regulated in autism and whether abnormal FAK-Src signaling leads to defects in B-lymphoblast adhesion, migration, proliferation, and IgG production. To our knowledge, for the first time, we show that protein expression levels of both integrin β1 and FAK are significantly decreased in autistic lymphoblasts and that Src protein expression and the phosphorylation of an active site (Y416) are also significantly decreased. We also found that lymphoblasts from autistic subjects exhibit significantly decreased migration, increased adhesion properties, and an impaired capacity for IgG production. The overexpression of FAK in autistic lymphoblasts countered the adhesion and migration defects. In addition, we demonstrate that FAK mediates its effect through the activation of Src, phosphatidylinositol 3-kinase-Akt, and mitogen-activated protein kinase signaling cascades and that paxillin is also likely involved in the regulation of adhesion and migration in autistic lymphoblasts. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  14. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

    DEFF Research Database (Denmark)

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid

    2016-01-01

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not unde...

  15. Regulation of the viability of Nf1 deficient cells by PKC isoforms.

    Science.gov (United States)

    Zhou, Xiaodong; Shen, Ling; Parris, Toshima; Huang, Junchi; Yi, Bo; Helou, Khalil; Chen, Changyan

    2014-11-15

    Suppression of protein kinase C (PKC) is known to be synthetically lethal with ras mutations in various types of cancer cells. The studies also showed that blockade of PKC affected the viability of Nf1 deficient cells. Since PKC family consists of more than 10 isoforms, our study aimed at identifying which isoform(s) played the crucial role in sensitizing Nf1 deficient cells to apoptosis. Using genetic and chemical PKC inhibitors, we demonstrated that the concurrent inhibition of PKC α and β induced Nf1 deficient ST or 96.2 cells, but not SNF02.2 cells with a normal Nf1 or ST cells ectopically expressing Nf1 effective domain gene, to apoptosis. In this process, PKC δ in Nf1 deficient cells, but not in ST/Nf1 cells, was upregulated and translocated to the nucleus. Furthermore, caspase 3 was cleaved and cytochrome c was released to the cytosol. Thus, it appeared that PKC δ and α/β are the crucial components for sustaining the aberrant Ras signaling and further viability of Nf1 deficient cells. The abrogation of these two isoforms activated their opponent PKC δ for switching on the caspase 3-governed apoptotic machinery.

  16. Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

    Science.gov (United States)

    Cannons, Jennifer L.; Wu, Julie Z.; Gomez-Rodriguez, Julio; Zhang, Jinyi; Dong, Baoxia; Liu, Yin; Shaw, Stephen; Siminovitch, Katherine A.; Schwartzberg, Pamela L.

    2012-01-01

    SAP, an adaptor molecule that recruits Fyn to the SLAM-family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4+ T cells from SAP-deficient mice have defective TCR-induced IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4+ T cells exhibit decreased PKC-θ recruitment upon TCR stimulation. We demonstrate here using GST-pulldowns and co-immunoprecipitation studies that SAP constitutively associates with PKC-θ in T cells. SAP-PKC-θ interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP’s interactions with PKC-θ occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-θ recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-θ, like SAP, was required for SLAM-mediated increases in IL-4 production and conversely, membrane-targeted PKC-θ mutants rescued IL-4 expression in SAP−/− CD4+ T cells, providing genetic evidence that PKC-θ is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production. PMID:20668219

  17. Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells

    Directory of Open Access Journals (Sweden)

    Zhang Yumin

    2011-04-01

    Full Text Available Abstract Background Excessive production of nitric oxide (NO by inducible nitric oxide synthase (iNOS in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia. Methods Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF-κB in activated microglia was examined by determining NF-κB transcriptional response element- (TRE- driven, promoter-mediated luciferase activity. Results Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC β attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF-κB activation that leads to the expression of iNOS in reactive microglia. Conclusions Our results identify PKC δ and β as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF-κB. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.

  18. Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

    Energy Technology Data Exchange (ETDEWEB)

    Matsuoka, Hiroshi [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-Osaka 577-8502 (Japan); Department of Pharmacy, Nara Hospital, Kinki University School of Medicine, 1248-1 Ikoma, Nara 630-0293 (Japan); Tsubaki, Masanobu [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-Osaka 577-8502 (Japan); Yamazoe, Yuzuru [Department of Pharmacy, Kinki University Hospital, Osakasayama, Osaka 589-8511 (Japan); Ogaki, Mitsuhiko [Department of Pharmacy, Higahiosaka City General Hospital, Higashi-osaka, Osaka 578-8588 (Japan); Satou, Takao; Itoh, Tatsuki [Department of Pathology, Kinki University School of Medicine, Osakasayama, Osaka 589-8511 (Japan); Kusunoki, Takashi [Department of Otolaryngology, Juntendo University School of Medicine, Tokyo (Japan); Nishida, Shozo, E-mail: nishida@phar.kindai.ac.jp [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-Osaka 577-8502 (Japan)

    2009-07-15

    In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC{alpha} and PKC{delta} phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.

  19. Effect of PKC pathway on G1/S progression control in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The effect of PKC activity on G1/S progression in HeLa cells has been studied.The result shows that (ⅰ) PKC activity alteration in G1 phase affects G1/S progression in HeLa cells.It has been observed that G1/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X.(ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G1 phase.(ⅲ) During G1/S progression,the expression of CyclinD1 is stimulated by TPA treatment and inhibited by GF-109203X treatment.There is no effect on the expression of CDK4.It is likely that PKC pathway regulates G1/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.

  20. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    Science.gov (United States)

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  1. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    Science.gov (United States)

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  2. Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Anindya Chatterjee

    2014-11-01

    Full Text Available Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML and myeloproliferative neoplasms (MPNs, and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.

  3. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M

    1998-01-01

    B-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.......beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...... experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fak...

  4. MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner

    Science.gov (United States)

    Moncayo, Gerald; Lin, Da; McCarthy, Michael T.; Watson, Aleksandra A.; O’Callaghan, Christopher A.

    2017-01-01

    MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell–cell contact or loss of cell–matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis. PMID:28154561

  5. A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

    Science.gov (United States)

    Bae, Yong Ho; Mui, Keeley L; Hsu, Bernadette Y; Liu, Shu-Lin; Cretu, Alexandra; Razinia, Ziba; Xu, Tina; Puré, Ellen; Assoian, Richard K

    2014-06-17

    Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.

  6. Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src.

    Directory of Open Access Journals (Sweden)

    Maoyong Fu

    Full Text Available Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2, a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

  7. Live-imaging of PKC translocation in Sf9 cells and in aplysia sensory neurons.

    Science.gov (United States)

    Farah, Carole A; Sossin, Wayne S

    2011-04-06

    Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using

  8. Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole A Kaplan

    Full Text Available Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

  9. Translocation of classical PKC and cortical granule exocytosis of human oocyte in germinal vesicle and metaphase Ⅱ stage

    Institute of Scientific and Technical Information of China (English)

    Xue-qing WU; Xiao ZHANG; Xiao-hong LI; Hui-hong CHENG; Yan-rong KUAI; Song WANG; Ying-lu GUO

    2006-01-01

    Aim: Protein kinase C (PKC) is as a family of serine/threonine kinases that can be activated by Ca2+, phospholipid and diacylglycerol. PKC plays an important role in oocyte maturation and activation. This study was undertaken to investigate classical PKC (cPKC) in human oocyte maturation and activation. Methods: Germinal vesicle (GV) and metaphase Ⅱ (MII) stage oocytes were collected from healthy women. The expression and distribution of cPKC were investigated by immunoflourescence. MII oocytes were treated with PKC activator or inhibitor and imaged using a laser confocal scanning microscope (LCSM). Results: In GV oocytes, PKC α,β1 and γ were localized to the germinal vesicles, with a weak expression in ooplasm. In MII oocytes, PKCα, β1 and γ were distributed evenly in ooplasm. After treatment with PKC activator, phorbol 12-myristate 13-acetate (PMA), cPKC translocated to the periphery of oocyte, and cortical granules (CG) exocytosis was found. When the oocytes were treated with PKC inhibitor, staurosporine, no translocation of cPKC and CG exocytosis were found. Conclusion: PKCα, β1 and γ exist in human oocytes and activation of these subunits could induce CG exocytosis in MII stage.

  10. Regulation of Transcriptional Networks by PKC Isozymes: Identification of c-Rel as a Key Transcription Factor for PKC-Regulated Genes.

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    Rachana Garg

    Full Text Available Activation of protein kinase C (PKC, a family of serine-threonine kinases widely implicated in cancer progression, has major impact on gene expression. In a recent genome-wide analysis of prostate cancer cells we identified distinctive gene expression profiles controlled by individual PKC isozymes and highlighted a prominent role for PKCδ in transcriptional activation.Here we carried out a thorough bioinformatics analysis to dissect transcriptional networks controlled by PKCα, PKCδ, and PKCε, the main diacylglycerol/phorbol ester PKCs expressed in prostate cancer cells. Despite the remarkable differences in the patterns of transcriptional responsive elements (REs regulated by each PKC, we found that c-Rel represents the most frequent RE in promoters regulated by all three PKCs. In addition, promoters of PKCδ-regulated genes were particularly enriched with REs for CREB, NF-E2, RREB, SRF, Oct-1, Evi-1, and NF-κB. Most notably, by using transcription factor-specific RNAi we were able to identify subsets of PKCδ-regulated genes modulated by c-Rel and CREB. Furthermore, PKCδ-regulated genes condensed under the c-Rel transcriptional regulation display significant functional interconnections with biological processes such as angiogenesis, inflammatory response, and cell motility.Our study identified candidate transcription factors in the promoters of PKC regulated genes, in particular c-Rel was found as a key transcription factor in the control of PKCδ-regulated genes. The deconvolution of PKC-regulated transcriptional networks and their nodes may greatly help in the identification of PKC effectors and have significant therapeutics implications.

  11. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  12. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

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    Vita M. Golubovskaya

    2014-01-01

    Full Text Available Focal Adhesion Kinase (FAK is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53+/+ and p53−/− cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05 in HCT116 p53+/+ cells but not in p53−/− cells. Among up-regulated genes in HCT p53+/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53+/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  13. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  14. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    Science.gov (United States)

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

  15. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta, E-mail: etta@bgu.ac.il

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  16. Small Review: Strategies for Palm Kernel Cake (PKC) As a New Potential Substrate in Biofuel Production

    OpenAIRE

    Hafiza Shukor; Mohd Sahaid Kalil; Nurina Anuar; Aidil Abdul Hamid; Asmidar Hanan

    2013-01-01

    The economic dependency on fossil fuels and the resulting effects on climate and environment have put tremendous focus on utilizing fermentable sugars from lignocellulose, the largest known renewable carbohydrate source. Palm kernel cake (PKC) is a residue from palm oil extraction presently only used as a low protein feed supplement. It’s contains 50% fermentable hexose sugars present in the form of glucan and mainly galactomannan. This makes PKC an interesting feedstock for processing into b...

  17. Synthesis and biological activities of simplified analogs of the natural PKC ligands, bryostatin-1 and aplysiatoxin.

    Science.gov (United States)

    Irie, Kazuhiro; Yanagita, Ryo C

    2014-04-01

    Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads. Copyright © 2014 The Chemical Society of Japan and Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. DNA damage targets PKC{eta} to the nuclear membrane via its C1b domain

    Energy Technology Data Exchange (ETDEWEB)

    Tamarkin, Ana; Zurgil, Udi; Braiman, Alex; Hai, Naama; Krasnitsky, Ella; Maissel, Adva; Ben-Ari, Assaf; Yankelovich, Liat; Livneh, Etta, E-mail: etta@bgumail.bgu.ac.il

    2011-06-10

    Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKC{eta}, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKC{eta} is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKC{eta} expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKC{eta}, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKC{eta} to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.

  19. Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells.

    Science.gov (United States)

    Zhou, Xiaodong; Kim, Sung-Hoon; Shen, Ling; Lee, Hyo-Jung; Chen, Changyan

    2014-01-01

    Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.

  20. A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

    Science.gov (United States)

    Gamblin, Clémence L.; Hardy, Émilie J.-L.; Chartier, François J.-M.; Bisson, Nicolas

    2014-01-01

    During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells. PMID:24515345

  1. Bile acid deoxycholate induces differential subcellular localisation of the PKC isoenzymes beta 1, epsilon and delta in colonic epithelial cells in a sodium butyrate insensitive manner.

    Science.gov (United States)

    Looby, Eileen; Long, Aideen; Kelleher, Dermot; Volkov, Yuri

    2005-05-10

    Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta. DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 microM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered. Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

  2. Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

    Science.gov (United States)

    Semaan, Noha; Alsaleh, Ghada; Gottenberg, Jacques-Eric; Wachsmann, Dominique; Sibilia, Jean

    2008-03-01

    MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.

  3. Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells

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    Matias Julian Stagno

    2017-07-01

    Full Text Available Background/Aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

  4. 1-o-acetylbritannilactone (ABL) inhibits angiogenesis and lung cancer cell growth through regulating VEGF-Src-FAK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zhengfu, He; Hu, Zhang; Huiwen, Miao; Zhijun, Li [Department of Thoracic Surgery, Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China); Jiaojie, Zhou [Zhejiang University School of Medicine, Hangzhou (China); Xiaoyi, Yan, E-mail: xiaoyiyan163@163.com [Zhejiang University School of Medicine, Hangzhou (China); Xiujun, Cai, E-mail: xiujuncaomaj@163.com [Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China)

    2015-08-21

    The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs. Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.

  5. DIFFERENTIAL DISTRIBUTION OF PROTEIN-KINASE-C (PKC-ALPHA-BETA AND PKC-GAMMA) ISOENZYME IMMUNOREACTIVITY IN THE CHICK BRAIN

    NARCIS (Netherlands)

    VANDERZEE, EA; BOLHUIS, JJ; SOLOMONIA, RO; HORN, G; LUITEN, PGM

    1995-01-01

    Protein kinase C (PKC) is involved in neural plasticity. The phosphorylation of the myristoylated alanine-rich protein kinase C substrate (MARCKS) in the left intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain has been shown previously to correlate significantly with the streng

  6. Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

    Directory of Open Access Journals (Sweden)

    McCarthy John J

    2005-08-01

    Full Text Available Abstract Background Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. Results We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. Conclusions Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

  7. Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor.

    Science.gov (United States)

    Premkumar, Louis S; Qi, Zhan-Heng; Van Buren, Jeremy; Raisinghani, Manish

    2004-03-01

    The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.

  8. PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

    Science.gov (United States)

    Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

    2011-06-01

    The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

  9. Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Pei-Jie Shi

    2016-02-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK down-regulation in the treatment of ALL. Methods The effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID mouse model. Results When combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2 gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis. Conclusions FAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel

  10. PKC{eta} confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara [The Schraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben - Gurion University, Beer Sheva 84105 (Israel); Livneh, Etta, E-mail: etta@bgumail.bgu.ac.il [The Schraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben - Gurion University, Beer Sheva 84105 (Israel)

    2009-09-10

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKC{eta}, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKC{eta} in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKC{eta}. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKC{eta} expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKC{eta} is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKC{eta} could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  11. Increased aPKC Expression Correlates with Prostatic Adenocarcinoma Gleason Score and Tumor Stage in the Japanese Population

    Directory of Open Access Journals (Sweden)

    Anthony S. Perry

    2014-01-01

    Full Text Available Background. Levels of the protein kinase aPKC have been previously correlated with prostate cancer prognosis in a British cohort. However, prostate cancer incidence and progression rates, as well as genetic changes in this disease, show strong ethnic variance, particularly in Asian populations. Objective. The aim of this study was to validate association of aPKC expression with prostatic adenocarcinoma stages in a Japanese cohort. Methods. Tissue microarrays consisting of 142 malignant prostate cancer cases and 21 benign prostate tissues were subject to immunohistological staining for aPKC. aPKC staining intensity was scored by three independent pathologists and categorized as absent (0, dim (1+, intermediate (2+, and bright (3+. aPKC staining intensities were correlated with Gleason score and tumor stage. Results. Increased aPKC staining was observed in malignant prostate cancer, in comparison to benign tissue. Additionally, aPKC staining levels correlated with Gleason score and tumor stage. Our results extend the association of aPKC with prostate cancer to a Japanese population and establish the suitability of aPKC as a universal prostate cancer biomarker that performs consistently across ethnicities.

  12. ETV6-NTRK3 as a therapeutic target of small molecule inhibitor PKC412

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Hoang Thanh, E-mail: kk086406@mgs.k.u-tokyo.ac.jp [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Ly, Bui Thi Kim [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Kano, Yasuhiko [Division of Hematology and Medical Oncology, Tochigi Cancer Center, Tochigi 321-0293 (Japan); Tojo, Arinobu [Division of Molecular Therapy, Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Watanabe, Toshiki [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Sato, Yuko [Musashimurayama Hospital, Musashimurayama, Tokyo 208-0011 (Japan)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer ETV6-NTRK3 is an oncogene with transformation activity in multiple cell lineages. Black-Right-Pointing-Pointer PKC412 could block ETV6-NTRK3 activation. Black-Right-Pointing-Pointer Loss of ETV6-NTRK3 phosphorylation leads to inactivation of its downstream signaling pathway. Black-Right-Pointing-Pointer Inhibition of ETV6-NTRK3 activation by PKC412 could be a novel strategy for the treatment. -- Abstract: The ETV6-NTRK3 (EN) fusion gene which encodes a chimeric tyrosine kinase was first identified by cloning of the t(12;15)(p13;q25) translocation in congenital fibrosarcoma (CFS). Since then, EN has been also found in congenital mesoblastic nephroma (CMN), secretory breast carcinoma (SBC) and acute myelogenous leukemia (AML). Using IMS-M2 and M0-91 cell lines harboring the EN fusion gene, and Ba/F3 cells stably transfected with EN, we demonstrated that PKC412, also known as midostaurin, is an inhibitor of EN. Inhibition of EN activity by PKC412 suppressed the activity of it downstream molecules leading to inhibition of cell proliferation and induction of apoptosis. Our data for the first time suggested that PKC412 could serve as therapeutic drug for treatment of patients with this fusion.

  13. Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

    Science.gov (United States)

    Morais-de-Sá, Eurico; Mukherjee, Avik; Lowe, Nick; St Johnston, Daniel

    2014-08-01

    The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

  14. PKC-theta in regulatory and effector T-cell functions

    Directory of Open Access Journals (Sweden)

    Vedran eBrezar

    2015-10-01

    Full Text Available One of the major goals in immunology research is to understand the regulatory mechanisms that underpin the rapid switch on/off of robust and efficient effector (Teff or regulatory (Tregs T-cell responses. Understanding the molecular mechanisms underlying the regulation of such responses is critical for the development of effective therapies. T-cell activation involves the engagement of T-cell receptor and co-stimulatory signals, but the subsequent recruitment of serine/threonine-specific protein Kinase C-theta (PKC-θ to the immunological synapse is instrumental for the formation of signalling complexes, that ultimately lead to a transcriptional network in T cells. Recent studies demonstrated that major differences between Teffs and Tregs occurred at the immunological synapse where its formation induces altered signalling pathways in Tregs. These pathways are characterized by reduced recruitment of PKC-θ, suggesting that PKC-θ inhibits Tregs suppressive function in a negative feedback loop. As the balance of Teffs and Tregs has been shown to be central in several diseases, it was not surprising that some studies revealed that PKC-θ plays a major role in the regulation of this balance.This review will examine recent knowledge on the role of PKC-θ in T-cell transcriptional responses and how this protein can impact on the function of both Tregs and Teffs.

  15. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  16. Role of PKC isozymes in low-power light-stimulated proliferation of cultured skin cells

    Science.gov (United States)

    Grossman, Nili; Kleitman, Vered; Meller, Julia; Kaufmann, Roland; Akgun, Nermin; Ruck, Angelika; Livneh, Etta; Lubart, Rachel

    2000-11-01

    Exposure of cultured skin cells to low power visible light leads to a transiently stimulated proliferation. Facilitation of this response requires the presence of active PKC, elevation of intracellular calcium, and involves reactive oxygen species. In the present study, the role of PKC(alpha) and PCK(eta) was examined using paired murine fibroblasts, differing in the level of these isozymes expression. The ability of the cells to respond to low power UVA light or HeNe laser by stimulated proliferation was correlated with an active state or overexpression of PKC(alpha) , but not PKC(eta) . A parallel response was obtained in cells that were loaded with A1PcS4 before photosensitization. Whenever this latter treatment caused a light-stimulated inhibition, it was accompanied by the intracellular calcium and photosensitizer dynamics typical of the effect of PDT on rate epithelial cells. Accordingly, added antioxidants that suppressed light-stimulated proliferation also suppressed this light-stimulated inhibition. The model systems employed in this study are the first to demonstrate the specific effect of PKC isozymes on light-stimulated proliferation, in relation to oxidative stress, and indicate their dual role in light-tissue interaction.

  17. The Expression of Integrinβ1 and FAK in Pituitary Adenomas and Their Correlation with Invasiveness

    Institute of Scientific and Technical Information of China (English)

    Feng WAN; Kai SHU; Ting LEI; Delin XUE

    2008-01-01

    Summary: The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 (INTβ1) and focal adhesion kinase (FAK) by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was analyzed. The results showed that INTβ1 was expressed in 46 cases (93.9%) and FAK in 36 cases (73.5%), respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

  18. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

    Energy Technology Data Exchange (ETDEWEB)

    Golubovskaya, Vita M., E-mail: Vita.Golubovskaya@roswellpark.org; Ho, Baotran [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Conroy, Jeffrey [Genomics Shared Resource, Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Liu, Song; Wang, Dan [Bioinformatics Core Facility, Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Cance, William G. [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States)

    2014-01-21

    Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53{sup +/+} and p53{sup −/−} cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53{sup +/+} cells but not in p53{sup −/−} cells. Among up-regulated genes in HCT p53{sup +/+} cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53{sup +/+} colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  19. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

    Science.gov (United States)

    Cremona, M Laura; Matthies, Heinrich J G; Pau, Kelvin; Bowton, Erica; Speed, Nicole; Lute, Brandon J; Anderson, Monique; Sen, Namita; Robertson, Sabrina D; Vaughan, Roxanne A; Rothman, James E; Galli, Aurelio; Javitch, Jonathan A; Yamamoto, Ai

    2011-04-01

    Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

  1. Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

    Science.gov (United States)

    Miao, Lei; Xin, Xiaoming; Xin, Hong; Shen, Xiaoyan; Zhu, Yi-Zhun

    2016-03-01

    Myocardial infarction (MI) triggers an inflammatory reaction, in which macrophages are of key importance for tissue repairing. Infiltration and/or migration of macrophages into the infarct area early after MI is critical for infarct healing, vascularization, and cardiac function. Hydrogen sulfide (H2S) has been demonstrated to possess cardioprotective effects post MI and during the progress of cardiac remodeling. However, the specific molecular and cellular mechanisms involved in macrophage recruitment by H2S remain to be identified. In this study, the NaHS (exogenous sources of H2S) treatment exerted an increased infiltration of macrophages into the infarcted myocardium at early stage of MI cardiac tissues in both wild type (WT) and cystathionine-γ-lyase-knockout (CSE-KO) mice. And NaHS accelerated the migration of macrophage cells in vitro. While, the inhibitors not only significantly diminished the migratory ability in response to NaHS, but also blocked the activation of phospho-Src, -Pyk2, -FAK397, and -FAK925. Furthermore, NaHS induced the internalization of integrin β1 on macrophage surface, but, integrin β1 silencing inhibited macrophage migration and Src signaling activation. These results indicate that H2S may have the potential as an anti-infarct of MI by governing macrophage migration, which was achieved by accelerating internalization of integrin β1 and activating downstream Src-FAK/Pyk2-Rac pathway.

  2. EFFECTS OF INTEGRIN ALPHA ⅡbR995A MUTATION ON RECEPTOR AFFINITY AND pp 125 (FAK) PHOSPHORYLATION

    Institute of Scientific and Technical Information of China (English)

    Xue-yuan Tang; Zai-fu Jian; Guo-ping Wang; Hong-hui Yang; Wei Liu

    2004-01-01

    Objective To investigate the role of cytoplasmic domain of integrin alpha Ⅱb in platelet signal transduction.Methods Binding capacity of integrin alpha ⅡbR995Ato antibody platelet activation complex-1 (PAC-1) and pp125focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.Results Without activation, wild-type alpha Ⅱ bbeta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha ⅡbR995Aeta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK)in wild-type alpha Ⅱbbeta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.Conclusion The mutation in integrin alpha ⅡbR995Aalters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

  3. Kibra and aPKC regulate starvation-induced autophagy in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Ahrum [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Neufeld, Thomas P. [Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States); Choe, Joonho, E-mail: jchoe@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-04

    Autophagy is a bulk degradation system that functions in response to cellular stresses such as metabolic stress, endoplasmic reticulum stress, oxidative stress, and developmental processes. During autophagy, cytoplasmic components are captured in double-membrane vesicles called autophagosomes. The autophagosome fuses with the lysosome, producing a vacuole known as an autolysosome. The cellular components are degraded by lysosomal proteases and recycled. Autophagy is important for maintaining cellular homeostasis, and the process is evolutionarily conserved. Kibra is an upstream regulator of the hippo signaling pathway, which controls organ size by affecting cell growth, proliferation, and apoptosis. Kibra is mainly localized in the apical membrane domain of epithelial cells and acts as a scaffold protein. We found that Kibra is required for autophagy to function properly. The absence of Kibra caused defects in the formation of autophagic vesicles and autophagic degradation. We also found that the well-known cell polarity protein aPKC interacts with Kibra, and its activity affects autophagy upstream of Kibra. Constitutively active aPKC decreased autophagic vesicle formation and autophagic degradation. We confirmed the interaction between aPKC and Kibra in S2 cells and Drosophila larva. Taken together, our data suggest that Kibra and aPKC are essential for regulating starvation-induced autophagy. - Highlights: • Loss of Kibra causes defects in autophagosome formation and autophagic degradation. • Constitutively-active aPKCs negatively regulate autophagy. • Kibra interacts with aPKC in vitro and in vivo. • Kibra regulates autophagy downstream of aPKC.

  4. The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation.

    Science.gov (United States)

    Santos, J M; Benite-Ribeiro, S A; Queiroz, G; Duarte, J A

    2014-12-01

    Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3-kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin-activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho-aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho-aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle.

  5. γIsoform-Selective Changes in PKC Immunoreactivity after Trace Eyeblink Conditioning in the Rabbit Hippocampus

    NARCIS (Netherlands)

    Zee, E.A. van der; Kronforst-Collins, M.A.; Maizels, E.T.; Hunzicker-Dunn, M.; Disterhoft, J.F.

    1997-01-01

    An immunocytochemical examination of the rabbit hippocampus was done to determine which of the Ca2+-dependent protein kinase C (PKC) isoforms (PKCα, -βI, -βII, or -γ) are involved in associative learning. The hippocampally dependent trace eyeblink conditioning task was used for behavioral training,

  6. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.

    Science.gov (United States)

    Ren, L; Hong, S H; Cassavaugh, J; Osborne, T; Chou, A J; Kim, S Y; Gorlick, R; Hewitt, S M; Khanna, C

    2009-02-12

    Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

  7. Lidocaine-induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity.

    Science.gov (United States)

    Villarruel, Emmanuel Quinteros; Borda, Enri; Sterin-Borda, Leonor; Orman, Betina

    2011-08-01

    Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.

  8. Tyrosinase kinetics in epidermal melanocytes: analysis of DAG-PKC-dependent signaling pathway

    Science.gov (United States)

    Stolnitz, Mikhail M.; Peshkova, Anna Y.

    2001-05-01

    Tyrosinase is the key enzyme of melanogenesis with unusual enzyme kinetics. Protein kinase C plays an important role in regulating of tyrosinase activity. In the paper the mathematical model of PKC-DAG-dependent signal transduction pathway for UV-radiation is presented.

  9. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    Science.gov (United States)

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR.

  10. NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

    Directory of Open Access Journals (Sweden)

    Fabio eCavaliere

    2013-12-01

    Full Text Available Multipotent cells from the juvenile subventricular zone (SVZ possess the ability to differentiate into new neural cells. Depending on local signals, SVZ can generate new neurons, astrocytes or oligodendrocytes. We previously demonstrated that activation of NMDA receptors in SVZ progenitors increases the rate of oligodendrocyte differentiation. Here we investigated the mechanisms involved in NMDA receptor-dependent differentiation. Using functional studies performed with the reporter gene luciferase we found that activation of NMDA receptor stimulates PKC. In turn, stimulation of PKC precedes the activation of NADPH oxidase (NOX as demonstrated by translocation of the p67phox subunit to the cellular membrane. We propose that NOX2 is involved in the transduction of the signal from NMDA receptors through PKC activation as the inhibitor gp91 reduced their pro-differentiation effect. In addition, our data and that from other groups suggest that signaling through the NMDA receptor/PKC/NOX2 cascade generates ROS that activate the PI3/mTOR pathway and finally leads to the generation of new oligodendrocytes.

  11. Protein kinase C-beta II (PKC-betaII) expression in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise; Lindebjerg, Jan; Lahn, Michael;

    2009-01-01

    PURPOSE: Current development of targeted agents for the treatment of colorectal cancer include the clinical evaluation of kinase inhibitors, such as enzastaurin, a serine/threonine kinase inhibitor designed to suppress signaling through Protein Kinase C (PKC) and AKT pathways. Little is known abo...

  12. Systems Biology Strategy Reveals PKC-delta is Key for Sensitizing TRAIL-Resistant Human Fibrosarcoma

    Directory of Open Access Journals (Sweden)

    Kentaro eHayashi

    2015-01-01

    Full Text Available Cancer cells are highly variable and resistant to therapeutic intervention. Recently, the use of the tumor necrosis factor related apoptosis-inducing ligand (TRAIL induced treatment is gaining momentum, due to TRAIL’s ability to specifically target cancers with limited effect on normal cells. However, several malignant cancer types still remain non-sensitive to TRAIL. Previously, we developed a dynamic computational model, based on perturbation-response approach, and predicted protein kinase C (PKC as the most effective target, with over 95% capacity to kill human fibrosarcoma (HT1080 in TRAIL stimulation (Piras, V. et al. 2011, Scientific Reports. Here, to validate the model prediction, which has significant implications for cancer treatment, we conducted experiments on two TRAIL-resistant cancer cell lines (HT1080 and HT29. Using PKC inhibitor Bisindolylmaleimide I, we first demonstrate, as predicted by our previous model, cell viability is significantly impaired with over 95% death of both cancer types. Next, to identify crucial PKC isoform from 10 known members, we analyzed their mRNA expressions in HT1080 cells and shortlisted 4 isoforms for siRNA knock-down (KD experiments. From these KDs, PKC-delta produced the most cancer cell death in conjunction with TRAIL. Overall, systems biology approach, combining model prediction with experimental validation, holds promise for TRAIL-based cancer therapy.

  13. PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hyunhee Kim

    Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

  14. The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Ayden Gouveia

    2016-10-01

    Full Text Available While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.

  15. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

    Science.gov (United States)

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F G; Sørensen, Jakob B; Verhage, Matthijs

    2016-05-03

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

  16. Does PKC activation increase the homologous desensitization of μ opioid receptors?

    Science.gov (United States)

    Arttamangkul, Seksiri; Birdsong, William; Williams, John T

    2015-01-01

    This study examined the role of agents known to activate PKC on morphine-induced desensitization of μ-opioid receptors (MOP receptors) in brain slices containing locus coeruleus neurons. Intracellular recordings were obtained from rat locus coeruleus neurons. Two measurements were used to characterize desensitization, the decline in hyperpolarization induced by application of a saturating concentration of agonist (acute desensitization) and the decrease in hyperpolarization induced by a subsaturating concentration of [Met](5) enkephalin (ME) following washout of the saturating concentration (sustained desensitization). Internalization of MOP receptors was studied in brain slices prepared from transgenic mice expressing Flag-MOP receptors. The subcellular distribution of activated PKC was examined using a novel fluorescent sensor of PKC in HEK293 cells. The phorbol esters (PMA and PDBu) and muscarine increased acute desensitization induced by a saturating concentration of morphine and ME. These effects were not sensitive to staurosporine. Staurosporine did not block the decline in hyperpolarization induced by muscarine. PDBu and muscarine did not affect sustained desensitization induced by ME nor did phorbol esters or muscarine change the trafficking of MOP receptors induced by morphine or ME. The distribution of activated PKC measured in HEK293 cells differed depending on which phorbol ester was applied. This study demonstrates a distinct difference in two measurements that are often used to evaluate desensitization. The measure of decline correlated well with the reduction in peak amplitudes caused by PKC activators implicating the modification of other factors rather than MOP receptors. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  17. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways.

    Science.gov (United States)

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-09

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

  18. Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin αVβ3-FAK cascade.

    Science.gov (United States)

    Matsuda, Shinji; Fujita, Tsuyoshi; Kajiya, Mikihito; Takeda, Katsuhiro; Shiba, Hideki; Kawaguchi, Hiroyuki; Kurihara, Hidemi

    2012-05-01

    Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal-regulated kinase (ERK), integrin α(V)β(3), and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α(V)β(3) and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti-integrin α(V)β(3) antibody suppressed the BDNF-induced migration. BDNF increased the levels of integrin α(V)β(3) and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α(V)β(3) and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α(V)β(3)/FAK, and this may help to enhance the regeneration of periodontal tissue.

  19. FAK tyrosine 407 organized with integrin αVβ5 in Hs578Ts(i)8 advanced triple-negative breast cancer cells.

    Science.gov (United States)

    Payan, Iliet; McDonnell, Susan; Torres, Haydee M; Steelant, Wim F A; Van Slambrouck, Séverine

    2016-05-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase known to promote cell migration and invasiveness. Overexpression and increased activity of FAK are closely associated with metastatic breast tumors and are linked to poor prognosis. This study discovered an inverse correlation between FAK activity and migratory and invasive behavior. We show decreased phosphorylation levels of FAK at tyrosine residues 397 and 861, and most prominently at Y407, in the more invasive Hs578Ts(i)8 subclone of the Hs578T breast cancer progression model. There is limited information available on FAK Y407, and here we demonstrate its presence in triple-negative breast cancer (TNBC) cell lines. Furthermore, our studies propose that localization of FAK Y407, rather than FAK expression and overall FAK Y407 phosphorylation levels, is crucial for the control of cell motility. FAK Y407 is found extensively at the cell periphery in focal adhesion-like structures at each end of actin stress fibers and organized with integrin αVβ5 receptors, linking the αVβ5 integrin-mediated migratory behavior of Hs578Ts(i)8 cells to FAK Y407. These data suggest that subcellular localization, next to expression and activity levels, are important for understanding TNBC progression. Such an approach opens new avenues for further studies and may provide novel insight for the classification of TNBC and facilitate the discovery of effective biomarkers for diagnosis and therapy of TNBC.

  20. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M;

    1998-01-01

    experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB......-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB....... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...

  1. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

    Science.gov (United States)

    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  2. Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

    Science.gov (United States)

    Shen, Yang; Wang, Guixue; Huang, Xianliang; Zhang, Qin; Wu, Jiang; Tang, Chaojun; Yu, Qingsong; Liu, Xiaoheng

    2012-01-01

    Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiOx:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiOx:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events. PMID:21715399

  3. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells

    Science.gov (United States)

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada

    2014-01-01

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  4. αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

    Directory of Open Access Journals (Sweden)

    Murilo F Roggia

    Full Text Available To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α by photoreceptor outer segments (POS and its effects on retinal pigment epithelium (RPE.PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK, and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS, mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

  5. FAK interacts with MEF2 and drives the stretch-induced activation of an intronic enhancer of phospholamban gene in cardiomyocytes

    OpenAIRE

    2008-01-01

    Resumo: Estudos anteriores demonstraram que em miócitos cardíacos submetidos a estímulos mecânicos ocorre pronta fosforilação e ativação da FAK. Resultados de estudos recentes, realizados em corações de ratos indicaram que em resposta a estímulos mecânicos, a FAK, além de ser ativada, localiza-se no núcleo dos miócitos cardíaco. Estudos conduzidos em corações de ratos Wistar adultos, utilizando a técnica de Imunoprecipitação de cromatina (ChIP) com anticorpo anti- FAK, identificaram uma seqüê...

  6. FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡%FAK Regulates the Balance between MAPK and Akt Signal Pathways through Interaction with EGFR

    Institute of Scientific and Technical Information of China (English)

    李永林; 祝梅香; 张连峰; 蔡永; 刘先菊; R.M.Lafranie; 沈洁; 王嫣; 张璐; 董伟; 张扬清

    2005-01-01

    目的研究上皮生长因子受体和FAK的相互作用以及对下游信号的影响.方法建立聚集粘连激酶(FAK)缺失突变和绿色荧光蛋白(GFP)融合基因del 1-693FAK-GFP、del 1-100FAK-GFP和FAK-GFP稳定表达细胞系.结果同野生型FAK-GFP相比,N-端1-100氨基酸残基的缺失突变体,缺失1-693氨基酸残基的突变体结合在黏附点的能力被完全抑制.应用等电聚焦和SDS-PAGE双向电泳证明,EGF和纤维连接蛋白诱导FAK磷酸化的位点不同,进一步证实del 1-693FAK-GFP、del 1-100FAK-GFP,抑制MAPK的磷酸化,增强Akt的磷酸化;而FAK-GFP增强MAPK磷酸化,抑制Akt磷酸化.结论FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡.

  7. Research progress on focal adhesion kinase in malignant tumors%FAK 在恶性肿瘤中的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵美娜(综述); 陈公琰(审校)

    2016-01-01

    Focal adhesion kinase( FAK) ,a cytoplasmic non-receptor protein tyrosine kinase,serves as both a molecular scaffold and a mediator participating in multiple signal transduction pathways.FAK is involved in tumor cell survival,proliferation,migration and metastasis.Recent studies have shown that FAK is expressed in many tumor cells.Currently,FAK has been regarded as a potential target for cancer therapy.This review is to summarize the relationship between FAK and tumor progression.%黏着斑激酶( Focal adhesion kinase,FAK)是细胞内重要的骨架蛋白,属于一种非受体型酪氨酸蛋白激酶,也是多种信号通路的关键性分子。在肿瘤发生、发展、迁移以及侵袭的各个阶段FAK都具有重要作用。近年来,人们对FAK的研究越来越多,综合国内外研究资料表明,FAK在许多肿瘤组织中表达增高,提示FAK可能与肿瘤的发生发展密切相关,可能是肿瘤治疗的潜在靶点。该综述将对FAK分子结构及功能特点,与肿瘤的关系进行系统阐述。

  8. Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

    Directory of Open Access Journals (Sweden)

    Ting-Lin Yen

    2014-01-01

    Full Text Available Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (MAPKs. It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  9. Biobutanol Production from Palm Kernel Cake (PKC) using Clostridium saccharoperbutylacetonicum N1-4 in Batch Culture Fermentation

    National Research Council Canada - National Science Library

    Hafiza Shukor; Najeeb Kaid Nasser Al-Shorgani; Peyman Abdeshahian; Aidil Abdul Hamid; Nurina Anuar; Norliza Abd. Rahman; Mohd Hafez Bin Mohd Isa; Mohd Sahaid Kalil

    2014-01-01

    .... This study was performed to determine the feasibility of using PKC as a lignocellulosic substrate for biobutanol production by Clostridium saccharoperbutylacetonicum N1-4 in an acetone-butanol-ethanol (ABE...

  10. Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

    Science.gov (United States)

    McGrail, Daniel J; Khambhati, Niti N; Qi, Mark X; Patel, Krishan S; Ravikumar, Nithin; Brandenburg, Chandler P; Dawson, Michelle R

    2015-04-17

    Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

  11. Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Wei-Hong [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Yang, Li-Yun [Department of Blood Transfusion, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Cao, Zhong-Yi, E-mail: m18070383032@163.com [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China); Qian, Yong, E-mail: yfykqkqy@163.com [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital, Nanchang University, Nanchang 330006 (China)

    2015-02-20

    Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. In all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC. - Highlights: • Tivantinib suppresses OSCC cell growth independent of the inhibition of HGF/MET signaling pathway. • Tivantinib blocks cell cycle and induces caspases-mediated apoptosis. • Tivantinib elicits its anti-tumor activity with the inhibition of FAK signaling pathway.

  12. A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice.

    Science.gov (United States)

    Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui

    2016-06-01

    Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel

  13. Inflammation induces multinucleation of Microglia via PKC inhibition of cytokinesis, generating highly phagocytic multinucleated giant cells.

    Science.gov (United States)

    Hornik, Tamara C; Neniskyte, Urte; Brown, Guy C

    2014-03-01

    Microglia are brain macrophages, which can undergo multinucleation to give rise to multinucleated giant cells that accumulate with ageing and some brain pathologies. However, the origin, regulation and function of multinucleate microglia remain unclear. We found that inflammatory stimuli, including lipopolysaccharide, amyloid β, α-synuclein, tumour necrosis factor-α and interferon γ, but not interleukin-4, induced multinucleation of cultured microglia: primary rat cortical microglia and the murine microglial cell line BV-2. Inflammation-induced multinucleation was prevented by a protein kinase C (PKC) inhibitor Gö6976 (100 nM) and replicated by a PKC activator phorbol myristate acetate (160 nM). Multinucleation was reversible and not because of cell fusion or phagocytosis, but rather failure of cytokinesis. Time-lapse imaging revealed that some dividing cells failed to abscise, even after formation of long cytoplasmic bridges, followed by retraction of bridge and reversal of cleavage furrow to form multinucleate cells. Multinucleate microglia were larger and 2-4 fold more likely to phagocytose large beads and both dead and live PC12 cells. We conclude that multinucleate microglia are reversibly generated by inflammation via PKC inhibition of cytokinesis, and may have specialized functions/dysfunctions including the phagocytosis of other cells. Inflammation resulted in the accumulation of multiple nuclei per cell in cultured microglia. This multinucleation was reversible and due to a PKC-dependent block of the last step of cell division. Multinucleate microglia were larger and had a greater capacity to phagocytose other cells, suggesting they might remove neurons in the brain. © 2013 International Society for Neurochemistry.

  14. Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle.

    Science.gov (United States)

    Singh, Jagmohan; Rattan, Satish

    2013-04-15

    The role of PKC and RhoA/ROCK pathways in the phasic activities in the rectal smooth muscles (RSM) in the basal state is not known. We examined this issue by determining the effects of PKC inhibitors (calphostin C and Gö-6850) and a ROCK inhibitor (Y-27632) on the slow-rate (~3/min) and fast-rate (~25/min) phasic activities. We also examined the corresponding signal transduction cascades and the PKC and ROCK enzymatic activities in the RSM in the basal state. PKC inhibition with calphostin C and Gö-6850 (10(-5) M) caused a significant decrease (~25%) in slow-rate (but not fast-rate) phasic activity (monitored by frequency and amplitude of contractions) of the RSM. Conversely, ROCK inhibition with Y-27632 (10(-5) M) caused a significant decrease not only in slow-rate, but also fast-rate, phasic activity caused by ROCK inhibition in the RSM. Western blot analysis revealed that the PKC inhibition-induced decrease in RSM phasic activity was associated with decreases in PKCα translocation, phosphorylated (Thr(38)) PKC-potentiated inhibitor (CPI-17), and phosphorylated (Thr(18)/Ser(19)) 20-kDa myosin regulatory light chain. Conversely, decreases in the phasic activity in the RSM by ROCK inhibition were accompanied by the additional decrease in phosphorylated (Thr(696)) myosin phosphatase target subunit 1. Data show that while PKC and RhoA/ROCK pathways play a significant role in slow-rate high-amplitude spontaneous phasic activity, only the RhoA/ROCK pathway primarily mediates fast-rate low-amplitude phasic activity, in the RSM. Such knowledge is important in the understanding of the pathophysiology of large intestinal motility disorders. Relative contributions of the PKC vs. the RhoA/ROCK pathway in the phasic activity remain to be determined.

  15. Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents.

    Science.gov (United States)

    Harmati, Gábor; Papp, Ferenc; Szentandrássy, Norbert; Bárándi, László; Ruzsnavszky, Ferenc; Horváth, Balázs; Bányász, Tamás; Magyar, János; Panyi, György; Krasznai, Zoltán; Nánási, Péter P

    2011-02-01

    Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 μM) suppressed I(Kr) in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 μM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 μM bisindolylmaleimide I or 0.1 μM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution of PKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.

  16. LeY oligosaccharide upregulates DAG/PKC signaling pathway in the human endometrial cells.

    Science.gov (United States)

    Li, Yali; Ma, Keli; Sun, Ping; Liu, Shuai; Qin, Huamin; Zhu, Zhengmei; Wang, Xiaoqi; Yan, Qiu

    2009-11-01

    LeY oligosaccharide is stage specifically expressed by the embryo and uterine endometrium, and it plays important roles in embryo implantation. In addition to participating in the recognition and adhesion on fetal-maternal interface, LeY potentially regulates the expression of some implantation-related factors. However, it remains elusive whether it can mediate the involved signaling pathway. In this study, agarose-LeY beads were used to mimic the embryos, and the effects of LeY oligosaccharide on DAG/PKC signaling pathway was studied in human endometrial epithelial cells. Results showed that LeY could significantly trigger the activation of cPKCalpha and cPKCbeta2, and their translocation from the cytosol to the plasma membrane. The cellular DAG content was also upregulated, and the activation of PLCgamma1 was promoted. On the contrary, DAG/PKC signaling pathway was significantly inhibited when anti-LeY antibody was used after confirmation of LeY expression in human endometrial epithelial cells by immunohistochemistry and flow cytometry. These results suggest that LeY oligosaccharide acts as a signal molecule to modulate DAG/PKC signaling pathway.

  17. Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity.

    Science.gov (United States)

    Wender, Paul A; Baryza, Jeremy L; Brenner, Stacey E; DeChristopher, Brian A; Loy, Brian A; Schrier, Adam J; Verma, Vishal A

    2011-04-26

    Modern methods for the identification of therapeutic leads include chemical or virtual screening of compound libraries. Nature's library represents a vast and diverse source of leads, often exhibiting exquisite biological activities. However, the advancement of natural product leads into the clinic is often impeded by their scarcity, complexity, and nonoptimal properties or efficacy as well as the challenges associated with their synthesis or modification. Function-oriented synthesis represents a strategy to address these issues through the design of simpler and therefore synthetically more accessible analogs that incorporate the activity-determining features of the natural product leads. This study illustrates the application of this strategy to the design and synthesis of functional analogs of the bryostatin marine natural products. It is specifically directed at exploring the activity-determining role of bryostatin A-ring functionality on PKC affinity and selectivity. The resultant functional analogs, which were prepared by a flexible, modular synthetic strategy, exhibit excellent affinity to PKC and differential isoform selectivity. These and related studies provide the basic information needed for the design of simplified and thus synthetically more accessible functional analogs that target PKC isoforms, major targets of therapeutic interest.

  18. Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner.

    Science.gov (United States)

    Cijsouw, Tony; Weber, Jens P; Broeke, Jurjen H; Broek, Jantine A C; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid; Verhage, Matthijs; Toonen, Ruud F

    2014-03-03

    Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon-synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.

  19. Targeting PKC: a novel role for beta-catenin in ER stress and apoptotic signaling.

    Science.gov (United States)

    Raab, Marc S; Breitkreutz, Iris; Tonon, Giovanni; Zhang, Jing; Hayden, Patrick J; Nguyen, Thu; Fruehauf, Johannes H; Lin, Boris K; Chauhan, Dharminder; Hideshima, Teru; Munshi, Nikhil C; Anderson, Kenneth C; Podar, Klaus

    2009-02-12

    Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising preclinical activity in a wide range of tumor cells. We further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of beta-catenin in regulating growth and survival of tumor cells. Specifically, inhibition of PKC leads to rapid accumulation of beta-catenin by preventing the phosphorylation required for its proteasomal degradation. Microarray analysis and small-interfering RNA (siRNA)-mediated gene silencing in MM cells revealed that accumulated beta-catenin activates early endoplasmic reticulum stress signaling via eIF2alpha, C/EBP-homologous protein (CHOP), and p21, leading to immediate growth inhibition. Furthermore, accumulated beta-catenin contributes to enzastaurin-induced cell death. Sequential knockdown of beta-catenin, c-Jun, and p73, as well as overexpression of beta-catenin or p73 confirmed that accumulated beta-catenin triggers c-Jun-dependent induction of p73, thereby conferring MM cell apoptosis. Our data reveal a novel role of beta-catenin in endoplasmic reticulum (ER) stress-mediated growth inhibition and a new proapoptotic mechanism triggered by beta-catenin on inhibition of PKC isoforms. Moreover, we identify p73 as a potential novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies, including MM.

  20. Blockade of PKC-beta protects HUVEC from advanced glycation end products induced inflammation.

    Science.gov (United States)

    Xu, Youhua; Wang, Shanshan; Feng, Liang; Zhu, Quan; Xiang, Ping; He, Bao

    2010-12-01

    Advanced glycation end products (AGEs) have been recognized as a pivotal inducer in diabetes and kinds of aging-related vasculopathy. Endothelial dysfunction and inflammatory cells adhesion to endothelium have been regarded as important and early factors in the pathogenesis of vascular complications in diabetic patients. Owing to the key role of PKC-beta in AGEs-induced vascular dysfunction, we investigated effects of blocking PKC-beta by LY333531 on macrophage adhesion to HUVEC and the related mechanism. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion ability. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions by SABC or SABC-AP method; mRNA expression of TGF-beta1, ICAM-1 and RAGE was determined by real-time RT-PCR. SOD and MDA levels in culture supernatant were detected. We found that LY333531 significantly reduced AGEs-induced macrophage adhesion to HUVEC. Blockade of PKC-beta strikingly decreased HUVEC TGF-beta1 and ICAM-1 expression in both protein and mRNA levels, RAGE protein level was also down-regulated. Furthermore, the anti-oxidative stress index, SOD/MDA was dramatically elevated on LY333531 application. Therefore we conclude that LY333531 can reduce AGEs-induced macrophage adhesion to endothelial cells and relieve the local inflammation, this was realized by its effect on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative ability.

  1. Insulin induces PKC-dependent proliferation of mesenteric vascular smooth muscle cells from hypertensive patients

    Institute of Scientific and Technical Information of China (English)

    Xukai WANG; Yan WANG; Chenming YANG; Ying WAN; Xianwen JI

    2006-01-01

    Background and objectives Proliferation of human vascular smooth muscle cells (VSMCs) induced by hyperinsulinemia is a very common clinical pathology. Extensive research has focused on PKC (Protein kinase C)-MAPK (mitogen-activated protein kinase)intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs.Methods DNA synthesis, signaling of ERK1/2 MAPKs, and changes in α-smooth muscle (SM) actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor, GF109203X.Results Differences among cell types in MAPK signaling, α-SM actin, and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension (EH) and normotension (NT) were identified in response to insulin treatment. Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs. Insulin exposure decreased expression of α-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs, especially in the EH group. These effects were reversed by treatment with the PKC inhibitor. Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation, MAPK signaling, and degree of changes in α-SM actin and F-actin isoforms immediately following insulin exposure in vitro.

  2. Small Review: Strategies for Palm Kernel Cake (PKC As a New Potential Substrate in Biofuel Production

    Directory of Open Access Journals (Sweden)

    Hafiza Shukor

    2013-01-01

    Full Text Available The economic dependency on fossil fuels and the resulting effects on climate and environment have put tremendous focus on utilizing fermentable sugars from lignocellulose, the largest known renewable carbohydrate source. Palm kernel cake (PKC is a residue from palm oil extraction presently only used as a low protein feed supplement. It’s contains 50% fermentable hexose sugars present in the form of glucan and mainly galactomannan. This makes PKC an interesting feedstock for processing into biofuel or in other biorefinery processes. This article reviews biotechnological innovation on Palm Kernel Cake (PKC as new potential of fermentable sugar for biofuel production. Strategies for biofuel production by utilizing palm kernel cake by several pretreatment processes to convert glucan and especially galactomanan into fermentable hexose sugar and further requirements to make fermentative biofuel production a successful industrial process are also discussed. This material recovery especially from lignocellulose agricultural wastes by product of palm oil mill industry into this potential bioproducts has not only benefited in oil palm planted but also to the environment and helps preserve natural resource.

  3. mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis.

    Directory of Open Access Journals (Sweden)

    Meghan M Morrison

    2015-07-01

    Full Text Available Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2. We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.

  4. PKC-mediated potentiation of morphine analgesia by St. John's Wort in rodents and humans.

    Science.gov (United States)

    Galeotti, Nicoletta; Farzad, Mersedeh; Bianchi, Enrica; Ghelardini, Carla

    2014-01-01

    Our purpose was to combine the use of morphine with clinically available inhibitors of protein kinase C (PKC), finally potentiating morphine analgesia in humans. Thermal tests were performed in rodents and humans previously administered with acute or chronic morphine combined or not with increasing doses of the PKC-blocker St. John's Wort (SJW) or its main component hypericin. Phosphorylation of the γ subunit of PKC enzyme was assayed by western blotting in the periaqueductal grey matter (PAG) from rodents co-administered with morphine and hypericin and was prevented in rodent PAG by SJW or hypericin co-administration with morphine, inducing a potentiation of morphine analgesia in thermal pain. The score of pain assessment in healthy volunteers were decreased by 40% when morphine was co-administered with SJW at a dose largely below those used to obtain an antidepressant or analgesic effect in both rodents and humans. The SJW/hypericin potentiating effect lasted in time and preserved morphine analgesia in tolerant mice. Our findings indicate that, in clinical practice, SJW could reduce the dose of morphine obtaining the same analgesic effect. Therefore, SJW and one of its main components, hypericin, appear ideal to potentiate morphine-induced analgesia.

  5. Structure-based modelling, scoring, screening, and in vitro kinase assay of anesthetic pkc inhibitors against a natural medicine library.

    Science.gov (United States)

    Shi, B X; Chen, F R; Sun, X

    2017-02-01

    Protein kinase C (PKC) is an intracellular effector of the inositol phosphate-mediated signal transduction pathway. Evidence is emerging that certain general anaesthetics can influence the activity of PKC by interacting with the regulatory domain of the enzyme, and targeting PKC kinase domain is considered as a strategy to modulate the anaesthetic effects. Here, an integrated method was used to perform virtual screening against a large library of natural compounds for the discovery of new and potent PKC modulators. A number of hits were identified and their inhibitory activity against PKC kinase domain was measured by using a standard kinase assay protocol. Three and five compounds were determined to have high and moderate activities with IC50 values at nanomolar and micromolar levels, respectively. These compounds can be considered as promising lead molecular entities to develop efficacious anaesthetic modulators. Structural examination revealed a variety of nonbonded interactions such as hydrogen bonds, cation-π contacts, and hydrophobic forces across the complex interface of PKC with the identified compounds. This study helps to establish an integrative approach to rational kinase inhibitor discovery by efficiently exploiting various existing natural products.

  6. Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release.

    Science.gov (United States)

    Genc, Ozgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf

    2014-02-11

    Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001.

  7. aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation.

    Science.gov (United States)

    Iden, Sandra; Misselwitz, Steve; Peddibhotla, Swetha S D; Tuncay, Hüseyin; Rehder, Daniela; Gerke, Volker; Robenek, Horst; Suzuki, Atsushi; Ebnet, Klaus

    2012-03-05

    The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.

  8. Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity.

    Science.gov (United States)

    Ogawa, Kota; Seki, Takahiro; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2013-10-11

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins' aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant's degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant's cytotoxicity by enhancing its degradation.

  9. Activation of JNK by TPA promotes apoptosis via PKC pathway in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Qiao Wu; Si-Yang Song; Wen-Jin Su

    2002-01-01

    AIM: JNK cascade plays an important role in cell proliferation, differentiation and apoptosis. However, the exact function of JNK cascade for apoptosis induction remains largely unknown. In this study, the role of JNK activation stimulated by TPA in the process of apoptosis induction and its signaling transduction pathway in gastric cancer cells were investigated and determined.METHODS: Expressions of mRNA and protein were detected by Northern blot and Western blot. Transcription activity was measured by transient transfection and CAT assay. Apoptotic cells were displayed through staining the nucleus with DAPI and were observed under fluorescence microscope. The apoptotic index was determined by counting 1000 cells randomly.RESULTS: JNK protein was stimulated rapidly by TPA, and reached its highest peak within 3 hr, then decreased in a time-dependent manner, but the expression level of JNK protein induced by TPA was always keeping higher than that in untreated cells. Similar pattern was seen in c-jun mRNA level induced by TPA. TPA significantly activated the transcriptional activity of activator protein-1 with a TPA-closedependent manner. Furthermore, activation of JNK was mediated through PKC pathway. Treatment of cells with PKC specific inhibitor, Wortmannin, led to repression of JNK even in the presence of TPA. More importantly, all these effects were associated with induction of apoptosis in gastric cancer cells. TPA inducted apoptosis obviously in gastric cancer cells. The apoptotic cells became smaller and rounded, and their nuclei became condensation and fragmentation with brightly stained chromatin. However, suppression of JNK by PKC specific inhibitor, Wortmannin, resulted in the decrease of apoptosis induced by TPA in a time-dependent manner, apoptotic index dramatically decreased from 32.56 % to 8.71%.CONCLUSION: TPA stimulates JNK cascade, including upregulation of JNK protein expression level and c-jun mRNA expression level, and activation of activator

  10. Anti-tumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGF-IR in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Kurio, Naito [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Shimo, Tsuyoshi, E-mail: shimotsu@md.okayama-u.ac.jp [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Fukazawa, Takuya; Takaoka, Munenori [Department of General Surgery, Kawasaki Medical School, Okayama, 700-0821 (Japan); Okui, Tatsuo; Hassan, Nur Mohammad Monsur; Honami, Tatsuki [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan); Hatakeyama, Shinji [Novartis Institutes for BioMedical Research, Basel (Switzerland); Ikeda, Masahiko [Department of Surgery, Fukuyama City Hospital, Fukuyama, 720-8511 (Japan); Naomoto, Yoshio [Department of General Surgery, Kawasaki Medical School, Okayama, 700-0821 (Japan); Sasaki, Akira [Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8525 (Japan)

    2011-05-01

    Focal adhesion kinase (FAK) is a 125-kDa non-receptor type tyrosine kinase that localizes to focal adhesions. FAK overexpression is frequently found in invasive and metastatic cancers of the breast, colon, thyroid, and prostate, but its role in osteolytic metastasis is not well understood. In this study, we have analyzed anti-tumor effects of the novel FAK Tyr{sup 397} inhibitor TAE226 against bone metastasis in breast cancer by using TAE226. Oral administration of TAE226 in mice significantly decreased bone metastasis and osteoclasts involved which were induced by MDA-MB-231 breast cancer cells and increased the survival rate of the mouse models of bone metastasis. TAE226 also suppressed the growth of subcutaneous tumors in vivo and the proliferation and migration of MDA-MB-231 cells in vitro. Significantly, TAE226 inhibited the osteoclast formation in murine pre-osteoclastic RAW264.7 cells, and actin ring and pit formation in mature osteoclasts. Moreover, TAE226 inhibited the receptor activator for nuclear factor {kappa} B Ligand (RANKL) gene expression induced by parathyroid hormone-related protein (PTHrP) in bone stromal ST2 cells and blood free calcium concentration induced by PTHrP administration in vivo. These findings suggest that FAK was critically involved in osteolytic metastasis and activated in tumors, pre-osteoclasts, mature osteoclasts, and bone stromal cells and TAE226 can be effectively used for the treatment of cancer induced bone metastasis and other bone diseases.

  11. Genotypic and phenotypic characterization of garlic-fermenting lactic acid bacteria isolated from som-fak, a Thai low-salt fermented fish product

    DEFF Research Database (Denmark)

    Paludan-Müller, Christine; Valyasevi, R.; Huss, Hans Henrik

    2002-01-01

    AIMS: To evaluate the importance of garlic for fermentation of a Thai fish product, and to differentiate among garlic-/inulin-fermenting lactic acid bacteria (LAB) at strain level. METHODS AND RESULTS: Som-fak was prepared by fermentation of a mixture of fish, salt, rice, sucrose and garlic. pH d...

  12. aPKC-ι/P-Sp1/Snail signaling induces epithelial-mesenchymal transition and immunosuppression in cholangiocarcinoma.

    Science.gov (United States)

    Qian, Yawei; Yao, Wei; Yang, Tao; Yang, Yan; Liu, Yan; Shen, Qi; Zhang, Jian; Qi, Weipeng; Wang, Jianming

    2017-10-01

    Cholangiocarcinoma (CCA) is a highly malignant bile duct cancer that tends to invade and metastasize early. The epithelial-mesenchymal transition (EMT) has been implicated in cancer cell invasion and metastasis, as well as in cancer cell evasion of host immunity. In this study, we investigated the interaction between atypical protein kinase C-iota (aPKC-ι) and Snail in the regulation of EMT and its relationship to CCA immunosuppression. Our results demonstrated that aPKC-ι, Snail, and infiltrated immunosuppressive cells were significantly up-regulated in CCA tumor tissues and linked to poor prognosis. aPKC-ι induced EMT and immunosuppression by regulating Snail in vitro and in vivo, although aPKC-ι did not directly interact with Snail in coimmunoprecipitation experiments. To further clarify the molecular interaction between aPKC-ι and Snail in relation to EMT, quantitative iTRAQ-based phosphoproteomic analysis and liquid chromatography-tandem mass spectrometry were conducted to identify the substrates of aPKC-ι-dependent phosphorylation. Combined with coimmunoprecipitation, we showed that specificity protein 1 (Sp1) was directly phosphorylated by aPKC-ι on Ser59 (P-Sp1). Both Sp1 and P-Sp1 were up-regulated in CCA tumor tissues and associated with clinicopathological features and poor prognosis in CCA patients. Moreover, using chromatin immunoprecipitation assays, we found that P-Sp1 regulated Snail expression by increasing Sp1 binding to the Snail promoter. P-Sp1 also regulated aPKC-ι/Snail-induced EMT-like changes and immunosuppression in CCA cells. Our findings further indicated that CCA cells with EMT-like features appear to generate immunosuppressive natural T regulatory-like cluster of differentiation 4-positive (CD4(+) )CD25(-) cells rather than to increase CD4(+) CD25(+) natural T regulatory cells, in part by mediating T regulatory-inducible cytokines such as transforming growth factor β1 and interleukin 2. These results demonstrate that aPKC

  13. Ang II-AT2R increases mesenchymal stem cell migration by signaling through the FAK and RhoA/Cdc42 pathways in vitro.

    Science.gov (United States)

    Xu, Xiu-Ping; He, Hong-Li; Hu, Shu-Ling; Han, Ji-Bin; Huang, Li-Li; Xu, Jing-Yuan; Xie, Jian-Feng; Liu, Ai-Ran; Yang, Yi; Qiu, Hai-Bo

    2017-07-12

    Mesenchymal stem cells (MSCs) migrate via the bloodstream to sites of injury and are possibly attracted by inflammatory factors. As a proinflammatory mediator, angiotensin II (Ang II) reportedly enhances the migration of various cell types by signaling via the Ang II receptor in vitro. However, few studies have focused on the effects of Ang II on MSC migration and the underlying mechanisms. Human bone marrow MSCs migration was measured using wound healing and Boyden chamber migration assays after treatments with different concentrations of Ang II, an AT1R antagonist (Losartan), and/or an AT2R antagonist (PD-123319). To exclude the effect of proliferation on MSC migration, we measured MSC proliferation after stimulation with the same concentration of Ang II. Additionally, we employed the focal adhesion kinase (FAK) inhibitor PF-573228, RhoA inhibitor C3 transferase, Rac1 inhibitor NSC23766, or Cdc42 inhibitor ML141 to investigate the role of cell adhesion proteins and the Rho-GTPase protein family (RhoA, Rac1, and Cdc42) in Ang II-mediated MSC migration. Cell adhesion proteins (FAK, Talin, and Vinculin) were detected by western blot analysis. The Rho-GTPase family protein activities were assessed by G-LISA and F-actin levels, which reflect actin cytoskeletal organization, were detected by using immunofluorescence. Human bone marrow MSCs constitutively expressed AT1R and AT2R. Additionally, Ang II increased MSC migration in an AT2R-dependent manner. Notably, Ang II-enhanced migration was not mediated by Ang II-mediated cell proliferation. Interestingly, Ang II-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased Talin and Vinculin expression. Moreover, RhoA and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. Furthermore, FAK, Talin, and Vinculin activation and F-actin reorganization in response to Ang II were prevented by PD-123319 but

  14. 粘着斑激酶在前列腺癌中表达的研究%Research on Expression of FAK in Prostate Carcinoma

    Institute of Scientific and Technical Information of China (English)

    廖洪利; 米叶赛尔·阿不都拉

    2016-01-01

    目的:探讨粘着斑激酶(FAK)表达与前列腺癌发病的关系。方法前列腺癌患者60例,经手术切除的癌组织及相应的癌旁组织行免疫组化检测 FAK表达情况。Trizol法提取组织总 RNA,RT-PCR检测 FAK mRNA表达量,并进行统计学分析。结果 FAK 在60例前列腺癌组织中表达明显比癌旁组织高,两者差异有统计学意义(χ2=72.55,P<0.01),FAK的mRNA水平在前列腺癌组织表达显著比癌旁组织高(t=30.51,P<0.01)。淋巴结转移情况看 pN0M0表达阳性例数比pN3M1少,pN0M0的mRNA表达量比pN3M1的表达量低,差异有统计学意义(t=25.43,P<0.01)。结论前列腺癌细胞FAK表达与侵袭转移相关。%Objective To investigate the relationships between expression of FAK and Prostate Carcinoma (PC)morbidity. Methods By surgery,get cancer tissue and para-carcinama tissue from 60 cases PC.To detect FAK expression by immuno-histochemical.To extract total RNA by Trizol.To detect the FAK mRNA by RT-PCR,and analysis these data.Results FAK expression level in cancer tissue was higher than that in para-carcinama tissue.There was statistical difference between them (χ2=72.55,P<0.01).mRNA expression levels of FAK in cancer tissue showed significant higher than the levels in para-carcinama tissue (t=30.51,P<0.01).According to lymphatic metastasis,the expression positive cases of FAK in pN0M0 classification were lower than these in pN3M1 classification,and mRNA expression levels of FAK were the same re-sults.There were clearly statistical distinctive (t=25.43,P<0.01).Conclusion The expression of FAK in PC cells was as-sociated with tumor invasion.

  15. Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

    Science.gov (United States)

    Chou, Wan-Yu; Chuang, Kun-Han; Sun, David; Lee, Yu-Hsiu; Kao, Pu-Hong; Lin, Yen-Yu; Wang, Hsei-Wei; Wu, Yuh-Lin

    2015-09-01

    Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

  16. Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Min WANG; Wen-bin ZHANG; Jun-hui ZHU; Guo-sheng FU; Bin-quan ZHOU

    2009-01-01

    Aim: To investigate the influence of breviscapine on high glucose-induced hypertrophy of cardiomyocytes and the relevant mechanism in vitro and in vivo.Methods: Cultured neonatal cardiomyocytes were divided into ⅰ) control; ⅱ) high glucose concentrations; ⅲ) high glucose+PKC inhibi-tor Ro-31-8220; ⅳ) high glucose+breviscapine; or ⅴ) high glucose+NF-κB inhibitor BAY11-7082. Cellular contraction frequency and volumes were measured; the expression of protein kinase C (PKC), NF-κB, TNF-α, and c-los were assessed by Western blot or reverse transcription-polymerase chain reaction (RT-PCR). Diabetic rats were induced by a single intraperitoneal injection of streptozotocin,and randomly divided into ⅰ) control rats; ⅱ) diabetic rats; or ⅲ) diabetic rats administered with breviscapine (10 or 25 mg·kg-1·d-1). After treatment with breviscapine for six weeks, the echocardiographic parameters were measured. All rats were then sacrificed and heart tissue was obtained for microscopy. The expression patterns of PKC, NF-κB, TNF-α, and c-los were measured by Western blot or RT-PCR.Results: Cardiomyocytes cultured in a high concentration of glucose showed an increased pulsatile frequency and cellular volume, as well as a higher expression of PKC, NF-κB, TNF-α, and c-fos compared with the control group. Breviscapine could partly prevent these changes. Diabetic rats showed relative cardiac hypertrophy and a higher expression of PKC, NF-κB, TNF-α, and c-los; treatment with breviscapine could ameliorate these changes in diabetic cardiomyopathy.Conclusion: Breviscapine prevented cardiac hypertrophy in diabetic rats by inhibiting the expression of PKC, which may have a protec-tive effect in the pathogenesis of diabetic cardiomyopathy via the PKC/NF-κB/C-fos signal transduction pathway.

  17. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    Science.gov (United States)

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  18. Cloning and expression pattern analysis of a cDNA of pkc-2 gene in Caenorhabditis elegans%秀丽小杆线虫Caenorhabditis elegans pkc-2基因cDNA的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    钱雨; Laurent SEGALAT

    2009-01-01

    蛋白激酶C在秀丽小杆线虫中具有调节肌细胞渐进性萎缩的功能.为了揭示它的调节机制,本研究克隆了秀丽小杆线虫中蛋白激酶C pkc-2基因的cDNA pkc-2-c,构建了含该pkc-2 基因cDNA亚型的重组质粒pPD 118.20-pKG 63;揭示了该cDNA在秀丽小杆线虫体壁肌细胞中的定位.%Protein kinase C regulates the progressive muscle degeneration in Caenorhabditis elegans. In order to investigate the function of PKC involved in muscle degeneration, this paper cloned a cDNA isoform of pkc-2 gene of C. elegans and constructed the recombinant plasmids pPD118.20-pKG63 containing the isoform. The new isoform was then further studied for gene expression pattern. Immunocytochemistry experiment showed that this cDNA isoform expressed in body-wall muscle cells and located near the dense body.

  19. A protein kinase C-encoding gene, pkcA, is essential to the viability of the filamentous fungus Aspergillus nidulans.

    Science.gov (United States)

    Ichinomiya, Masayuki; Uchida, Hirotaka; Koshi, Yukako; Ohta, Akinori; Horiuchi, Hiroyuki

    2007-11-01

    A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.

  20. Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons.

    Science.gov (United States)

    Dumitriu, Bogdan; Cohen, Jonathan E; Wan, Qin; Negroiu, Andreea M; Abrams, Thomas W

    2006-04-01

    Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

  1. Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC.

    Science.gov (United States)

    Hoffman, D A; Johnston, D

    1998-05-15

    We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.

  2. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    Science.gov (United States)

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  3. Translocation of PKC-betaII is mediated via RACK-1 in the neuronal cells following dioxin exposure.

    Science.gov (United States)

    Lee, Hyun-Gyo; Kim, Sun-Young; Choi, Eun-Jung; Park, Ki-Yeon; Yang, Jae-Ho

    2007-03-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce neurotoxic effects. However, the mechanism of TCDD-mediated signaling pathways and its possible molecular targets in neurons remains unknown. In this study, we analyzed effects of TCDD on neurofilament subunits, receptor for activated C kinase-1 (RACK-1), and PKC-betaII activity in developing neuronal cells. TCDD induced a significant increase of RACK-1, an adaptor protein for protein kinase C (PKC), in cerebellar granule cells in both dose- and time-dependent manner, indicating that RACK-1 is a sensitive molecular target in neuronal cells for TCDD exposure. TCDD induced a dose-dependent translocation of PKC-betaII from cytosol to membrane fractions. However, when RACK-1 induction was blocked by antisense oligonucleotide or alpha-naphthoflavone, Ah receptor (AhR) inhibitor, the translocation of PKC-betaII was inhibited. Our data suggests that TCDD activates PKC-betaII via RACK-1 in an AhR-dependent manner. This is the first report identifying RACK-1 as a target molecule involved in TCDD-mediated signaling pathways. TCDD exposure also increased the level of neurofilament-H mRNA. These results suggest that identification of target molecules may contribute to improve our understanding of TCDD-mediated signaling pathway and the risk assessment of TCDD-induced neurotoxicities.

  4. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination.

    Directory of Open Access Journals (Sweden)

    Susana P Barrera

    Full Text Available Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1. Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.

  5. Role of Protein Kinase C (PKC in Podocytes and Development of Glomerular Damage in Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Beina eTeng

    2014-11-01

    Full Text Available The early glomerular changes in diabetes include a podocyte phenotype with loss of slit diaphragm proteins, changes in the actin cytoskeleton and foot process architecture. This review focusses on the role of the Protein Kinase C family in podocytes and points out the differential roles of classical, novel and atypical PKCs in podocytes. Some PKC-isoforms are indispensable for proper glomerular development and slit diaphragm maintenance whereas others might be harmful when activated in the diabetic milieu. Therefore some might be interesting treatment targets in the early phase of diabetes.

  6. PKC and AMPK regulation of Kv1.5 potassium channels

    DEFF Research Database (Denmark)

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells......-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems....

  7. Phosphorylation of h1 Calponin by PKC epsilon may contribute to facilitate the contraction of uterine myometrium in mice during pregnancy and labor

    Directory of Open Access Journals (Sweden)

    Li Lesai

    2012-05-01

    Full Text Available Abstract Background The timely onset of powerful uterine contractions during parturition occurs through thick and thin filament interactions, similar to other smooth muscle tissues. Calponin is one of the thin filament proteins. Phosphorylation of calponin induced by PKC-epsilon can promote the contraction of vascular smooth muscle. While the mechanism by which calponin regulates the contraction of pregnant myometrium has rarely been explored. Here, we explore whether PKC-epsilon/h1 calponin pathway contribute to regulation of myometrial contractility and development of parturition. Methods We detected the expression of h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon in the different stages of mice during pregnancy and in labor by the method of western blot and recorded the contraction activity of myometrium strips at the 19th day during pregnancy with different treatments by the organ bath experiments. Results The level of the four proteins including h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice. The ratios of phosphorylated h1 calponin/h1 calponin and phosphorylated PKC-epsilon/PKC-epsilon were reached the peak after the onset of labor in myometrium in the mice. After the treatment of more than 10(9- mol/L Psi-RACK (PKC-epsilon activator, the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1 calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-epsilon. Meanwhile, the change of the ratio of phosphorylated h1 calponin/h1 calponin was consistent with that of contraction force of mice myometrium strips. Conclusions These data suggest that in mice myometrium, phosphorylation of h1 calponin induced by the PKC-epsilon might facilitate the contraction of uterine in labor and regulate pregnant

  8. PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila

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    Julien Colomb

    2016-04-01

    Full Text Available Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment, one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning, may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity.

  9. Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis.

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    Nataliya Kotelevets

    Full Text Available Sphingosine kinases (SK catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P, thereby promoting oncogenic processes. Breast (MDA-MB-231, lung (NCI-H358, and colon (HCT 116 carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

  10. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

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    Andrea Toledo

    Full Text Available MARCKS (Myristoylated Alanine-Rich C Kinase Substrate is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25 in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

  11. Signaling via ITGB1/FAK and microfilament rearrangement mediates the internalization of Leptospira interrogans in mouse J774A.1 macrophages

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    Zhao Xin

    2015-01-01

    Full Text Available Leptospirosis caused by pathogenic Leptospira species is a worldwide zoonotic 2 infectious disease, but the mechanisms of leptospiral internalization remain poorly understood. Here, we report that mouse J774A.1 macrophages expressed integrin-subfamily proteins (ITGB1, ITGB2 and ITGB3. Antibody blockage and siRNA-based knockdown of ITGB1 decreased the internalization of leptospires into mouse J774A.1 macrophage cells. The internalization required focal adhesion kinase (FAK activation in J774A.1 cells rather than phosphoinositide-3-kinase (PI3K, and microfilament rather than microtubule aggregation during infection. The data indicated that the ITGB1/FAK/microfilament signaling pathway is responsible for leptospiral internalization in mouse macrophages.

  12. The Microarray Gene Profiling Analysis of Glioblastoma Cancer Cells Reveals Genes Affected by FAK Inhibitor Y15 and Combination of Y15 and Temozolomide

    OpenAIRE

    2014-01-01

    Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarr...

  13. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

    Science.gov (United States)

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

  14. Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells.

    Science.gov (United States)

    Alisi, Anna; Arciello, Mario; Petrini, Stefania; Conti, Beatrice; Missale, Gabriele; Balsano, Clara

    2012-01-01

    Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

  15. Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion.

    Science.gov (United States)

    Van Slambrouck, Severine; Grijelmo, Clara; De Wever, Olivier; Bruyneel, Erik; Emami, Shahin; Gespach, Christian; Steelant, Wim F A

    2007-12-01

    Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

  16. Sodium butyrate-induced death-associated protein kinase expression promote Raji cell morphological change and apoptosis by reducing FAK protein levels

    Institute of Scientific and Technical Information of China (English)

    Hai-tao ZHANG; Zhe-ling FENG; Jun WU; Ya-jun WANG; Xia GUO; Nian-ci LIANG; Zhen-yu ZHU; Jian-quan MA

    2007-01-01

    Aim:To investigate the role of death-associated protein kinase (DAPK) on the apoptosis of Raji cells induced by sodium butyrate. Methods:The apoptosis of Raji cells were induced by sodium butyrate for 2,4,6,8,and 10 d. Simultaneity,the Raji cells were inhibited to adhere on culture flask by polyHEME. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and the cell apoptosis percentage was estimated by flow cytometry. DAPK and focal adhesion kinase (FAK) expression were measured by Western blotting.Coding sequence on the C-terminal of DAPK,which can suppress the function of DAPK,was tranfected into the Raji cells to investigate whether the C-terminal of DAPK could inhibit the apoptosis of Raji cells induced by sodium butyrate. Results:After being treated with sodium butyrate,the Raji cells expressed DAPK and displayed many protrusions to adhere onto the culture flask. The Raji cells were susceptive to apoptosis when they were inhibited adhesion by polyHEME. At that time,the cell viability decreased,the cell apoptosis percentage increased and the protein levels of total FAK were reduced. The Raji cells,which were transfected with the coding region on the C-terminal of DAPK,sustained apoptosis and the FAK protein level when treated with sodium butyrate. Conclusion:Sodium butyrate induced DAPK expression. It caused the Raji cells to display many protrusions all around the cells and adhere onto the culture flask. DAPK expression prompted apoptosis by reducing the FAK protein level in sodium butyrate induced Raji cells.

  17. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    Science.gov (United States)

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Ganoderiol A-enriched extract suppresses migration and adhesion of MDA-MB-231 cells by inhibiting FAK-SRC-paxillin cascade pathway.

    Directory of Open Access Journals (Sweden)

    Guo-Sheng Wu

    Full Text Available Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA, dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

  19. Simultaneous determination of multi-mycotoxins in palm kernel cake (PKC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Yibadatihan, S; Jinap, S; Mahyudin, N A

    2014-01-01

    Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.

  20. Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

    Science.gov (United States)

    Tang, Jun; Bouyer, Patrice; Mykoniatis, Andreas; Buschmann, Mary; Matlin, Karl S; Matthews, Jeffrey B

    2010-10-29

    The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCε- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCε-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCε, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

  1. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner.

    Directory of Open Access Journals (Sweden)

    Rajeev Mehla

    Full Text Available HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC -alpha and -delta, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC involving stress induced AMP Kinase (AMPK inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs.

  2. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner.

    Science.gov (United States)

    Mehla, Rajeev; Bivalkar-Mehla, Shalmali; Zhang, Ruonan; Handy, Indhira; Albrecht, Helmut; Giri, Shailendra; Nagarkatti, Prakash; Nagarkatti, Mitzi; Chauhan, Ashok

    2010-06-16

    HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -alpha and -delta, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs.

  3. Knockout of the predominant conventional PKC isoform, PKCalpha, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake

    DEFF Research Database (Denmark)

    Jensen, Thomas E; Maarbjerg, Stine J; Rose, Adam J;

    2009-01-01

    for approximately 97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCalpha knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction...

  4. Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation.

    Science.gov (United States)

    Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar

    2012-07-15

    Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

  5. Combination treatments with the PKC inhibitor, enzastaurin, enhance the cytotoxicity of the anti-mesothelin immunotoxin, SS1P.

    Directory of Open Access Journals (Sweden)

    Abid R Mattoo

    Full Text Available Activated protein kinase C (PKC contributes to tumor survival and proliferation, provoking the development of inhibitory agents as potential cancer therapeutics. Immunotoxins are antibody-based recombinant proteins that employ antibody fragments for cancer targeting and bacterial toxins as the cytotoxic agent. Pseudomonas exotoxin-based immunotoxins act via the ADP-ribosylation of EF2 leading to the enzymatic inhibition of protein synthesis. Combining PKC inhibitors with the immunotoxin SS1P, targeted to surface mesothelin, was undertaken to explore possible therapeutic strategies. Enzastaurin but not two other PKC inhibitors combined with SS1P to produce synergistic cell death via apoptosis. Mechanistic insights of the synergistic killing centered on the complete loss of the prosurvival Bcl2 protein, Mcl-1, the loss of AKT and the activation of caspase 3/7. Synergy was most evident when cells exhibited resistance to the immunotoxin alone. Further, because PKC inhibition by itself was not sufficient to enhance SS1P action, enzastaurin must target other kinases that are involved in the immunotoxin pathway.

  6. Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation.

    Science.gov (United States)

    Petpiroon, Nareerat; Suktap, Chalermlat; Pongsamart, Sunanta; Chanvorachote, Pithi; Sukrong, Suchada

    2015-07-01

    The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

  7. Anti-tumor activity of cabozantinib by FAK down-regulation in human oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Da-Lu Li

    2016-03-01

    Full Text Available Cabozantinib is a tyrosine kinase inhibitor involved in inhibition of cell proliferation and colony formation. We studied anti-cancer properties of cabozantinib in oral squamous cell carcinoma cells. The viability of BHY and HSC-3 cells decreased with increase in cabozantinib concentration and time. The proliferation of cell lines was affected by increasing concentration of cabozantinib from 0.3 to 1.2 μM after 48 hours of treatment. The expression of MET and phosphorylated MET was not affected by cabozantinib treatment. Cabozantinib-treated cells when compared to control, showed concentration-dependent increase in BHY and HSC-3 cells during G2/M phase and decrease in S phase with increase in cabozantinib concentration. Annexin-V/propidium iodide double staining showed that cells with annexin-V increased with the increase in cabozantinib concentration. The expression of apoptosis related proteins cleaved caspase-3 and cleaved-PARP were increased with increase in cabozantinib concentration. It was also found that suppression of FAK activation and expression was dose dependent. The results from this study revealed that cabozantinib can be useful in developing a drug for effective treatment of oral squamous cell carcinoma cells.

  8. Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Shi

    Full Text Available BACKGROUND: Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP expression in rat vascular smooth muscle cells (SMCs and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D. METHODOLOGY/PRINCIPAL FINDINGS: Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS chains from proteoglycan (PG core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1 suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13 expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity. CONCLUSIONS/SIGNIFICANCE: We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D

  9. TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    kappaB inhibition and in the presence of p38 blockers. Akt/ERK signalling but not p38 activation was abolished in the presence of the iron chelator desferroxamine that blocks formation of hydroxyl ( OH) radicals. Thus, the TNF-alpha signalling in keratinocytes seems to bifurcate into an aPKC-, NFk......Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine involved in the pathogenesis of inflammatory skin diseases and cutaneous squamous cell carcinoma. Some of these effects are mediated by the stimulatory effect of this cytokine on the Akt signalling pathway, which...... renders keratinocytes less susceptible to proapoptotic stimuli and enhances cell growth. We have recently shown that TNF-alpha-induced Akt activation may promote the early stages of skin cancer. In this work, we demonstrate that in the premalignant keratinocyte cell line HaCaT, TNF-alpha activates Akt...

  10. Absence of PKC-alpha attenuates lithium-induced nephrogenic diabetes insipidus.

    Directory of Open Access Journals (Sweden)

    Jae H Sim

    Full Text Available Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1 and water channel (AQP2 expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO and strain-matched wild type (WT controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.

  11. Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKC.

    Science.gov (United States)

    Seroussi, Yaron; Brosh, Inbar; Barkai, Edi

    2002-09-01

    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.

  12. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

    Science.gov (United States)

    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

  13. ErbB receptors and PKC regulate PC12 neuronal-like differentiation and sodium current elicitation.

    Science.gov (United States)

    García, L; Castillo, C; Carballo, J; Rodríguez, Y; Forsyth, P; Medina, R; Martínez, J C; Longart, M

    2013-04-16

    Excitability, neurite outgrowth and their specification are very important features in the establishment of neuronal differentiation. We have studied a conditioned medium (CM) from sciatic nerve which is able to induce a neuronal-like differentiation of PC12 cells. Previously, we have demonstrated that supplementing this CM with a generic inhibitor (k252a), which mainly inhibits tropomyosin-related kinase receptors (Trk receptors) and protein kinase C (PKC), caused neurite elongation, sodium current induction and axon development. In the present work, we are showing that the enhancement of neurite length and induction of sodium currents induced by CM+k252a were prevented by ErbB receptor inhibition. Additionally, we demonstrated that specific inhibition of PKC produced a similar effect to that exerted by k252a in CM-treated cells, specifically by increasing the percentage of differentiated cells with long neurites and inducing sodium currents. Moreover, CM changed the mRNA levels for ErbB2 and ErbB3 increasing them 6- and 36-folds respectively compared to their control. The inclusion of k252a with CM changed the ErbB1, ErbB2 and ErbB3 mRNA proportions increasing those eight-, seven- and fivefolds respectively. From this point, it is clear that appropriate ErbB receptor levels and PKC inhibition are necessary to enhance the effect of the CM in inducing the neuronal-like differentiation of PC12 cells. In summary, we demonstrated the involvement of ErbB receptors in the regulation of neurite elongation and sodium current induction in PC12 cells and propose that these processes could be initiated by ErbB receptors followed by a fine regulation of PKC signaling. These findings might implicate a novel interplay between ErbB receptors and PKC in the regulation of these molecular mechanisms.

  14. Widdrol-induced lipolysis is mediated by PKC and MEK/ERK in 3T3-L1 adipocytes.

    Science.gov (United States)

    Jeong, Hyun Young; Yun, Hee Jung; Kim, Byung Woo; Lee, Eun Woo; Kwon, Hyun Ju

    2015-12-01

    Obesity is a serious medical condition causing various diseases such as heart disease, type-2 diabetes, and cancer. Fat cells (adipocytes) play an important role in the generation of energy through hydrolysis of lipids they accumulate. Therefore, induction of lipolysis (breakdown of lipids into fatty acids and glycerol), is one of the ways to treat obesity. In the present study, we investigated the lipolytic effect of widdrol in 3T3-L1 adipocytes and its mechanism. Widdrol considerably increased the amount of glycerol released from 3T3-L1 adipocytes into the medium in a time- and dose-dependent manner. To determine the mechanism of this effect, we investigated the alterations in glycerol release and protein expression in 3T3-L1 adipocytes treated with widdrol alone or widdrol and inhibitors of proteins involved in the cAMP-dependent pathway or cAMP-independent PKC-MAPK pathway, which are known to induce lipolysis in adipocytes. The adenylyl cyclase inhibitor SQ-22536, PLA2 inhibitor dexamethasone, PI3K inhibitor wortmannin, and PKA inhibitor H-89, which were used to investigate the involvement of the cAMP-dependent pathway, did not affect the lipolytic effect of widdrol. Widdrol-induced phosphorylation of PKC, MEK, and ERK, which are related to the PKC-MAPK pathway, and their phosphorylation was inhibited by their inhibitors (H-7, U0126, and PD-98059, respectively). Moreover, the increase in glycerol release induced by widdrol was almost completely blocked by PKC, MEK, and ERK inhibitors. These results suggest that widdrol induces lipolysis through activation of the PKC-MEK-ERK pathway.

  15. Bryostatin-1 vs. TPPB: dose-dependent APP processing and PKC-α, -δ, and -ε isoform activation in SH-SY5Y neuronal cells.

    Science.gov (United States)

    Yi, P; Schrott, L; Castor, T P; Alexander, J S

    2012-09-01

    Activation of the α-secretase processing pathway of amyloid precursor protein (APP) is recognized as an important mechanism which diverts APP processing from production of beta-amyloid (Aβ) to non toxic sAPPα, decreasing Alzheimer's disease (AD) plaque formation and AD-associated cognitive deficits. Two potent classes of PKC modulators can activate the α-secretase pathway, the benzo/indolactams and bryostatin/bryologues. While both modulate PKC-dependent APP processing, no direct comparisons of their relative pharmacological potencies have been accomplished which could assist in the development of AD therapies. In this study, we measured the activation of α-secretase APP processing and PKC-α, -δ, and -ε induced by the benzolactam-APP modulator TPPB and bryostatin-1 in the neuroblastoma cell line SH-SY5Y which expresses APP and α- and β-secretase processing mechanisms. Bryostatin-1 produced a more rapid, potent, and sustained activation of α-secretase APP processing than TPPB and selectively activated PKC-δ and PKC-ε. Although TPPB also activated α-secretase, its potency was approximately 10- to 100-fold lower, possibly reflecting lower PKC-δ and -ε activation. Because bryostatin-1 is a highly potent PKC-δ and -ε activator which activates α-secretase APP processing, further characterization of bryostatin-1/bryologues may help refine their use as important tools for the clinical management of AD.

  16. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

    Directory of Open Access Journals (Sweden)

    Shantelle L LaFayette

    Full Text Available Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC, which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which

  17. Different roles of PKC and PKA in effect of interferon-γ on proliferation and collagen synthesis of fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Xuan-fen ZHANG; Shu-zhong GUO; Kai-hua LU; Hui-yuan LI; Xiang-dong LI; Lin-xi ZHANG; Li YANG

    2004-01-01

    AIM: To study the signal roles of protein kinase C (PKC) and protein kinase A (PKA) in the influence of interferony (IFN-γ) on proliferation and collagen synthesis of fibroblasts derived from hypertrophic scar (HS-FB) and normal skin (NS-FB). METHODS: HS-FB and NS-FB were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Activity of PKC and PKA were assayed by transferring phosphorus (32p) into substrate after treatment with IFN-γ 1000 kU/L at 10, 30, 60, and 120 min. Cell proliferation was determined with MTT assay.The collagen synthesis was measured with [3H]proline incorporation and Type III pre-collagen was determined with radioimmunoassay. RESULTS: After exposure to IFN-γ 1000 kU/L for 30 min, PKC activity of HS-FB and respectively (P<0.05). After exposure to IFN-y 1000 kU/L for 60 and 120 min, PKA activities of HS-FB increased The PKA activities of NS-FB also increased from 0.52+0.03 nmol.min-1.g-1of control to 0.68±0.03 and 0.89±0.05 nmol.min-1.g-1, respectively (P<0.05). The proliferation and collagen synthesis were enhanced by PKC activator (containing phosphatidylserine, diacylglycerol and Ca2+) and PKA inhibitor [H7250 μmol/L, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine], and inhibited by PKC inhibior (GF109 250 μmol/L) and PKA activator (cAMP 25 rmol/L)(P<0.01). GF109 abrogated increased proliferation and collagen synthesis by IFN-y but it did not affect the inhibitory effects of IFN-γ. At 120 min H7 reversed the inhibitory functions of IFN-γ. CONCLUSION: IFN-γ transiently increased proliferation and collagen synthesis of HS-FB and NS-FB by activation of PKC and subsequently inhibited proliferation and collagen synthesis by activation of PKA.

  18. FAK-related nonkinase attenuates hypertrophy induced by angiotensin-Ⅱ in cultured neonatal rat cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Jin QIN; Zheng-xiang LIU

    2006-01-01

    Aim: To examine the inhibitory effect of FAK-related nonkinase (FRNK) in cardiac hypertrophy in vitro and investigate the possible mechanisms. Methods: A functional fragment of FRNK cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into the vector pcDNA3.1. Hypertrophy in neonatal rat cardiac myocytes was established with angiotensin-Ⅱ stimulation. The pcDNA3.1-FRNK or pcDNA3.1 was respectively transfected into cardiomyocytes by Lipofectamine 2000. The surface area and mRNA expression of atrial natriuretic peptide (ANP) of myocytes were employed to detect cardiac hypertrophy. NF-κB p65 protein in nuclear extracts, phosphorylation levels of ERK1/2 (p-ERK1/2) and AKT (p-AKT), as well as total ERK1/2, and AKT in variant treated cardiomyocytes were determined by Western blot. Results: Under the stimulation of angiotensin Ⅱ, the surface area of myocytes and levels of ANP mRNA were significantly increased. But transient transfection with pcDNA3.1-FRNK in advance may reduce the surface area and expression of ANP mRNA of hypertrophic myocytes. The protein levels of NF-κB p65 in nuclear extracts and p-ERK1/2, p-AKT in FRNK treated cardiomyocytes were significantly decreased compared with that in angiotensin-Ⅱ induced cardiomyocytes, while different treatments had little effect on total ERK1/2 and AKT. Conclusion: FRNK may inhibit angiotensin-Ⅱ-induced cardiomyocyte hypertrophy via decreasing phosphorylation levels at ERK1/2 and AKT, consequently downregulating nuclear translocation of NF-κB p65.

  19. Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells.

    Science.gov (United States)

    Lin, Su-Hsuan; Shih, Yuan-Wei

    2014-06-01

    Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.

  20. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica

    Directory of Open Access Journals (Sweden)

    Andrew T Kempsell

    2015-09-01

    Full Text Available Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature versus advanced age Aplysia. Glutamate- (L-Glu- evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT in sensory neurons (SN isolated from mature but not aged animals. Activation of PKA and PKC signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems

  1. NMDAR NR2A and NR2B specific PKC-dependent regulation of mGluR is defective in the Fragile X Syndrome mouse model

    DEFF Research Database (Denmark)

    Banke, Tue G.; Toft, Anna Karina; Lundbye, Camilla Johanne

    ). Similar results were observed when mature KO slices were preincubated with the protein kinase C activator (PMA), or inactivator (chelerythrine), suggesting that NMDAR activation leads to PKC regulation of mGluR-LTD. Surprisingly, preincubation WT with the NR2B specific NMDAR inhibitor CP-101.......606 completely blocked DHPG-induced LTD. This blockage was reversed by either preincubation with PMA or by co-application of the NR2A antagonist TCN-201. In contrast, application of TCN-201 alone had no apparent effects. Our data suggest a model where NMDARs regulate mGluR-LTD through regulation of PKC....... Furthermore, in this model it appears that NR2B activation stimulates PKC, while NR2A activation halts or reverses this effect. In addition, in the KO mice, the coupling between specific NMDAR subunits and mGluR-LTD activity through PKC seems defective in an age-dependent manner. These findings suggest strong...

  2. Focal adhesion kinase (FAK mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV-infected cells.

    Directory of Open Access Journals (Sweden)

    Anna Alisi

    Full Text Available Hepatitis C Virus (HCV infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC. The pivotal role of hepatic stellate cells (HCSs and extracellular matrix (ECM in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP. These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in

  3. 大麻素受体1、FAK mRNA在小鼠肝纤维化形成过程中肝组织中的表达及相互关系%Hepatic expression of CB1 in rats with fibrosis and the relationship with FAK

    Institute of Scientific and Technical Information of China (English)

    杨莉; 赵召霞; 侯军良; 刘玉珍; 姜惠卿; 戴二黑

    2011-01-01

    Objective To evaluate hepatic expression of CBl mRNA in rats during liver fibrogenesis and study the relationship between CB1 and FAK. Methods Liver fibrosis model was prepared by intraperitoneal injection of carbon tetrachloride ( 10% ). Liver tissues and serum samples were collected at 2, 4, 6 and 8 week. The scores of fibrosis stage (S) were performed. The mRNA levels of CB1 mRNA and FAK were determined by quantitative RT - PCR. The levels of serum TGFβ1 were detected by ELISA. Results Compared with normal control group, CBI mRNA and FAK mRNA levels in every model group were significantly increased ( P < O.05 ). With the modeling time prolonged, CB1 and FAK mRNA levels gradually increased (P <0.05 ). CB1 mRNA level in liver tissue not only had correlation with the degree of liver fibrosis ( r = O. 747, P < 0.01 ), but also with FAK mRNA level in liver tissue ( r = 0.907, P < O. 01 ). With the modeling time prolonged, serum TGFβ1 level gradually increased (P < O. 05 ). CBI mRNA level in liver tissue had positive correlation with serum TGFβ1 level ( r = O. 542, P < 0. O1 ). Conclusion CB1 may promote the activation of FAK in rats with fibrosis, and induce HSC proliferation and secrete plentiful TGFβ1 by PI3K signal transduction. CB1 may promote progression of liver fibrosis.%目的 研究大麻素受体1(CB1)mRNA在肝纤维化形成过程中表达的变化,从基因转录水平探讨其与黏着斑激酶(FAK)的关系.方法 采用10%四氯化碳腹腔注射制备肝纤维化模型,分别于造模第2、4、6、8周留取小鼠的肝组织及血清.通过肝组织病理对肝纤维化程度进行评分,荧光定量PCR检测CB1 mRNA和FAK mRNA水平,ELISA方法检测血清中转化生长因子(TGF)β1的含量.结果 与正常对照组相比,各模型组小鼠肝组织中CBI mRNA和FAK mRNA含量显著升高(P<0.05),而且随造模时间的延长,CB1 mRNA和FAK mRNA含量亦逐渐增高,各组之间差异有统计学意义(P<0.05).CBI mRNA的含

  4. Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Neves-dos-Santos Sandra

    2010-01-01

    Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

  5. NOC/oFQ PKC-dependent superoxide generation contributes to hypoxic-ischemic impairment of NMDA cerebrovasodilation.

    Science.gov (United States)

    Armstead, W M

    2000-12-01

    This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O(2)(-)) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O(2)(-) generation. Under non-H-I conditions, topical NOC/oFQ (10(-10) M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 20 +/- 3 pmol/mm(2). PKC inhibitors staurosporine and chelerythrine (10(-7) M) blunted NBT reduction (1 +/- 1 to 7 +/- 2 pmol/mm(2) for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH(2) (10(-6) M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH(2) and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10(-8), 10(-6) M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 +/- 1 and 16 +/- 1; H-I: -5 and -10 +/- 1; H-I staurosporine pretreated: 3 +/- 1 and 6 +/- 1%). These data show that NOC/oFQ increased O(2)(-) production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O(2)(-) generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.

  6. miR-486 suppresses the development of osteosarcoma by regulating PKC-δ pathway.

    Science.gov (United States)

    He, Ming; Wang, Guangbin; Jiang, Linlin; Qiu, Chuang; Li, Bin; Wang, Jiashi; Fu, Yonghui

    2017-05-01

    Osteosarcoma is one of the most highly malignant types of cancer in adolescents and young adults with a high mortality rate. Despite advances in surgery, radiation therapy and chemotherapy, the prognosis for patients with osteosarcoma has not significantly improved over the past several decades. It is necessary to find new indicators of prognosis and therapeutic targets of osteosarcoma. Through the analysis of 40 osteosarcoma tissues, we found that the expression of miR‑486 was low and the expression of PKC‑δ was high in osteosarcoma. Median survival of patients with low expression of miR-486 (30 months) was shorter than the patients with higher expression of miR‑486 (40 months). We further found that miR-486 can inhibit the targeting of PKC‑δ signaling pathways, and this inhibition can inhibit the growth and invasion of osteosarcoma cells. After transfection of miR‑486 for 24 h, the proliferation of osteosarcoma cells was inhibited by ~20%, and the migration was inhibited by ~15%. In the present investigation, we demonstrated that miR‑486 is negatively associated with the expression of PKC-δ and could regulate the development of osteosarcoma. miR-486 may be a potential target for the treatment of osteosarcoma.

  7. The 5-HT7 receptor triggers cerebellar long-term synaptic depression via PKC-MAPK.

    Science.gov (United States)

    Lippiello, Pellegrino; Hoxha, Eriola; Speranza, Luisa; Volpicelli, Floriana; Ferraro, Angela; Leopoldo, Marcello; Lacivita, Enza; Perrone-Capano, Carla; Tempia, Filippo; Miniaci, Maria Concetta

    2016-02-01

    The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

  8. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate.

    Science.gov (United States)

    Vorhagen, Susanne; Niessen, Carien M

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway

    Directory of Open Access Journals (Sweden)

    In Kyoung Mah

    2015-11-01

    Full Text Available The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

  10. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  11. The participation of NMDA receptors, PKC, and MAPK in the formation of memory following operant conditioning in Lymnaea

    Directory of Open Access Journals (Sweden)

    Rosenegger David

    2010-08-01

    Full Text Available Abstract Background Memory is the ability to store, retain, and later retrieve information that has been learned. Intermediate term memory (ITM that persists for up to 3 h requires new protein synthesis. Long term memory (LTM that persists for at least 24 h requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. It has been shown in a number of different model systems that NMDA receptors, protein kinase C (PKC and mitogen activated protein kinase (MAPK are all involved in the memory formation process. Results Here we show that snails trained in control conditions are capable of forming, depending on the training procedure used, either ITM or LTM. However, blockage of NMDA receptors (MK 801, inhibition of PKC (GF109203X hydrochloride and MAPK activity (UO126 prevent the formation of both ITM and LTM. Conclusions The injection of either U0126 or GF109203X, which inhibit MAPK and PKC activity respectively, 1 hour prior to training results in the inhibition of both ITM and LTM formation. We further found that NMDA receptor activity was necessary in order for both ITM and LTM formation.

  12. Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo.

    Science.gov (United States)

    Spivak, Adam M; Larragoite, Erin T; Coletti, McKenna L; Macedo, Amanda B; Martins, Laura J; Bosque, Alberto; Planelles, Vicente

    2016-12-20

    Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation. We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants. We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo. The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

  13. Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

    Science.gov (United States)

    He, Ying; Wang, Zaijie Jim

    2015-03-18

    As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

  14. Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling.

    Science.gov (United States)

    Dia, Vermont P; Gonzalez de Mejia, Elvira

    2011-12-27

    The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α5β1 integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P cancer cells by direct binding with α5β1 integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.

  15. MicroRNA-151 and its hosting gene FAK (focal adhesion kinase) regulate tumor cell migration and spreading of hepatocellular carcinoma.

    Science.gov (United States)

    Luedde, Tom

    2010-09-01

    Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

  16. Ruthenium Polypyridyl Complex Inhibits Growth and Metastasis of Breast Cancer Cells by Suppressing FAK signaling with Enhancement of TRAIL-induced Apoptosis

    Science.gov (United States)

    Cao, Wenqiang; Zheng, Wenjie; Chen, Tianfeng

    2015-03-01

    Ruthenium-based complexes have emerged as promising antitumor and antimetastatic agents during the past decades. However, the limited understanding of the antimetastatic mechanisms of these agents is a roadblock to their clinical application. Herein, we reported that, RuPOP, a ruthenium polypyridyl complex with potent antitumor activity, was able to effectively inhibit growth and metastasis of MDA-MB-231 cells and synergistically enhance TRAIL-induced apoptosis. The selective intracellular uptake and cytotoxic effect of RuPOP was found associated with transferring receptor (TfR)-mediated endocytosis. Further investigation on intracellular mechanisms reveled that RuPOP notably suppressed FAK-mediated ERK and Akt activation. Pretreatment of cells with ERK inhibitor (U0126) and PI3K inhibitor (LY294002) significantly potentiated the inhibitory effect of RuPOP on cell growth, migration and invasion. Moreover, the alternation in the expression levels of metastatic regulatory proteins, including uPA, MMP-2/-9, and inhibition of VEGF secretion were also observed after RuPOP treatment. These results demonstrate the inhibitory effect of RuPOP on the growth and metastasis of cancer cells and the enhancement of TRAIL-induced apoptosis though suppression of FAK-mediated signaling. Furthermore, RuPOP exhibits the potential to be developed as a metal-based antimetastatic agent and chemosensitizer of TRAIL for the treatment of human metastatic cancers.

  17. The microarray gene profiling analysis of glioblastoma cancer cells reveals genes affected by FAK inhibitor Y15 and combination of Y15 and temozolomide.

    Science.gov (United States)

    Huang, Grace; Ho, Baotran; Conroy, Jeffrey; Liu, Song; Qiang, Hu; Golubovskaya, Vita

    2014-01-01

    Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and detected 8087 and 6555 genes, which were significantly either up- or down-regulated by Y15-treatment in DBTRG and U87 cells, respectively (ptemozolomide and by combination of Y15 and temozolomide treatment in U87 cells. Among genes up-regulated by Y15 and temozolomide more significantly than by each agent alone were: COX7B; interferon, gamma-inducible transcript: IFI16; DDIT4; GADD45G and down-regulated: KIF3A, AKT1; ABL; JAK1, GLI3 and ALDH1A3. Thus, microarray gene expression analysis can be effective in establishing genes affected in response to FAK inhibitor alone and in response to combination of Y15 with temozolomide that is important for glioblastoma therapy.

  18. Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK.

    Science.gov (United States)

    Lin, Sen-Sen; Li, Fang-Fang; Sun, Li; Fan, Wei; Gu, Ming; Zhang, Lu-Yong; Qin, Song; Yuan, Sheng-Tao

    2016-03-01

    Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

  19. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  20. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  1. Go-6976 Reverses Hyperglycemia-Induced Insulin Resistance Independently of cPKC Inhibition in Adipocytes

    Science.gov (United States)

    Robinson, Katherine A.; Hegyi, Krisztina; Hannun, Yusuf A.; Buse, Maria G.; Sethi, Jaswinder K.

    2014-01-01

    Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1) plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used “specific” inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not –β) was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway. PMID:25330241

  2. PKA, PKC, and AKAP localization in and around the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Newton Alexandra

    2001-10-01

    Full Text Available Abstract Background One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA and protein kinase C (PKC. A-kinase anchoring proteins (AKAPs can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIα in addition to the RII subunits. Results Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIα at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIα and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIα and RIIβ are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCβ appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. Conclusions The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIα and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIα in these regions. Likewise, gravin may be an anchor of PKCβ at the NMJ.

  3. Taurine Inhibits Myocardial Fibrosis via PKC-ERK1/2 Signaling Pathways

    Institute of Scientific and Technical Information of China (English)

    WANG Li-ying; LI Hong; YANG Shi-jie

    2012-01-01

    Previous studies have demonstrated the important role of taurine in inhibiting proliferation of myofibroblasts(myoFb) and myocardial fibrosis.However,the underlying mechanisms are unclear.The present study was designed to shed light on this issue through exploring the signal pathways via in vitro experiments.Angiotension Ⅱ (AngⅡ) treatment significantly increased myoFb proliferation and the levels of collagens Ⅰ and Ⅲ(P<0.05),whereas taurine,PKCαt(PKC:protein kinase C) specific inhibitor L-threo-dihydro-sphingosine(D4681),ERK1/2 inhibitor (PD98095) abrogated myoFb proliferation and collagen levels(P<0.05,P<0.01,respectively),and increased the G0/G1 phase rate and decreased S phase rate.Immunocytochemistry,confocal fluorescence staining and image analysis showed that taurine could inhibit the translocation and expression of p-PKCαtin membrane,and then inhibit nuclear translocation and expression of p-ERK1/2.These results have statistically significant differences compared with those of AngⅡ group(P<0.0l).Western blot results also show that taurine could inhibit the protein expression of p-PKCαt and p-ERK1/2.We used p-PKCα specific inhibitor D4681 in order to elucidate the relationship between p-PKCα and p-ERK1/2 in signal transduction pathways.Finally,the results show that the protein expression of p-ERK1/2 and nuclear translocation were suppressed in D4681 group.

  4. Go-6976 reverses hyperglycemia-induced insulin resistance independently of cPKC inhibition in adipocytes.

    Directory of Open Access Journals (Sweden)

    Katherine A Robinson

    Full Text Available Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1 plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used "specific" inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not -β was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway.

  5. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    Science.gov (United States)

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

  6. Quantification of PKC family genes in sporadic breast cancer by qRT-PCR: evidence that PKCι/λ overexpression is an independent prognostic factor.

    Science.gov (United States)

    Awadelkarim, Khalid Dafaallah; Callens, Céline; Rossé, Carine; Susini, Aurélie; Vacher, Sophie; Rouleau, Etienne; Lidereau, Rosette; Bièche, Ivan

    2012-12-15

    Drugs targeting protein kinase C (PKC) show promising therapeutic activity. However, little is known about the expression patterns of the 11 PKC genes in human tumors, and the clinical significance of most PKC genes is unknown. We used qRT-PCR assays to quantify mRNA levels of the 11 PKC genes in 458 breast tumors from patients with known clinical/pathological status and long-term outcome. The proportion of tumors in which the expression of the different genes was altered varied widely, from 9.6% for PKN2 to 40.2% for PKCι/λ. In breast tumors, overexpression was the main alteration observed for PKCι/λ (33.4%), PKCδ (29.5%) and PKCζ (9.6%), whereas underexpression was the main alteration observed for PKCα (27.3%), PKCε (11.6%), PKCη (8.7%) and PKN2 (8.1%). Both overexpression and underexpression were observed for PKCβ (underexpression 15.5%, overexpression 13.8%), PKCθ (underexpression 14.8%, overexpression 10.0%) and PKN1 (underexpression 6.6%, overexpression 7.4%). Several links were found between different PKC genes; and also between the expression patterns of PKC genes and several classical pathological and clinical parameters. PKCι/λ alone was found to have prognostic significance (p = 0.043), whereas PKCα showed a trend towards an influence on relapse-free survival (p = 0.052). PKCι/λ retained its prognostic significance in Cox multivariate regression analysis (p = 0.031). These results reveal very complex expression patterns of PKC genes in breast tumors, and suggest that their expression should be considered together when evaluating anti-tumoral drugs. PKCι/λ seems to be the most promising therapeutic target in breast cancer.

  7. Roundabout4 Suppresses Glioma-Induced Endothelial Cell Proliferation, Migration and Tube Formation in Vitro by Inhibiting VEGR2-Mediated PI3K/AKT and FAK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Heng Cai

    2015-03-01

    Full Text Available Background and Aims: Endothelial cell (EC proliferation, migration, and tube formation are the critical steps for tumor angiogenesis, which is involved in the formation of new tumor blood vessels. Roundabout4 (Robo4, a new member of Robo proteins family, is specifically expressed in endothelial cells. This study aimed to investigate the effects of Robo4 on glioma-induced endothelial cell proliferation, migration and tube formation in vitro. Methods and Results: We found that Robo4 was endogenously expressed in Human Brain Microvascular Endothelial Cells (HBMECs, while Robo4 was significantly down-regulated in endothelial cells cultured in glioma conditioned medium. Robo4 over-expression remarkably suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro. In addition, Robo4 influenced the glioma-induced angiogenesis via binding to its ligand Slit2. Further studies demonstrated that the knockdown of Robo4 up-regulated the phosphorylation of VEGFR2, PI3K, AKT and FAK in EC cultured in glioma conditioned medium. VEGFR2 inhibitor SU-1498, AKT inhibitor LY294002 and FAK inhibitor 14 (FAK inhibitor blocked the Robo4 knockdown-mediated alteration in glioma angiogenesis in vitro. Conclusion: Our results proved that Robo4 suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated activation of PI3K/AKT and FAK signaling pathways.

  8. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingyu [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Luo, Qing, E-mail: qing.luo@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Mao, Xinjian [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Xu, Baiyao [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Ju, Yang [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Song, Guanbin, E-mail: song@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the

  9. Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

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    Yan-Bo Zhang

    Full Text Available Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC and extracellular signal-regulated kinase1/2 (ERK1/2 in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6-S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling

  10. Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

    Science.gov (United States)

    Zhang, Yan-Bo; Guo, Zheng-Dong; Li, Mei-Yi; Fong, Peter; Zhang, Ji-Guo; Zhang, Can-Wen; Gong, Ke-Rui; Yang, Ming-Feng; Niu, Jing-Zhong; Ji, Xun-Ming; Lv, Guo-Wei

    2015-01-01

    Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6-S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our

  11. Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways

    Science.gov (United States)

    2010-01-01

    Background c-erbB2, a proto-oncogene coding epidermal growth factor receptor-like receptor, also as a chemosensitivity/prognosis marker for gynecologic cancer, may be involved in initiation of growth of rat primordial follicles. The aim of the present study is to investigate the role and signal pathway of c-erbB2 in onset of rat primordial follicle development. Methods The expression of c-erbB2 mRNA and protein in neonatal ovaries cultured 4 and 8 days with/without epidermal growth factor (EGF) were examined by in situ hybridization, RT-PCR and western blot. The function of c-erbB2 in the primordial folliculogenesis was abolished by small interfering RNA transfection. Furthermore, MAPK inhibitor PD98059 and PKC inhibitor calphostin were used to explore the possible signaling pathway of c-erbB2 in primordial folliculogenesis. Results The results showed that c-erbB2 mRNA was expressed in ooplasm and the expression of c-erbB2 decreased after transfection with c-erbB2 siRNA. Treatment with EGF at 50 ng/ml significantly increased c-erbB2 expression and primary and secondary follicle formation in ovaries. However, this augmenting effect was remarkably inhibited by c-erbB2 siRNA transfection. Furthermore, folliculogenesis offset was blocked by calphostin (5 × 10(-4) mmol/L) and PD98059 (5 × 10(-2) mmol/L), but both did not down-regulate c-erbB2 expression. In contrast, the expressions of p-ERK and p-PKC were decreased obviously by c-erbB2 siRNA transfection. Conclusions c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways, suggesting an important role of c-erbB2 in rat primordial follicle initiation and development. PMID:20565902

  12. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior

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    Ramsey Cara

    2008-07-01

    Full Text Available Abstract The laterocapsular division of the central nucleus of the amygdala (CeLC has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction. Membrane-permeable inhibitors of PKA (KT5720, 1 μM; cAMPS-Rp, 10 μM and ERK (U0126, 1 μM activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 μM and an inactive structural analogue of U0126 (U0124, 1 μM had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 μM, concentration in microdialysis probe or U0126 (100 μM into the CeLC, but not striatum (placement control, inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 μM and U0124 (100 μM did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.

  13. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    Science.gov (United States)

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-11-08

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

  14. Neurotensin Phosphorylates GSK-3α/β through the Activation of PKC in Human Colon Cancer Cells

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    Qingding Wang

    2006-09-01

    Full Text Available Neurotensin (NT, a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3 regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3α/β in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC, but not by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3β phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCα and PKCβ1 or downregulation of endogenous PKCα or PKCβ1 blocked NT-mediated GSK-3β (but not GSK-3α phosphorylation. Moreover, a selective PKCβ inhibitor, LY379196, reduced NT-mediated GSK-3β (but not GSK-3α phosphorylation, suggesting a role for PKCbβ in the NT-mediated phosphorylation of GSK-3β and an undefined kinase in the NT-mediated phosphorylation of GSK-3α. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.

  15. mGluR5 positive modulators both potentiate activation and restore inhibition in NMDA receptors by PKC dependent pathway

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    Liao Pei-Fei

    2011-02-01

    Full Text Available Abstract Background In order to understand the interaction between the metabotropic glutamate subtype 5 (mGluR5 and N-methyl-D-aspartate (NMDA receptors, the influence of mGluR5 positive modulators in the inhibition of NMDA receptors by the noncompetitive antagonist ketamine, the competitive antagonist D-APV and the selective NR2B inhibitor ifenprodil was investigated. Methods This study used the multi-electrode dish (MED system to observe field potentials in hippocampal slices of mice. Results Data showed that the mGluR5 agonist (RS-2-chloro-5-hydroxyphenylglycine (CHPG, as well as the positive allosteric modulators 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl benzamide (CDPPB and 3,3'-difluorobenzaldazine (DFB alone did not alter the basal field potentials, but enhanced the amplitude of field potentials induced by NMDA. The inhibitory action of ketamine on NMDA-induced response was reversed by CHPG, DFB, and CDPPB, whereas the blockade of NMDA receptor by D-APV was restored by CHPG and CDPPB, but not by DFB. Alternatively, activation of NMDA receptors prior to the application of mGluR5 modulators, CHPG was able to enhance NMDA-induced field potentials and reverse the suppressive effect of ketamine and D-APV, but not ifenprodil. In addition, chelerythrine chloride (CTC, a protein kinase C (PKC inhibitor, blocked the regulation of mGluR5 positive modulators in enhancing NMDA receptor activation and recovering NMDA receptor inhibition. The PKC activator (PMA mimicked the effects of mGluR5 positive modulators on enhancing NMDA receptor activation and reversing NMDA antagonist-evoked NMDA receptor suppression. Conclusion Our results demonstrate that the PKC-dependent pathway may be involved in the positive modulation of mGluR5 resulting in potentiating NMDA receptor activation and reversing NMDA receptor suppression induced by NMDA antagonists.

  16. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue.

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    Yong-Seok Oh

    Full Text Available Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

  17. PKC enhances the capacity for secretion by rapidly recruiting covert voltage-gated Ca2+ channels to the membrane.

    Science.gov (United States)

    Groten, Christopher J; Magoski, Neil S

    2015-02-11

    It is unknown whether neurons can dynamically control the capacity for secretion by promptly changing the number of plasma membrane voltage-gated Ca(2+) channels. To address this, we studied peptide release from the bag cell neurons of Aplysia californica, which initiate reproduction by secreting hormone during an afterdischarge. This burst engages protein kinase C (PKC) to trigger the insertion of a covert Ca(2+) channel, Apl Cav2, alongside a basal channel, Apl Cav1. The significance of Apl Cav2 recruitment to secretion remains undetermined; therefore, we used capacitance tracking to assay secretion, along with Ca(2+) imaging and Ca(2+) current measurements, from cultured bag cell neurons under whole-cell voltage-clamp. Activating PKC with the phorbol ester, PMA, enhanced Ca(2+) entry, and potentiated stimulus-evoked secretion. This relied on channel insertion, as it was occluded by preventing Apl Cav2 engagement with prior whole-cell dialysis or the cytoskeletal toxin, latrunculin B. Channel insertion reduced the stimulus duration and/or frequency required to initiate secretion and strengthened excitation-secretion coupling, indicating that Apl Cav2 accesses peptide release more readily than Apl Cav1. The coupling of Apl Cav2 to secretion also changed with behavioral state, as Apl Cav2 failed to evoke secretion in silent neurons from reproductively inactive animals. Finally, PKC also acted secondarily to enhance prolonged exocytosis triggered by mitochondrial Ca(2+) release. Collectively, our results suggest that bag cell neurons dynamically elevate Ca(2+) channel abundance in the membrane to ensure adequate secretion during the afterdischarge.

  18. Sonic Hedgehog gene delivery to the rodent heart promotes angiogenesis via iNOS/netrin-1/PKC pathway.

    Directory of Open Access Journals (Sweden)

    Rafeeq P H Ahmed

    Full Text Available BACKGROUND: We hypothesized that genetic modification of mesenchymal stem cells (MSCs with Sonic Hedgehog (Shh transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway. METHODS/PRINCIPAL FINDINGS: MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid ((ShhMSCs. Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in (ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in (ShhMSCs. Significantly improved migration and tube formation was seen in (ShhMSCs as compared to empty vector transfected MSCs ((EmpMSCs. Significant upregulation of netrin-1 and iNOS was observed in (ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 microl basal DMEM without cells (group-1 or containing 1x10(6 (EmpMSCs (group-2 and (ShhMSCs (group-3. Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of (ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with (ShhMSCs in group-3 animals. CONCLUSIONS/SIGNIFICANCE: Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.

  19. Comparative analysis of the anti-chikungunya virus activity of novel bryostatin analogs confirms the existence of a PKC-independent mechanism.

    Science.gov (United States)

    Abdelnabi, Rana; Staveness, Daryl; Near, Katherine E; Wender, Paul A; Delang, Leen; Neyts, Johan; Leyssen, Pieter

    2016-11-15

    Previously, we reported that salicylate-based analogs of bryostatin protect cells from chikungunya virus (CHIKV)-induced cell death. Interestingly, 'capping' the hydroxyl group at C26 of a lead bryostatin analog, a position known to be crucial for binding to and modulation of protein kinase C (PKC), did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a pathway independent of PKC. The work detailed in this study demonstrates that salicylate-derived analog 1 and two capped analogs (2 and 3) are not merely cytoprotective compounds, but act as selective and specific inhibitors of CHIKV replication. Further, a detailed comparative analysis of the effect of the non-capped versus the two capped analogs revealed that compound 1 acts both at early and late stages in the chikungunya virus replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with the PKC inhibitors sotrastaurin and Gö6976 counteracts the antiviral activity of compound 1 without affecting that of capped analogs 2 and 3, providing further evidence that the latter elicit their anti-CHIKV activity independently of PKC. Remarkably, treatment of CHIKV-infected cells with a combination of compound 1 and a capped analog resulted in a pronounced synergistic antiviral effect. Thus, these salicylate-based bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Inhibition of Chikungunya Virus-Induced Cell Death by Salicylate-Derived Bryostatin Analogues Provides Additional Evidence for a PKC-Independent Pathway.

    Science.gov (United States)

    Staveness, Daryl; Abdelnabi, Rana; Near, Katherine E; Nakagawa, Yu; Neyts, Johan; Delang, Leen; Leyssen, Pieter; Wender, Paul A

    2016-04-22

    Chikungunya virus (CHIKV) has been spreading rapidly, with over one million confirmed or suspected cases in the Americas since late 2013. Infection with CHIKV causes devastating arthritic and arthralgic symptoms. Currently, there is no therapy to treat this disease, and the only medications focus on relief of symptoms. Recently, protein kinase C (PKC) modulators have been reported to inhibit CHIKV-induced cell death in cell assays. The salicylate-derived bryostatin analogues described here are structurally simplified PKC modulators that are more synthetically accessible than the natural product bryostatin 1, a PKC modulator and clinical lead for the treatment of cancer, Alzheimer's disease, and HIV eradication. Evaluation of the anti-CHIKV activity of these salicylate-derived bryostatin analogues in cell culture indicates that they are among the most potent cell-protective agents reported to date. Given that they are more accessible and significantly more active than the parent natural product, they represent new therapeutic leads for controlling CHIKV infection. Significantly, these analogues also provide evidence for the involvement of a PKC-independent pathway. This adds a fundamentally distinct aspect to the importance or involvement of PKC modulation in inhibition of chikungunya virus replication, a topic of recent and growing interest.

  1. The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye.

    Science.gov (United States)

    Djiane, Alexandre; Yogev, Shaul; Mlodzik, Marek

    2005-05-20

    Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.

  2. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1

    Science.gov (United States)

    García-Ortiz, Almudena; Martín-Cofreces, Noa B.; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M.; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco

    2017-01-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. PMID:28394935

  3. Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl).

    Science.gov (United States)

    Podar, Klaus; Raab, Marc S; Zhang, Jing; McMillin, Douglas; Breitkreutz, Iris; Tai, Yu-Tzu; Lin, Boris K; Munshi, Nikhil; Hideshima, Teru; Chauhan, Dharminder; Anderson, Kenneth C

    2007-02-15

    In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester-induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCmu. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

  4. Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation

    Science.gov (United States)

    Li, Jasmine; Hardy, Kristine; Phetsouphanh, Chan; Tu, Wen Juan; Sutcliffe, Elissa L.; McCuaig, Robert; Sutton, Christopher R.; Zafar, Anjum; Munier, C. Mee Ling; Zaunders, John J.; Xu, Yin; Theodoratos, Angelo; Tan, Abel; Lim, Pek Siew; Knaute, Tobias; Masch, Antonia; Zerweck, Johannes; Brezar, Vedran; Milburn, Peter J.; Dunn, Jenny; Casarotto, Marco G.; Turner, Stephen J.; Seddiki, Nabila; Kelleher, Anthony D.

    2016-01-01

    ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. PMID:27149922

  5. JAM-A and aPKC: A close pair during cell-cell contact maturation and tight junction formation in epithelial cells.

    Science.gov (United States)

    Ebnet, Klaus

    2013-01-01

    Cell-cell adhesion plays a critical role in the formation of barrier-forming epithelia. The molecules which mediate cell-cell adhesion frequently act as signaling molecules by recruiting and/or assembling cytoplasmic protein complexes. Junctional Adhesion Molecule (JAM)-A interacts with the cell polarity protein PAR-3, a member of the PAR-3-aPKC-PAR-6 complex, which regulates the formation of cell-cell contacts and the development of tight junctions (TJs). In our recent study we found that JAM-A is localized at primordial, spot-like cell-cell junctions (pAJs) in a non-phosphorylated form. After the recruitment of the PAR-aPKC complex and its activation at pAJs, aPKC phosphorylates JAM-A at Ser285 to promote the maturation of immature junctions. In polarized epithelial cells, aPKC phosphorylates JAM-A selectively at the TJs to maintain the barrier function of TJs. Thus, through mutual regulation, JAM-A and aPKC form a functional unit that regulates the establishment of barrier-forming junctions in vertebrate epithelial cells.

  6. Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure.

    Science.gov (United States)

    Jozic, Ivan; Vukelic, Sasa; Stojadinovic, Olivera; Liang, Liang; Ramirez, Horacio A; Pastar, Irena; Tomic Canic, Marjana

    2017-05-01

    Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Role of the Chemokine MCP-1 in Sensitization of PKC-Mediated Apoptosis in Prostate Cancer Cells

    Science.gov (United States)

    2010-02-01

    275: 7574-7582 (2000). 5. Garcia -Bermejo, M.L., Leskow, F.C., Fujii, T., Wang, Q., Blumberg, P.M., Ohba, M., Kuroki, T., Han, K.C., Lee, J... Marquez , V.E., and Kazanietz, M.G. Diacylglycerol (DAG)- lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate...induced apoptosis and, conversely, a dominant-negative (kinase-deficient) PKCa mutant inhibits PMA-induced apoptosis ( Garcia -Bermejo et al., 2002). Sig

  8. Apm4, the mu subunit of yeast AP-2 interacts with Pkc1, and mutation of the Pkc1 consensus phosphorylation site Thr176 inhibits AP-2 recruitment to endocytic sites

    Science.gov (United States)

    Chapa-y-Lazo, Bernardo; Ayscough, Kathryn R

    2014-01-01

    The AP-2 endocytic adaptor has been extensively characterized in mammalian cells and is considered to play a role both in cargo binding and in formation of endocytic sites. However, despite our detailed knowledge of mechanistic aspects of endocytic complex assembly and disassembly in the model organism Saccharomyces cerevisiae, no function of AP-2 had been described in wild-type yeast under normal growth conditions. A recent study however revealed that disruption of the complex caused by deletion of the gene encoding its mu subunit (APM4) caused defects in cell polarity such that responses to pheromone, nutritional status and cell wall damage were affected. Furthermore, a homozygous deletion of the mu subunit gene in Candida albicans affected its ability to grow hyphae. Direct binding to the yeast cell wall stress sensor Mid2 was detected, and in an apm4 deletion strain Mid2 showed reduced re-localization to the mother bud neck region following cell wall damage with calcofluor or to the mating projection tip. Here we demonstrate an interaction between Apm4 and the yeast cell wall integrity pathway component Pkc1 and show that mutation of the predicted Pkc1 site in the Apm4 hinge region affects recruitment of the AP-2 complex to endocytic sites. PMID:25346786

  9. Genotypic and phenotypic characterization of garlic-fermenting lactic acid bacteria isolated from som-fak, a Thai low-salt fermented fish product

    DEFF Research Database (Denmark)

    Paludan-Müller, Christine; Valyasevi, R.; Huss, Hans Henrik

    2002-01-01

    AIMS: To evaluate the importance of garlic for fermentation of a Thai fish product, and to differentiate among garlic-/inulin-fermenting lactic acid bacteria (LAB) at strain level. METHODS AND RESULTS: Som-fak was prepared by fermentation of a mixture of fish, salt, rice, sucrose and garlic. p......H decreased to 4.5 in 2 days, but omitting garlic resulted in a lack of acidification. LAB were predominant and approximately one third of 234 isolated strains fermented garlic and inulin (the carbohydrate reserve in garlic). These strains were identified as Lactobacillus pentosus and Lact. plantarum...... AND IMPACT OF THE STUDY: The present study indicates the role of fructans (garlic/inulin) as carbohydrate sources for LAB. Fructan fermenters may have several biotechnological applications, for example, as probiotics....

  10. CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Vishnubalaji, Radhakrishnan; Hamam, Rimi;

    2015-01-01

    INTRODUCTION: Tumor microenvironment conferred by stromal (mesenchymal) stem cells (MSCs) plays a key role in tumor development, progression, and response to therapy. Defining the role of MSCs in tumorigenesis is crucial for their safe utilization in regenerative medicine. Herein, we conducted...... was dependent on direct cell-cell contact. Our data also revealed transfer of cellular components between cancer cells and hMSCs as one possible mechanism for intercellular communication. Global gene expression analysis of sorted hMSCs following co-culturing with MCF7 and BT-20 cells revealed enrichment...... in signaling pathways related to bone formation, FAK and MAPKK signaling. Co-culturing hMSCs with MCF7 cells increased their growth evidenced by increase in Ki67 and PCNA staining in tumor cells in direct contact with hMSCs niche. On the other hand, co-culturing hMSCs with FaDu, HT-29 or MDA-MB-231 cells led...

  11. Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach

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    Reddivari Lavanya

    2011-08-01

    Full Text Available Abstract Background We and others have previously reported that resveratrol (RSV suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM and RSV (150 μM using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology. Results Pentose phosphate pathway (PPP, a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting and transketolase, key enzymes of the PPP. RSV (150 μM suppressed, whereas IGF-1 (10 nM elevated focal adhesion complex (FAC proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively. Conclusions Proteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.

  12. Regulation of taurine transport at the blood-placental barrier by calcium ion, PKC activator and oxidative stress conditions

    Science.gov (United States)

    2010-01-01

    Background In the present study, we investigated the changes of uptake and efflux transport of taurine under various stress conditions using rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT cells), as in vitro blood-placental barrier (BPB) model. Methods The transport of taurine in TR-TBT cells were characterized by cellular uptake study using radiolabeled taurine. The efflux of taurine was measured from the amount of radiolabeled taurine remaining in the cells after the uptake of radiolabeled taurine for 60 min. Results Taurine uptake was significantly decreased by phosphorylation of protein kinase C (PKC) activator in TR-TBT cells. Also, calcium ion (Ca2+) was involved in taurine transport in TR-TBT cells. Taurine uptake was inhibited and efflux was enhanced under calcium free conditions in the cells. In addition, oxidative stress induced the change of taurine transport in TR-TBT cells, but the changes were different depending on the types of oxidative stress inducing agents. Tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS) and diethyl maleate (DEM) significantly increased taurine uptake, but H2O2 and nitric oxide (NO) donor decreased taurine uptake in the cells. Taurine efflux was down-regulated by TNF-α in TR-TBT cells. Conclusion Taurine transport in TR-TBT cells were regulated diversely at extracellular Ca2+ level, PKC activator and oxidative stress conditions. It suggested that variable stresses affected the taurine supplies from maternal blood to fetus and taurine level of fetus. PMID:20804613

  13. Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene.

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    Regenhard, P; Goethe, R; Phi-van, L

    2001-04-01

    The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.

  14. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism.

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    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2015-07-22

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1(NL4.3) were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection.

  15. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

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    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  16. Neuroprotective effects of nitric oxide donor NOC-18 against brain ischemia-induced mitochondrial damages: role of PKG and PKC.

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    Arandarcikaite, Odeta; Jokubka, Ramunas; Borutaite, Vilmante

    2015-01-23

    In this study we sought to determine whether NO donor NOC-18 can protect brain mitochondria against ischemia-induced dysfunction, particularly opening of mitochondrial permeability transition pore (MPTP), and cell death. We found that inhibition of respiration with NAD-dependent substrates, but not with succinate, was observed after 30 min ischemia indicating that complex I of the mitochondrial respiratory chain is the primary site affected by ischemia. There was no loss of mitochondrial cytochrome c during 30-120 min of brain ischemia. Prolonged, 90 min ischemia substantially decreased calcium retention capacity of brain mitochondria suggesting sensitization of mitochondria to Ca(2+)-induced MPTP opening, and this was prevented by NOC-18 infusion prior to ischemia. NOC-18 did not prevent ischemia-induced inhibition of mitochondrial respiration, however, it partially protected against ischemia-induced necrosis. Protective effects of NOC-18 were abolished in the presence of selective inhibitors of protein kinase G (PKG) and protein kinase C (PKC). These results indicate that pre-treatment with NOC-18 protected brain mitochondria against ischemia-induced MPTP opening by decreasing mitochondrial sensitivity to calcium and partly protected brain cells against necrotic death in PKG- and PKC-depending manner.

  17. Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

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    Laura Monturiol-Gross

    Full Text Available Clostridium perfringens phospholipase C (CpPLC, also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

  18. Breviscapine alleviates hepatic injury and inhibits PKC-mRNA and its protein expression in brain-dead BA-Ma mini pigs

    Institute of Scientific and Technical Information of China (English)

    Shui-Jun Zhang; Yan Song; Wen-Long Zhai; Ji-Hua Shi; Liu-Shun Feng; Yong-Fu Zhao; Shi Chen

    2007-01-01

    BACKGROUND:Brain-dead donors are the main sources for organ transplantation, but many studies show that brain-death affects the organ's function after transplantation. This study was undertaken to investigate liver injury after brain-death in BA-Ma mini pigs and the protective effects of breviscapine on hepatic function and on PKC-α mRNA and its protein expression. METHODS:Fifteen BA-Ma mini pigs were equally divided into 3 groups at random: brain-dead (group B), breviscapine pretreated (group P), and control (group C). The brain-dead model was established by increasing intracranial pressure in a modiifed, slow and intermittent way. At 3, 6, 12, 18 and 24 hours after the initial brain-death, the levels of serum AST, ALT, TNF-α, IL-1β, and IL-6 were determined. The changes in hepatic tissues were assessed, and the expression of PKC-α and PKC-αmRNA was detected by immunohistochemistry and RT-PCR, respectively. RESULTS:The levels of AST and ALT in groups B and P began to increase 12 hours after brain-death, while the values in group P were lower than those in group B (P<0.05). The levels of IL-1β, IL-6, and TNF-α in groups B and P at 3, 6, 12 and 18 hours were lower than those in group B (P<0.05). At 6, 12 and 24 hours, the expressions of PKC-α mRNA and PKC-α protein in group P were lower than those in group B (P<0.05). The degree of injury to hepatic cells in group P was milder than that in group B.CONCLUSIONS:Breviscapine inhibits the degree of PKC-αmRNA transcription and its protein translation, decreases the release of inlfammatory factors, and thus alleviates hepatic injury during brain-death.

  19. Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation.

    Science.gov (United States)

    Podar, Klaus; Tai, Yu-Tzu; Lin, Boris K; Narsimhan, Radha P; Sattler, Martin; Kijima, Takashi; Salgia, Ravi; Gupta, Deepak; Chauhan, Dharminder; Anderson, Kenneth C

    2002-03-08

    In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta(1) integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta(1) integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta(1) integrin. Moreover, we demonstrate that PKC alpha is constitutively associated with beta(1) integrin. These data are consistent with PKC alpha-dependent exocytosis of activated beta(1) integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC alpha-driven internalization of beta(1) integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta(1) integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC alpha. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.

  20. Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC

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    Galiñanes Manuel

    2002-07-01

    Full Text Available Abstract Background These studies investigate the role of mitoKATP channels, protein kinase C (PKC and Mitogen activated protein kinase (p38MAPK on the cardioprotection of ischemic (IP and pharmacological preconditioning (PP of the human myocardium and their sequence of activation. Results Right atrial appendages from patients undergoing elective cardiac surgery were equilibrated for 30 min and then subjected to 90 min of simulated ischemia followed by 120 min reoxygenation. At the end of each protocol creatinine kinase leakage (CK U/g wet wt and the reduction of MTT to formazan dye (mM/g wet wt were measured. Similar protection was obtained with α1 agonist phenylephrine, adenosine and IP and their combination did not afford additional cardioprotection. Blockade of mitoKATP channels with 5-hydroxydecanoate, PKC with chelerythrine, or p38MAPK with SB203580 abolished the protection of IP and of PP. In additional studies, the stimulation of mitoKATP channels with diazoxide or activation of PKC with PMA or p38MAPK with anisomycin induced identical protection to that of IP and PP. The protection induced by diazoxide was abolished by blockade of PKC and by blockade of p38MAPK. Furthermore, the protection induced by PMA was abolished by SB203580 but not by 5-hydroxydecanoate, whereas the protection induced by anisomycin was unaffected by either 5-hydroxydecanoate or chelerythrine. Conclusions Opening of mitoKATP channels and activation of PKC and p38MAPK are obligatory steps in the signal transduction cascade of IP and PP of the human myocardium with PKC activation being downstream of the opening of mitoKATP channels and upstream of p38MAPK activation.

  1. PI3K and PKC contribute to membrane depolarization mediated by α2-adrenoceptors in the canine isolated mesenteric vein

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    Mutafova-Yambolieva Violeta N

    2005-06-01

    Full Text Available Abstract Background Norepinephrine (NE, a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K and protein kinase C (PKC are involved in the electrical field stimulation (EFS-induced slow membrane depolarization (SMD in canine isolated mesenteric vein. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3 receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined. Results Contractile responses to NE and clonidine (0.05 – 10 μM were significantly diminished in the presence of yohimbine (0.1 μM. Exogenous NE (0.1 μM and clonidine (1 μM elicited SMD. The resting membrane potential of canine mesenteric vein smooth muscle cells was -68.8 ± 0.8 mV. EFS elicited a biphasic depolarization comprised of excitatory junction potentials and SMD that are purinergic and adrenergic in nature, respectively. The magnitude of the SMD in response to EFS at 0.5 Hz was 9.4 ± 0.7 mV. This response was reduced by 65–98% by the fast Na+ channel inhibitor tetrodotoxin (1 μM, by the inhibitor of N-type Ca2+ channels ω-conotoxin GVIA (5 nM, the non-selective α-adrenoceptor blocker phentolamine (1 μM, the selective α2-adrenoceptor blocker yohimbine (0.1 μM, the ion channel inhibitors niflumic acid (NFA, 100 μM, 5-nitro-2-(3-phenylpropylamino benzoic acid (NPPB, 30 μM, 4,4'-diisothiocyanatostilbene-2

  2. Post-conditioning protects cardiomyocytes from apoptosis via PKC(epsilon)-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk.

    Science.gov (United States)

    Dong, Shiyun; Teng, Zongyan; Lu, Fang-Hao; Zhao, Ya-Jun; Li, Hulun; Ren, Huan; Chen, He; Pan, Zhen-Wei; Lv, Yan-Jie; Yang, Bao-Feng; Tian, Ye; Xu, Chang-Qing; Zhang, Wei-Hua

    2010-08-01

    The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the

  3. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

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    Zhang, Chenyue; Wu, Ying; Xuan, Zinan; Zhang, Shujing; Wang, Xudan; Hao, Yu; Wu, Jun; Zhang, Shu

    2014-11-04

    Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615

  4. Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

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    Xu Zheng

    Full Text Available In response to interleukin 6 (IL-6 stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK, and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC

  5. Replacement of the Bryostatin A- and B-Pyran Rings With Phenyl Rings Leads to Loss of High Affinity Binding With PKC.

    Science.gov (United States)

    Petersen, Mark E; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Keck, Gary E

    2016-10-19

    We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring 'binding domain'.

  6. Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs

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    Lisienny C. T. Rempel

    2015-05-01

    Full Text Available Advanced glycation end products (AGEs are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs–endothelium interactions through the receptor for AGEs (RAGE in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs, monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-β inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-β inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-β inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.

  7. Different effect of chronic stress on learning and memory in BALB/c and C57BL/6 inbred mice: Involvement of hippocampal NO production and PKC activity.

    Science.gov (United States)

    Palumbo, María Laura; Zorrilla Zubilete, María Aurelia; Cremaschi, Graciela Alicia; Genaro, Ana María

    2009-07-01

    Nitric oxide (NO) has been involved in many pathophysiological brain processes. Recently, we showed that neuronal nitric oxide synthase (nNOS)-mediated decrease in NO production is involved in memory impairment induced by chronic mild stress (CMS) in BALB/c mice. Two genetically different inbred murine strains, C57BL/6 and BALB/c, show distinct behavioral responses, neurodevelopmental and neurochemical parameters. Here, we perform a comparative study on CMS effects upon learning and memory in both strains, analyzing the role of NO production and its regulation by protein kinase C (PKC). Stressed BALB/c, but not C57Bl/6 mice, showed a poor learning performance in both the open field and passive avoidance inhibitory tasks. Also, CMS induced a diminished NO production by nNOS, associated with an increment in gamma and zeta PKC isoenzymes in BALB/c mice. In C57BL/6 mice, CMS had no effect on NO production, but increased delta and decreased betaI PKC isoforms. In vivo administration of a NOS inhibitor induced behavioral alterations in both strains. These results suggest a differential effect of stress, with BALB/c being more vulnerable to stress than C57BL/6 mice. This effect could be related to a differential regulation of NOS and PKC isoenzymes, pointing to an important role of NO in learning and memory.

  8. Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis.

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    Song, Kyoung-Sub; Kim, Jong-Seok; Yun, Eun-Jin; Kim, Young-Rae; Seo, Kang-Sik; Park, Ji-Hoon; Jung, Yeon-Joo; Park, Jong-Il; Kweon, Gi-Ryang; Yoon, Wan-Hee; Lim, Kyu; Hwang, Byung-Doo

    2008-07-01

    Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.

  9. A PKM Generated by Calpain Cleavage of a Classical PKC Is Required for Activity-Dependent Intermediate-Term Facilitation in the Presynaptic Sensory Neuron of "Aplysia"

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    Farah, Carole A.; Hastings, Margaret H.; Dunn, Tyler W.; Gong, Katrina; Baker-Andresen, Danay; Sossin, Wayne S.

    2017-01-01

    Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that…

  10. Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs).

    Science.gov (United States)

    Rempel, Lisienny C T; Finco, Alessandra B; Maciel, Rayana A P; Bosquetti, Bruna; Alvarenga, Larissa M; Souza, Wesley M; Pecoits-Filho, Roberto; Stinghen, Andréa E M

    2015-05-14

    Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs-endothelium interactions through the receptor for AGEs (RAGE) in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-β inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-β inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-β inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.

  11. Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST.

    Science.gov (United States)

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X; Lu, Zhimin

    2011-11-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.

  12. A PKC-dependent recruitment of MMP-2 controls semaphorin-3A growth-promoting effect in cortical dendrites.

    Directory of Open Access Journals (Sweden)

    Bertrand Gonthier

    Full Text Available There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3. Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

  13. Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.

    Science.gov (United States)

    Schott, Micah B; Grove, Bryon

    2013-11-01

    A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC. © 2013.

  14. Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice.

    Directory of Open Access Journals (Sweden)

    Luiz O Leiria

    Full Text Available Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC and Ca(v1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Ca(v1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001-100 µM, α,β-methylene ATP (1-10 µM, KCl (1-300 mM, extracellular Ca(2+ (0.01-100 mM and phorbol-12,13-dibutyrate (PDBu; 0.001-3 µM all produced greater DSM contractions in obese mice, which were fully reversed by the Ca(v1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Ca(v1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca(v1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

  15. Douchi (fermented Glycine max Merr.) alleviates atopic dermatitis-like skin lesions in NC/Nga mice by regulation of PKC and IL-4.

    Science.gov (United States)

    Jung, A-Ram; Ahn, Sang-Hyun; Park, In-Sik; Park, Sun-Young; Jeong, Seung-Il; Cheon, Jin-Hong; Kim, Kibong

    2016-10-24

    Douchi (fermented Glycine max Merr.) is produced from fermented soybeans, which is widely used in traditional herbal medicine. In this study, we investigated whether Douchi attenuates protein kinase C (PKC) and interleukin (IL)-4 response and cutaneous inflammation in Atopic dermatitis (AD)-like NC/Nga mice. To induce AD-like skin lesions, D. farinae antigen was applied to the dorsal skin of 3-week-old NC/Nga mice. After inducing AD, Douchi extract was administered 20 mg/kg daily for 3 weeks to the Douchi-treated mice group. We identified the changes of skin barrier and Th2 differentiation through PKC and IL-4 by immunohistochemistry. Douchi treatment of NC/Nga mice significantly reduced clinical scores (p < 0.01) and histological features. The levels of PKC and IL-4 were significantly reduced in the Douchi-treated group (p < 0.01). The reduction of IL-4 and PKC led to decrease of inflammatory factors such as substance P, inducible nitric oxide synthase (iNOS) and Matrix metallopeptidase 9 (MMP-9) (all p < 0.01). Douchi also down-regulated Th1 markers (IL-12, TNF-α) as well as Th2 markers (IL-4, p-IκB) (p < 0.01). Douchi alleviates AD-like skin lesions through suppressing of PKC and IL-4. These results also lead to diminish levels of substance P, iNOS and MMP-9 in skin lesions. Therefore, Douchi may have potential applications for the prevention and treatment of AD.

  16. Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways.

    Science.gov (United States)

    Xiong, Niya; Li, Shun; Tang, Kai; Bai, Hongxia; Peng, Yueting; Yang, Hong; Wu, Chunhui; Liu, Yiyao

    2017-01-01

    Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.

  17. Decreased nuclear stiffness via FAK-ERK1/2 signaling is necessary for osteopontin-promoted migration of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Wang, Aoli; Shi, Yisong; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

    2017-04-06

    Migration of bone marrow-derived mesenchymal stem cells (BMSCs) plays an important role in many physiological and pathological settings, including wound healing. During the migration of BMSCs through interstitial tissues, the movement of the nucleus must be coordinated with the cytoskeletal dynamics, which in turn affects the cell migration efficiency. Our previous study indicated that osteopontin (OPN) significantly promotes the migration of rat BMSCs. However, the nuclear behaviors and involved molecular mechanisms in OPN-mediated BMSC migration are largely unclear. In the present study, using an atomic force microscope (AFM), we found that OPN could decrease the nuclear stiffness of BMSCs and reduce the expression of lamin A/C, which is the main determinant of nuclear stiffness. Increased lamin A/C expression attenuates BMSC migration by increasing nuclear stiffness. Decreased lamin A/C expression promotes BMSC migration by decreasing nuclear stiffness. Furthermore, OPN promotes BMSC migration by diminishing lamin A/C expression and decreasing nuclear stiffness via the FAK-ERK1/2 signaling pathway. This study provides strong evidence for the role of nuclear mechanics in BMSC migration as well as new insight into the molecular mechanisms of OPN-promoted BMSC migration.

  18. Lewis y enhances CAM-DR in ovarian cancer cells by activating the FAK signaling pathway and upregulating Bcl-2/Bcl-XL expression.

    Science.gov (United States)

    Yan, Limei; Wang, Changzhi; Lin, Bei; Liu, Juanjuan; Liu, Dawo; Hou, Rui; Wang, Yifei; Gao, Lili; Zhang, Shulan; Iwamori, Masao

    2015-06-01

    Oligosaccharides on the surface of adhesion molecules may contribute to the process of CAM-DR. To investigate the role of the Lewis y antigen in this process, we established a cell adhesion model mediated by the integrin α5β1-FN interaction in the ovarian cancer cell line, RMG-1-hFUT, which highly expresses Lewis y by transfection with α1,2-fucosyltransferase into RMG-1 cells. Our results indicate that the rates of carboplatin-induced apoptosis and necrosis are reduced in FN-adhered tumor cells, and carboplatin resistance is significantly decreased in the presence of anti-Lewis y antibody. CAM-DR in tumor cells has been correlated with elevated expression of the nuclear anti-apoptotic proteins Bcl-2 and Bcl-XL. Lewis y promotes the expression of the Bcl-2 and Bcl-XL genes by activating the focal adhesion kinase signaling pathway and accelerating their transcription. Thus, Lewis y leads to inhibition of apoptosis and enhancement of CAM-DR by activation of the FAK signaling pathway and upregulation of Bcl-2/Bcl-XL expression in ovarian cancer cell lines. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  19. D-pinitol Inhibits Prostate Cancer Metastasis through Inhibition of αVβ3 Integrin by Modulating FAK, c-Src and NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Chih-Hsin Tang

    2013-05-01

    Full Text Available Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145 at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK phosphorylation, c-Src kinase activity and NF-kB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.

  20. D-pinitol inhibits prostate cancer metastasis through inhibition of αVβ3 integrin by modulating FAK, c-Src and NF-κB pathways.

    Science.gov (United States)

    Lin, Tien-Huang; Tan, Tzu-Wei; Tsai, Tsung-Hsun; Chen, Chi-Cheng; Hsieh, Teng-Fu; Lee, Shang-Sen; Liu, Hsin-Ho; Chen, Wen-Chi; Tang, Chih-Hsin

    2013-05-08

    Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145) at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK) phosphorylation, c-Src kinase activity and NF-kB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.

  1. 2类多巴胺受体通过促进 PKC-ε转位参与心肌缺血后适应抑制细胞凋亡%Involvement of dopamine receptor-2 in myocardial ischemic postconditioning inhibited apop-tosis by promoting translocation of PKC

    Institute of Scientific and Technical Information of China (English)

    李鸿珠; 高君; 郝晓敏; 张丽敏; 陈俊亭

    2014-01-01

    目的:以蛋白激酶C-ε(PKC-ε)转位为切入点,探讨2类多巴胺受体(DR2)在心肌缺血后适应抑制细胞凋亡中的作用及可能机制。方法复制原代培养乳鼠心肌细胞缺氧/复氧和缺血后适应模型。 MTT检测心肌细胞的存活率;Hoechst 33342染色观察细胞凋亡;Western blotting检测Bcl-2、caspase-3、caspase-9、PKC-ε蛋白的表达和细胞色素C( Cyt c)的释放;免疫共沉淀检测PKC-ε和DR2的相互作用。结果与正常组比较,缺氧/复氧组细胞存活率降低,细胞凋亡增加,促凋亡因子( Cyt c、caspase-3、caspase-9)表达增加,抑凋亡因子(Bcl-2)表达亦增加,PKC-ε没有转位到细胞膜,PKC-ε和DR2不存在相互作用。与缺氧/复氧组比较,缺血后适应明显升高细胞存活率,降低细胞凋亡,抑制促凋亡因子(Cyt c、caspase-3、caspase-9)表达,促进抑凋亡因子(Bcl-2)表达,PKC-ε转位到细胞膜,PKC-ε和DR2存在相互作用。与缺血后适应组比较,DR2激动剂进一步增加缺血后适应的保护作用,而DR2抑制剂则取消了DR2激动剂的作用。结论 DR2参与心肌缺血后适应抑制细胞凋亡,其机制与DR2促进PKC-ε转位及DR2和PKC-ε存在相互作用有关。%Objective To investigate the effect and possible mechanism of dopamine receptor-2 (DR2) activation in ischemic postconditioning ( PC) inhibited cardiomyocytes apoptosis through translocation of PKC-ε. Method The hypoxia/reoxygenation ( H/R) injury and PC models was established using primarily cultured neonatal rat cardio-myocytes. The cell survival rate was detected by MTT. The cell apoptosis was observed using Hoechst 33342 sta-ning. The protein expression of Bcl-2, caspase-3, caspase-9, the release of cytochrome c( Cyt c) and translocation of PKC-εwere analyzed using Western blotting. The interaction of DR2 and PKC-εwas tested by co-immunoprecipi-tation. Result Compared with Control group, the cell survival rate was decreased, cardiomyocytes

  2. Activation of Protein Kinase C (PKC)α or PKCε as an Approach to Increase Morphine Tolerance in Respiratory Depression and Lethal OverdoseS⃞

    OpenAIRE

    2012-01-01

    Long-term use of opioids is hindered by respiratory depression and the possibility for fatal overdose in drug abusers. This is attributed to higher levels of tolerance that develops against antinociception than to respiratory depression. Identifying important mechanisms that would increase morphine respiratory depression and overdose tolerance could lead to the safer use of opioids. Because protein kinase C (PKC) activity mediates the development and maintenance of morphine antinociceptive to...

  3. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    Science.gov (United States)

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  4. Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway.

    Science.gov (United States)

    Bai, Xiaoming; Wang, Jie; Guo, Yan; Pan, Jinshun; Yang, Qinyi; Zhang, Min; Li, Hai; Zhang, Li; Ma, Juan; Shi, Feng; Shu, Wei; Wang, Yipin; Leng, Jing

    2014-10-07

    Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1-4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer.

  5. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

    Science.gov (United States)

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-12-01

    The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase

    Science.gov (United States)

    Ahn, Jee-Yin; Liu, Xia; Liu, Zhixue; Pereira, Lorena; Cheng, Dongmei; Peng, Junmin; Wade, Paul A; Hamburger, Anne W; Ye, Keqiang

    2006-01-01

    Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1. PMID:16642037

  7. LDB3 splicing abnormalities are specific to skeletal muscles of patients with myotonic dystrophy type 1 and alter its PKC binding affinity.

    Science.gov (United States)

    Yamashita, Yoshihiro; Matsuura, Tohru; Kurosaki, Tatsuaki; Amakusa, Yoshinobu; Kinoshita, Masanobu; Ibi, Tohru; Sahashi, Ko; Ohno, Kinji

    2014-09-01

    Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC.

  8. Ganoderma atrum polysaccharide evokes antitumor activity via cAMP-PKA mediated apoptotic pathway and down-regulation of Ca(2+)/PKC signal pathway.

    Science.gov (United States)

    Zhang, Shenshen; Nie, Shaoping; Huang, Danfei; Huang, Jianqin; Feng, Yanling; Xie, Mingyong

    2014-06-01

    Ganoderma atrum polysaccharide (PSG-1) has been commonly suggested as a candidate for prevention and therapy of cancer. We investigated the antitumor effect and the underlying molecular mechanisms of PSG-1. The results showed that PSG-1 inhibited tumor growth and resulted in tumor cell apoptosis in vivo. Here, the data revealed that PSG-1 caused a markedly increase in cAMP and PKA activities, rather than cGMP and PKC. Moreover, the treatment of PSG-1 induced a dramatic increase in the protein level of PKA. In contrast, the expression of PKC and intracellular [Ca(2+)]i were inhibited. Our study also revealed that treatment with PSG-1 increased the spleen and thymus weights, lymphocyte proliferation and macrophage phagocytic activity in tumor-bearing mice. Taken together, we conclude that PSG-1 could inhibit the tumor growth, possibly in part by enhancing the induction of apoptosis through cAMP-PKA signaling pathway and down-regulation of Ca(2+)/PKC signal pathway, activating host immune function in S180-bearing mice.

  9. Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate.

    Science.gov (United States)

    Gong, Chenyuan; Yao, Chao; Xu, Zihang; Ni, Zhongya; Zhu, Xiaowen; Wang, Lixin; Yan, Xuewei; Zhou, Wuxiong; Zhu, Shiguo

    2017-03-01

    The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

  10. Protein kinase C (PKC phosphorylates the system N glutamine transporter SN1 (slc38a3 and regulates its membrane trafficking and degradation

    Directory of Open Access Journals (Sweden)

    Lise Sofie H. Nissen-Meyer

    2013-10-01

    Full Text Available The system N transporter SN1 (also known as SNAT3 is enriched on perisynaptic astroglial cell membranes. SN1 mediates electroneutral and bidirectional glutamine transport, and regulates the intracellular as well as the extracellular concentrations of glutamine. We hypothesize that SN1 participates in the glutamate/GABA-glutamine cycle and regulates the amount of glutamine supplied to the nerve terminals for replenishment of the neurotransmitter pools of glutamate and GABA. We also hypothesize that its activity on the plasma membrane is regulated by PKC-mediated phosphorylation and that SN1 activity has an impact on synaptic plasticity. This review discusses inconcistencies reported in the regulation of SN1 by PKC and presents a consolidated model for regulation and degradation of SN1 and the subsequent functional implications. As SN1 function is likely also regulated by PKC-mediated phosphorylation in peripheral organs, the same mechanisms may, thus, have impact on e.g. pH regulation in the kidney, urea formation in the liver and insulin secretion in the pancreas.

  11. Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-α/NADPH Oxidase Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Lishan Zhou

    2013-01-01

    Full Text Available Hu-Lu-Ba-Wan (HLBW is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH and protein kinase C-alpha (PKC-α related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-α in renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-α/NADPH oxidase signaling pathway.

  12. 运动性心肌肥厚中PKC信号通路研究进展%Advance of PKC Signaling Pathway in Exercise -induced Cardiac Myocyte Hypertrophy

    Institute of Scientific and Technical Information of China (English)

    柳华; 杨翼

    2011-01-01

    心肌肥厚是心脏受到刺激后的一种代偿反应。心肌的生理性肥厚可提高心脏机能,但病理性肥厚则会引起心血管疾病心律失常发病和死亡,亦是运动员发生猝死的原因之一。明确运动性心肌肥厚的机制,可为保护运动员心脏提供理论依据。目前为止,形成心肌肥厚的信号通路包括PKC、蛋白激酶B(Akt)、钙调神经磷酸酶(CaM)、丝裂原活化蛋白激酶MAPK、酪氨酸激酶受体gp130/STAT、牵拉敏感离子通道以及细胞骨架等。其中PKC途径是调控心肌细胞的生长、分化以及肥厚的过程中信号转导通路的关键环节。心肌细胞可通过Gq/PLC/Ca2+/PKC、PKC/MAPK、PKC/NF-κB等相关途径促使活化晚期反应基因使心肌肥厚。在运动过程中,形成的生理性肥厚主要通过AkffmTOR途径。心肌细胞的机械牵拉亦可以通过PKC信号传导促使肥厚。PKC包括13种亚型,各亚型对心脏的作用不一。PKCa对多次运动后心肌有保护作用;PKCβ1对心肌可能有保护作用,B2则是相反的作用;PKC8有利于心肌细胞的适应;PKCs对心肌的保护作用亦有可能有相反的作用;PKCζ促使心肌肥厚。%Myocardial hypertrophy is a compensatory response after the heart stimulation. Myocardial physio- logic hypertrophy can improve heart function, but pathological hypertrophy may cause cardiovascular disease as onset of arrhythmia and death, and also one of the causes of sudden death in athletes. The clearing mecha- nism of exercise -induced cardiac hypertrophy can provide a theoretical basis for the protection of athlete' s heart. So far, formation of myocardial hypertrophy signaling pathways includes PKC, protein kinase B ( Akt), calcineurin ( CaM), activation of the mitogen - activated protein kinase MAPK, tyrosine kinase re- ceptor gpl30/STAT, and the stretch of sensitive ion channels and cytoskeletal etc.. The PKC pathway is the

  13. L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells.

    Science.gov (United States)

    Toselli, Mauro; Taglietti, Vanni

    2005-05-01

    Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

  14. CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt activation in a PI3K-dependent and FAK-independent fashion

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    Lillard James W

    2011-05-01

    Full Text Available Abstract Background Chemotherapy heavily relies on apoptosis to kill breast cancer (BrCa cells. Many breast tumors respond to chemotherapy, but cells that survive this initial response gain resistance to subsequent treatments. This leads to aggressive cell variants with an enhanced ability to migrate, invade and survive at secondary sites. Metastasis and chemoresistance are responsible for most cancer-related deaths; hence, therapies designed to minimize both are greatly needed. We have recently shown that CCR9-CCL25 interactions promote BrCa cell migration and invasion, while others have shown that this axis play important role in T cell survival. In this study we have shown potential role of CCR9-CCL25 axis in breast cancer cell survival and therapeutic efficacy of cisplatin. Methods Bromodeoxyuridine (BrdU incorporation, Vybrant apoptosis and TUNEL assays were performed to ascertain the role of CCR9-CCL25 axis in cisplatin-induced apoptosis of BrCa cells. Fast Activated Cell-based ELISA (FACE assay was used to quantify In situ activation of PI3Kp85, AktSer473, GSK-3βSer9 and FKHRThr24 in breast cancer cells with or without cisplatin treatment in presence or absence of CCL25. Results CCR9-CCL25 axis provides survival advantage to BrCa cells and inhibits cisplatin-induced apoptosis in a PI3K-dependent and focal adhesion kinase (FAK-independent fashion. Furthermore, CCR9-CCL25 axis activates cell-survival signals through Akt and subsequent glycogen synthase kinase-3 beta (GSK-3β and forkhead in human rhabdomyosarcoma (FKHR inactivation. These results show that CCR9-CCL25 axis play important role in BrCa cell survival and low chemotherapeutic efficacy of cisplatin primarily through PI3K/Akt dependent fashion.

  15. CCR9 interactions support ovarian cancer cell survival and resistance to cisplatin-induced apoptosis in a PI3K-dependent and FAK-independent fashion

    Directory of Open Access Journals (Sweden)

    Johnson Erica L

    2010-06-01

    Full Text Available Abstract Background Cisplatin is more often used to treat ovarian cancer (OvCa, which provides modest survival advantage primarily due to chemo-resistance and up regulated anti-apoptotic machineries in OvCa cells. Therefore, targeting the mechanisms responsible for cisplatin resistance in OvCa cell may improve therapeutic outcomes. We have shown that ovarian cancer cells express CC chemokine receptor-9 (CCR9. Others have also shown that CCL25, the only natural ligand for CCR9, up regulates anti-apoptotic proteins in immature T lymphocytes. Hence, it is plausible that CCR9-mediated cell signals might be involved in OvCa cell survival and inhibition of cisplatin-induced apoptosis. In this study, we investigated the potential role and molecular mechanisms of CCR9-mediated inhibition of cisplatin-induced apoptosis in OvCa cells. Methods Cell proliferation, vibrant apoptosis, and TUNEL assays were performed with or without cisplatin treatment in presence or absence of CCL25 to determine the role of the CCR9-CCL25 axis in cisplatin resistance. In situ Fast Activated cell-based ELISA (FACE assays were performed to determine anti-apoptotic signaling molecules responsible for CCL25-CCR9 mediated survival. Results Our results show interactions between CCR9 and CCL25 increased anti-apoptotic signaling cascades in OvCa cells, which rescued cells from cisplatin-induced cell death. Specifically, CCL25-CCR9 interactions mediated Akt, activation as well as GSK-3β and FKHR phosphorylation in a PI3K-dependent and FAK-independent fashion. Conclusions Our results suggest the CCR9-CCL25 axis plays an important role in reducing cisplatin-induced apoptosis of OvCa cells.

  16. CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt activation in a PI3K-dependent and FAK-independent fashion

    Science.gov (United States)

    2011-01-01

    Background Chemotherapy heavily relies on apoptosis to kill breast cancer (BrCa) cells. Many breast tumors respond to chemotherapy, but cells that survive this initial response gain resistance to subsequent treatments. This leads to aggressive cell variants with an enhanced ability to migrate, invade and survive at secondary sites. Metastasis and chemoresistance are responsible for most cancer-related deaths; hence, therapies designed to minimize both are greatly needed. We have recently shown that CCR9-CCL25 interactions promote BrCa cell migration and invasion, while others have shown that this axis play important role in T cell survival. In this study we have shown potential role of CCR9-CCL25 axis in breast cancer cell survival and therapeutic efficacy of cisplatin. Methods Bromodeoxyuridine (BrdU) incorporation, Vybrant apoptosis and TUNEL assays were performed to ascertain the role of CCR9-CCL25 axis in cisplatin-induced apoptosis of BrCa cells. Fast Activated Cell-based ELISA (FACE) assay was used to quantify In situ activation of PI3Kp85, AktSer473, GSK-3βSer9 and FKHRThr24 in breast cancer cells with or without cisplatin treatment in presence or absence of CCL25. Results CCR9-CCL25 axis provides survival advantage to BrCa cells and inhibits cisplatin-induced apoptosis in a PI3K-dependent and focal adhesion kinase (FAK)-independent fashion. Furthermore, CCR9-CCL25 axis activates cell-survival signals through Akt and subsequent glycogen synthase kinase-3 beta (GSK-3β) and forkhead in human rhabdomyosarcoma (FKHR) inactivation. These results show that CCR9-CCL25 axis play important role in BrCa cell survival and low chemotherapeutic efficacy of cisplatin primarily through PI3K/Akt dependent fashion. PMID:21539750

  17. Carbon nanotube-composite hydrogels promote intercalated disc assembly in engineered cardiac tissues through β1-integrin mediated FAK and RhoA pathway.

    Science.gov (United States)

    Sun, Hongyu; Tang, Jiajia; Mou, Yongchao; Zhou, Jing; Qu, Linlin; Duval, Kayla; Huang, Zhu; Lin, Ning; Dai, Ruiwu; Liang, Chengxiao; Chen, Zi; Tang, Lijun; Tian, Fuzhou

    2017-01-15

    Carbon nanotube (CNT)-based hydrogels have been shown to support cardiomyocyte growth and function. However, their role in cellular integrity among cardiomyocytes has not been studied in detail and the mechanisms underlying this process remain unclear. Here, single walled CNTs incorporated into gelatin with methacrylate anhydride (CNT/GelMA) hydrogels were utilized to construct cardiac tissues, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, transmission electron microscopy and intracellular calcium transient measurement, the incorporation of CNTs into the scaffolds was observed to markedly enhance the assembly and formation in the cardiac constructs. Importantly, we further explored the underlying mechanism behind these effects through the use of immunohistochemical staining and western blotting. The β1-integrin-mediated FAK and RhoA signaling pathways were found to be responsible for CNT-induced upregulation of electrical and mechanical junction proteins respectively. Together, our study provides new insights into the facilitative effects of CNTs on ID formation, which has important significance for improving the quality of engineered cardiac tissue and applying them to cardiac regenerative therapies. Currently, the bottleneck to engineering cardiac tissues (ECTs) for cardiac regeneration is the lack of efficient cellular integrity among adjacent cells, especially the insufficient remodeling of intercalated discs (IDs) in ECTs. Recently, carbon nanotube (CNT) hydrogels provide an advantageous supporting microenvironment and therefore benefit greatly the functional performance of ECTs. Although their beneficial effect in modulating ECT performance is evident, the influence of CNTs on structural integrity of ECTs has not been studied in detail, and the mechanisms underlying the process remain to be determined. Here, we utilized carbon nanotube incorporated into gelatin with methacrylate anhydride

  18. Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer.

    Science.gov (United States)

    Aponte, Margarita; Jiang, Wei; Lakkis, Montaha; Li, Ming-Jiang; Edwards, Dale; Albitar, Lina; Vitonis, Allison; Mok, Samuel C; Cramer, Daniel W; Ye, Bin

    2008-07-15

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

  19. Dokuz Eylül Üniversitesi Denizcilik Fakültesi Öğrencileri Arasında Obezite Araştırması

    Directory of Open Access Journals (Sweden)

    Selçuk NAS

    2014-12-01

    Full Text Available Bu çalışma Dokuz Eylül Üniversitesi Denizcilik Fakültesi öğrencilerinin “Beden Kitle Endekslerinin” (BKE belirlenmesi amacıyla yapılmıştır. Bu amaçla, 328 öğrencinin 2012 Aralık ayında boy ve vücut ağırlığı ölçümleri yapılmıştır. Daha sonra bu öğrencilerin fakülteye ilk kabullerindeki yapılan boy ve vücut ağırlığı ölçüm verilerini toplamak için arşiv çalışması yapılmıştır. Toplanan veriler kullanılarak hesaplanan, öğrencilerin BKE değerlerinin yıllara göre değişimleri incelenmiştir. Sonuç olarak öğrenciler ortalama 22,8 BKE değeri ile “normal kilolu” olarak Fakülteye kabul edilmektedir. 5 yıl sonra ise ortalama 24,3 BKE değeri ile “fazla kilolu” sınırına yakın olarak mezun olmaktadır.

  20. FAK及EphA2在人脑胶质瘤中的表达及其相关性研究%Expression characteristic and correlation of FAK and EphA2 in human gliomas

    Institute of Scientific and Technical Information of China (English)

    焦征; 李震; 张训; 张异春; 陈盛强; 杨宏

    2011-01-01

    目的 探讨FAK及EphA2在人脑胶质瘤不同病理级别中的表达,并阐述FAK及EphA2与胶质瘤发生、进展的关系.方法 收集广州医学院第二医院65例人脑胶质瘤标本其中Ⅰ-Ⅱ级(低级恶性组)38例,Ⅲ-Ⅳ级(高度恶性组)27例,男性34例,女性31例,年龄4~72岁;肿瘤直径>5cm 30例,肿瘤直径≤5cm 35例,所有胶质瘤标本均为第一次手术切除,术前均未行放、化疗;另取14例正常新鲜脑组织做对照组.应用免疫组化sp(链霉印白素-过氧化物酶法)测定FAK和EphA2蛋白表达水平并分析其相关性.结果 在人正常脑组织中未见EphA2蛋白表达,其在人脑胶质瘤高度恶性组中的表达显著高于低度恶性组(P<0.05);FAK蛋白在人正常脑组织中和脑胶质瘤中均可表达;且在人脑胶质瘤高、低度恶性组中的表达均显著高于正常脑组织(P<0.05);FAK蛋白在人脑胶质瘤高度恶性组表达显著高于低度恶性组(P<0.05).结论 FAK及EphA2与人脑胶质瘤恶性度有关,随着恶性度增高,FAK及EphA2的表达也增加.笔者认为FAK及EphA2的高表达与胶质瘤的发生及其侵袭性生物学有关,因此FAK及EphA2可以作为胶质瘤侵袭性的生物学指标.同时以FAK及EphA2为中心的靶向治疗,可能成为抑制胶质瘤的有潜力的途径之一.%Objective To explore the expression of FAK and EphA2 in various grade of human gliomas and their correlation, to explain the role of FAK and EphA2 in tumorigenesis and progression of human gliomas. Methods Totally 65 primary human gliomas were collected, including 38 grade Ⅰ-Ⅱ (low potential malignancy) ,27 grade Ⅲ - IV ( high potentiial malignancy). Male 34 cases and female 31 cases, age are from 4 to 72 years old, all the cases didn' t accept radio or chemical therapy before surgery; 14 normal fresh cerebral tissuses were used to compare. The expression of FAK protein and EphA2 protein were detected by SP immunohistochemistry, and the

  1. ARC (NSC 188491 has identical activity to Sangivamycin (NSC 65346 including inhibition of both P-TEFb and PKC

    Directory of Open Access Journals (Sweden)

    Hollingshead Melinda G

    2009-02-01

    Full Text Available Abstract Background The nucleoside analog, ARC (NSC 188491 is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines. Methods Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays. Results Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1 cytotoxicity assays, 2 ability to induce a G2/M block, 3 inhibitory effects on RNA/DNA/protein synthesis, 4 transcriptomic response to treatment, 5 inhibition of protein kinase C, 6 inhibition of positive transcription elongation factor b (P-TEFb, 7 inhibition of VEGF secretion, and 8 activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance. Conclusion Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

  2. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D₂ receptors mediated by GRK and PKC in transfected cells.

    Science.gov (United States)

    Cho, D I; Zheng, M; Min, C; Kwon, K J; Shin, C Y; Choi, H K; Kim, K M

    2013-03-01

    GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D₂ receptor were investigated. All of the S/T residues located within the intracellular loops of D₂ receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D₂ receptors were investigated in the transfected cells. T134, T225/S228/S229 and S325 were involved in PKC-mediated D₂ receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D₂ receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D₂ receptors, which induced receptor resensitization. ARF6 mediated the recycling of D₂ receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D₂ receptors internalized in a PKC-dependent manner. GRK- and PKC-mediated internalizations of D₂ receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D₂ receptors and different sorting proteins are involved in the dissimilar regulation of D₂ receptors by GRK2 and PKC. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  3. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

    Science.gov (United States)

    Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

    2013-01-01

    Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

  4. Activation of protein kinase C (PKC)α or PKCε as an approach to increase morphine tolerance in respiratory depression and lethal overdose.

    Science.gov (United States)

    Lin, Hong-Yiou; Law, Ping-Yee; Loh, Horace H

    2012-04-01

    Long-term use of opioids is hindered by respiratory depression and the possibility for fatal overdose in drug abusers. This is attributed to higher levels of tolerance that develops against antinociception than to respiratory depression. Identifying important mechanisms that would increase morphine respiratory depression and overdose tolerance could lead to the safer use of opioids. Because protein kinase C (PKC) activity mediates the development and maintenance of morphine antinociceptive tolerance, we hypothesized that activating PKCα or PKCε at the pre-Bötzinger complex (preBötC) can increase morphine tolerance in respiration and overdose. Laser microdissection and quantitative reverse transcriptase-polymerase chain reaction were used to compare the relative mRNA abundances of PKCα, γ, and ε between ventrolateral periaqueductal gray (vlPAG) and preBötC. To test whether PKCα or ε could enhance morphine tolerance in respiratory depression and overdose, lentivirus carrying the wild type, constitutively activated mutants, and small interference RNA against PKCα or ε was stereotaxically injected into the preBötC. Expression of constitutively active PKC (CAPKC) α or ε, but not wild-type PKC (WTPKC) α or ε, at the preBötC allowed rats to develop tolerance to morphine respiratory depression. In terms of lethality, expression of WTPKCε, CAPKCα, or CAPKCε at preBötC increased morphine tolerance to lethal overdose. CAPKCε-expressing rats developed the highest level of respiratory depression tolerance. Furthermore, when CAPKCε lentivirus was injected into the vlPAG, rats were able to develop significant antinociceptive tolerance at low doses of morphine that normally do not cause tolerance. The approach of increasing morphine respiratory depression and lethality tolerance by increasing PKCα or ε activity at preBötC could be used to make opioids safer for long-term use.

  5. Fargesin exerts anti-inflammatory effects in THP-1 monocytes by suppressing PKC-dependent AP-1 and NF-ĸB signaling.

    Science.gov (United States)

    Pham, Thu-Huyen; Kim, Man-Sub; Le, Minh-Quan; Song, Yong-Seok; Bak, Yesol; Ryu, Hyung-Won; Oh, Sei-Ryang; Yoon, Do-Young

    2017-01-15

    Fargesin is a lignan from Magnolia fargesii, an oriental medicine used in the treatment of nasal congestion and sinusitis. The anti-inflammatory properties of this compound have not been fully elucidated yet. This study focused on assessing the anti-inflammatory effects of fargesin on phorbal ester (PMA)-stimulated THP-1 human monocytes, and the molecular mechanisms underlying them. Cell viability was evaluated by MTS assay. Protein expression levels of inflammatory mediators were analyzed by Western blotting, ELISA, Immunofluorescence assay. mRNA levels were measured by Real-time PCR. Promoter activities were elucidated by Luciferase assay. It was found that pre-treatment with fargesin attenuated significantly the expression of two major inflammatory mediators, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Fargesin also inhibited the production of pro-inflammation cytokines (IL-1β, TNF-α) and chemokine (CCL-5). Besides, nuclear translocation of transcription factors nuclear factor-kappa B (NF-ĸB) and activator protein-1 (AP-1), which regulate multiple pro-inflammatory genes, was suppressed by fargesin in a PKC-dependent manner. Furthermore, among the mitogen-activated protein kinases (MAPKs), only c-Jun N-terminal kinase (JNK) was downregulated by fargesin in a PKC-dependent manner, and this reduction was involved in PMA-induced AP-1 and NF-ĸB nuclear translocation attenuation, demonstrated using a specific JNK inhibitor. Taken together, our results found that fargesin exhibits anti-inflammation effects on THP-1 cells via suppression of PKC pathway including downstream JNK, nuclear factors AP-1 and NF-ĸB. These results suggest that fargesin has anti-inflammatory properties with potential applications in drug development against inflammatory disorders. Copyright © 2016. Published by Elsevier GmbH.

  6. Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phase II detoxification gene expression via activation of the PI3K and PKC signaling pathways.

    Science.gov (United States)

    Lin, Musen; Zhai, Xiaohan; Wang, Guangzhi; Tian, Xiaofeng; Gao, Dongyan; Shi, Lei; Wu, Hang; Fan, Qing; Peng, Jinyong; Liu, Kexin; Yao, Jihong

    2015-02-01

    Acetaminophen (APAP) is used drugs worldwide for treating pain and fever. However, APAP overdose is the principal cause of acute liver failure in Western countries. Salvianolic acid B (SalB), a major water-soluble compound extracted from Radix Salvia miltiorrhiza, has well-known antioxidant and anti-inflammatory actions. We aimed to evaluate the ability of SalB to protect against APAP-induced acute hepatotoxicity by inducing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. SalB pretreatment ameliorated acute liver injury caused by APAP, as indicated by blood aspartate transaminase levels and histological findings. Moreover, SalB pretreatment increased the expression of Nrf2, Heme oxygenase-1 (HO-1) and glutamate-l-cysteine ligase catalytic subunit (GCLC). Furthermore, the HO-1 inhibitor zinc protoporphyrin and the GCLC inhibitor buthionine sulfoximine reversed the protective effect of SalB. Additionally, siRNA-mediated depletion of Nrf2 reduced the induction of HO-1 and GCLC by SalB, and SalB pretreatment activated the phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) signaling pathways. Both inhibitors (PI3K and PKC) blocked the protective effect of SalB against APAP-induced cell death, abolishing the SalB-induced Nrf2 activation and decreasing HO-1 and GCLC expression. These results indicated that SalB induces Nrf2, HO-1 and GCLC expression via activation of the PI3K and PKC pathways, thereby protecting against APAP-induced liver injury.

  7. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  8. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    Science.gov (United States)

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  9. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC-ERK1/2-mTOR signaling pathway.

    Science.gov (United States)

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W

    2015-03-17

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

  10. The PDK1 master kinase is over-expressed in acute myeloid leukemia and promotes PKC-mediated survival of leukemic blasts.

    Science.gov (United States)

    Zabkiewicz, Joanna; Pearn, Lorna; Hills, Robert K; Morgan, Rhys G; Tonks, Alex; Burnett, Alan K; Darley, Richard L

    2014-05-01

    PDK1 is a master kinase that activates at least six protein kinase groups including AKT, PKC and S6K and is a potential target in the treatment of a range of malignancies. Here we show overexpression of PDK1 in over 40% of myelomonocytic acute leukemia patients. Overexpression of PDK1 occurred uniformly throughout the leukemic population, including putative leukemia-initiating cells. Clinical outcome analysis revealed PDK1 overexpression was associated with poorer treatment outcome. Primary acute myeloid leukemia blasts over-expressing PDK1 showed improved in vitro survival and ectopic expression of PDK1 promoted the survival of myeloid cell lines. Analysis of PDK1 target kinases revealed that PDK1 overexpression was most closely associated with increased phosphorylation of PKC isoenzymes and inhibition of PKC strongly inhibited the survival advantage of PDK1 over-expressing cells. Membrane localization studies implicated PKCα as a major target for PDK1 in this disease. PDK1 over-expressing blasts showed differential sensitivity to PDK1 inhibition (in the low micromolar range) suggesting oncogene addiction, whilst normal bone marrow progenitors were refractory to PDK1 inhibition at effective inhibitor concentrations. PDK1 inhibition also targeted subpopulations of leukemic blasts with a putative leukemia-initiating cell phenotype. Together these data show that overexpression of PDK1 is common in acute myelomonocytic leukemia and is associated with poorer treatment outcome, probably arising from the cytoprotective function of PDK1. We also show that therapeutic targeting of PDK1 has the potential to be both an effective and selective treatment for these patients, and is also compatible with current treatment regimes.

  11. Increased PKC activity and altered GSK3β/NMDAR function drive behavior cycling in HINT1-deficient mice: bipolarity or opposing forces.

    Science.gov (United States)

    Garzón-Niño, Javier; Rodríguez-Muñoz, María; Cortés-Montero, Elsa; Sánchez-Blázquez, Pilar

    2017-02-27

    Mice with histidine triad nucleotide-binding protein 1 (HINT1) deletion exhibit manic-like symptoms that evolve into depressive-like behavior in response to stressful paradigms. Molecular and electrophysiological studies have indicated that HINT1(-/-) mice exhibit increased PKC, PKA, and GSK3β activities, as well as glutamate N-methyl-D-aspartate receptor (NMDAR)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and NR2B/NR2A subunit ratios. Pharmacological interventions stabilized their behavior but through different mechanisms. GSK3β inhibitors and valproate directly attenuated the expression of the manic-like symptoms, whereas PKC inhibition, lamotrigine, or risperidone promoted NMDAR-mediated depressive-like behaviors that counterbalanced the preexisting manic-like symptoms. Naïve HINT1(-/-) mice exposed to stressful paradigms rapidly manifested depressive-like behaviors in subsequent stressful situations, a capacity that persisted for a couple of weeks thereafter. During the depressive-like phase, citalopram, amitriptyline and MK801 precipitated manic-like behaviors in stressed HINT1(-/-) mice. Notably, the antagonism of NMDARs prevented HINT1(-/-) mice from alternating behaviors in response to stress. A comparison with "manic" Black Swiss mice indicated that in HINT1(-/-) mice, PKC supports manic-like symptoms and reduces the expression of depressive-like behaviors via activation of GSK3β and regulation of NR2B-enriched NMDARs. HINT1(-/-) mice represent a suitable model for studying human BPD and may facilitate the identification of novel targets and drugs to treat this mental disorder.

  12. Impact of Rosuvastatin Treatment on HDL-Induced PKC-βII and eNOS Phosphorylation in Endothelial Cells and Its Relation to Flow-Mediated Dilatation in Patients with Chronic Heart Failure

    Directory of Open Access Journals (Sweden)

    Ephraim B. Winzer

    2016-01-01

    Full Text Available Background. Endothelial function is impaired in chronic heart failure (CHF. Statins upregulate endothelial NO synthase (eNOS and improve endothelial function. Recent studies demonstrated that HDL stimulates NO production due to eNOS phosphorylation at Ser1177, dephosphorylation at Thr495, and diminished phosphorylation of PKC-βII at Ser660. The aim of this study was to elucidate the impact of rosuvastatin on HDL mediated eNOS and PKC-βII phosphorylation and its relation to endothelial function. Methods. 18 CHF patients were randomized to 12 weeks of rosuvastatin or placebo. At baseline, 12 weeks, and 4 weeks after treatment cessation we determined lipid levels and isolated HDL. Human aortic endothelial cells (HAEC were incubated with isolated HDL and phosphorylation of eNOS and PKC-βII was evaluated. Flow-mediated dilatation (FMD was measured at the radial artery. Results. Rosuvastatin improved FMD significantly. This effect was blunted after treatment cessation. LDL plasma levels were reduced after rosuvastatin treatment whereas drug withdrawal resulted in significant increase. HDL levels remained unaffected. Incubation of HAEC with HDL had no impact on phosphorylation of eNOS or PKC-βII. Conclusion. HDL mediated eNOS and PKC-βII phosphorylation levels in endothelial cells do not change with rosuvastatin in CHF patients and do not mediate the marked improvement in endothelial function.

  13. Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention.

    Science.gov (United States)

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2015-11-01

    Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton's lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.

  14. Searching for disease modifiers-PKC activation and HDAC inhibition - a dual drug approach to Alzheimer's disease that decreases Abeta production while blocking oxidative stress.

    Science.gov (United States)

    Kozikowski, Alan P; Chen, Yihua; Subhasish, Tapadar; Lewin, Nancy E; Blumberg, Peter M; Zhong, Zhenyu; D'Annibale, Melissa A; Wang, Weng-Long; Shen, Yong; Langley, Brett

    2009-07-01

    A series of benzolactam compounds were synthesized, some of which caused a concentration-dependent increase in sAPPalpha and decrease in Abeta production in the concentration range of 0.1-10 microM. Moreover, some compounds showed neuroprotective effects in the 10-20 microM 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 microM). 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 alpha-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 sAPPalpha and decrease in beta-amyloid (Abeta) production in the concentration range of 0.1-10 microM, consistent with a shift of APP metabolism toward the alpha-secretase-processing pathway. Moreover, compounds 9-14 showed neuroprotective effects in the 10-20 microM 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

  15. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi-Feng [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Yu, Hong-Wei [Department of Cardiology, Jinzhou Central Hospital, Jinzhou 121001 (China); Sun, Li-Li [Department of Ophthalmology, The Third Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); You, Lu; Tao, Gui-Zhou [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Qu, Bao-Ze, E-mail: qubaoze1971@hotmail.com [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China)

    2015-12-25

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  16. 2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles: Design and synthesis of a potent and isoform selective PKC-[zeta] inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Trujillo, John I.; Kiefer, James R.; Huang, Wei; Thorarensen, Atli; Xing, Li; Caspers, Nicole L.; Day, Jacqueline E.; Mathis, Karl J.; Kretzmer, Kuniko K.; Reitz, Beverley A.; Weinberg, Robin A.; Stegeman, Roderick A.; Wrightstone, Ann; Christine, Lori; Compton, Robert; Li, Xiong; (Pfizer)

    2009-03-16

    The inhibition of PKC-{zeta} has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-{zeta}). The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and analysis of the series. The most selective and potent analog was identified by variation of the aryl substituent at the 6-position of the indazole template to give a 4-NH{sub 2} derivative. The analog displays good selectivity over other PKC isoforms ({alpha}, {beta}II, {gamma}, {delta}, {epsilon}, {mu}, {theta}, {eta} and {ell}/{lambda}) and CDK-2, however it displays marginal selectivity against a panel of other kinases (37 profiled).

  17. The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development.

    Science.gov (United States)

    Neto, Ana; Mercader, Nadia; Gómez-Skarmeta, José Luis

    2012-01-01

    Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.

  18. Structural insight with mutational impact on tyrosinase and PKC-β interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-10-30

    Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, β-subunit (PKC-β) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCβ complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-β, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact.

  19. Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons.

    Science.gov (United States)

    Sheng, Hui; Xu, Yongjun; Chen, Yanming; Zhang, Yanmin; Ni, Xin

    2012-10-15

    Corticotropin-releasing hormone (CRH) has been shown to modulate dendritic development in hippocampus. Mitotic kinesin-like protein 1 (MKLP1) plays key roles in dendritic differentiation. In the present study, we examined the effects of CRH on MKLP1 expression in cultured hippocampal neurons and determine subsequent signaling pathways involved. CRH dose-dependently increased MKLP1 mRNA and protein expression. This effect can be reversed by CRHR1 antagonist but not by CRHR2 antagonist. CRHR1 knockdown impaired this effect of CRH. CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C (PLC)-β3 expression, which were blocked by CRHR1 antagonist. Transfection of GP antagonist-2A, an inhibitory peptide of Gαq protein, blocked CRH-induced phosphorylated PLC-β3 expression. PLC and PKC inhibitors completely blocked whereas adenylyl cyclase (AC) and PKA inhibitors did not affect CRH-induced MKLP1 expression. Our results indicate that CRH act on CRHR1 to induce MKLP1 expression via PLC/PKC signaling pathway. CRH may regulate MKLP1 expression, thereby modulating dendritic development.

  20. Allium cepa Extract and Quercetin Protect Neuronal Cells from Oxidative Stress via PKC-ε Inactivation/ERK1/2 Activation

    Science.gov (United States)

    2016-01-01

    Oxidative stress plays an important role in the pathophysiology of various neurologic disorders. Allium cepa extract (ACE) and their main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress. We investigated the underlying molecular mechanisms, particularly those linked to the antioxidant effects of the ACE. Primary cortical neuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30 min and exposed to L-buthionine sulfoximine for 4~24 h. We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced by L-buthionine-S, R-sulfoximine (BSO) in a concentration-dependent manner. Furthermore, ACE and QCT activated extracellular signal-regulated kinase 1/2 (ERK1/2), leading to downregulation of protein kinase C-ε (PKC-ε) in BSO-stimulated neuronal cells. In addition, ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase. Our results provide new insight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells. The results suggest that the inactivation of PKC-ε induced by phosphorylating ERK1/2 is responsible for the neuroprotective effect of ACE and QCT against BSO-induced oxidative stress. PMID:27668036

  1. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-{alpha} and NF-{kappa}B pathways in lipopolysaccharide-stimulated mouse macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tzung-Yan, E-mail: joyamen@mail.cgu.edu.tw [Graduate Institute of Traditional Chinese Medicine, Chang Gung University, No. 259, Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 333, Taiwan (China); Lee, Ko-Chen [School of Traditional Chinese Medicine, Chang Gung University, Taiwan (China); Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, Shih-Yuan [Graduate Institute of Traditional Chinese Medicine, Chang Gung University, No. 259, Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 333, Taiwan (China); Chang, Hen-Hong [Graduate Institute of Traditional Chinese Medicine, Chang Gung University, No. 259, Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 333, Taiwan (China); Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan (China)

    2009-04-24

    Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-{alpha} expression through suppression of I-{kappa}B{alpha} phosphorylation, NF-{kappa}B nuclear activation and PKC-{alpha} translocation, which in turn inhibits Ca{sup 2+} mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-{kappa}B and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.

  2. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    Science.gov (United States)

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  3. Protection against Ischemia-Induced Oxidative Stress Conferred by Vagal Stimulation in the Rat Heart: Involvement of the AMPK-PKC Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Jin Zang

    2012-11-01

    Full Text Available Reactive oxygen species (ROS production is an important mechanism in myocardial ischemia and nicotinamide adenine dinucleotide phosphate (NADPH oxidase is one of major sources of ROS in the heart. Previous studies showed that vagus nerve stimulation (VNS is beneficial in treating ischemic heart diseases. However, the effect of VNS on ROS production remains elusive. In this study, we investigated the role of VNS onischemia-induced ROS production. Our results demonstrated that VNS alleviated the myocardial injury, attenuated the cardiac dysfunction, reserved the antioxidant enzyme activity and inhibited the formation of ROS as evidenced by the decreased NADPH oxidase (Nox activity and superoxide fluorescence intensity as well as the expression of p67phox, Rac1 and nitrotyrosine. Furthermore, VNS resulted in the phosphorylation and activation of adenosine monophosphate activated protein kinase (AMPK, which in turn led to an inactivation of Nox by protein kinase C (PKC; however, the phenomena were repressed by the administration of a muscarinic antagonist atropine. Taken together, these data indicate that VNS decreases ROS via AMPK-PKC-Nox pathway; this may have potential importance for the treatment of ischemic heart diseases.

  4. Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway.

    Science.gov (United States)

    Yang, Jing; Zhang, Dan; Yu, Ying; Zhang, Run-Ju; Hu, Xiao-Ling; Huang, He-Feng; Lu, Yong-Chao

    2015-01-01

    Fibroblast growth factors (FGF1, FGF2 and FGF4) and fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3 and FGFR4) have been reported to be expressed in preimplantation embryos and be required for their development. However, the functions of these molecules in trophectoderm cells (TEs) that lead to the formation of the blastocyst as well as the underlying mechanism have not been elucidated. The present study has demonstrated for the first time that endogenous FGF2 secreted by TEs can regulate protein expression and distribution in TEs via the FGFR2-mediated activation of PKC and p38, which are important for the development of expanded blastocysts. This finding provides the first explanation for the long-observed phenomenon that only high concentrations of exogenous FGFs have effects on embryonic development, but in vivo the amount of endogenous FGFs are trace. Besides, the present results suggest that FGF2/FGFR2 may act in an autocrine fashion and activate the downstream PKC/p38 pathway in TEs during expanded blastocyst formation.

  5. Role of mitochondrial ATP-sensitive potassium channel-mediated PKC-ε in delayed protection against myocardial ischemia/reperfusion injury in isolated hearts of sevoflurane-preconditioned rats

    Directory of Open Access Journals (Sweden)

    C. Wang

    2015-06-01

    Full Text Available This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP channels and protein kinase C (PKC-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9. The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group or 33% oxygen inhalation (I/R group 24 h before coronary occlusion. The control group (CON and the sevoflurane group (SEVO group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD was given 30 min before sevoflurane preconditioning (5-HD+SWOP group. Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05 in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05 compared with the SWOP group. The results suggest that mitoKATP channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.

  6. Role of mitochondrial ATP-sensitive potassium channel-mediated PKC-ε in delayed protection against myocardial ischemia/reperfusion injury in isolated hearts of sevoflurane-preconditioned rats

    Energy Technology Data Exchange (ETDEWEB)

    Wang, C. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Institute of Neuroscience, Soochow University, Suzhou (China); Hu, S.M. [Institute of Neuroscience, Soochow University, Suzhou (China); Xie, H.; Qiao, S.G. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Liu, H. [Department of Anesthesiology and Pain Medicine, University of California Davis Health System, Davis, CA (United States); Liu, C.F. [Institute of Neuroscience, Soochow University, Suzhou (China)

    2015-03-27

    This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoK{sub ATP}) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoK{sub ATP} channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoK{sub ATP} channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.

  7. Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells.

    Science.gov (United States)

    Santofimia-Castaño, Patricia; Clea Ruy, Deborah; Garcia-Sanchez, Lourdes; Jimenez-Blasco, Daniel; Fernandez-Bermejo, Miguel; Bolaños, Juan P; Salido, Gines M; Gonzalez, Antonio

    2015-10-01

    The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca(2+) in the extracellular medium, induced a slow and progressive increase of [Ca(2+)](c) toward a stable level. Melatonin did not inhibit the typical Ca(2+) response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca(2+) in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl(3), to inhibit Ca(2+) entry, we could not detect any change in [Ca(2+)](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca(2+). When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca(2+) in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca(2+)]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca(2+). Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and

  8. Gazi Üniversitesi Tip Fakültesi Pediatrik Endokrinoloji polikliniğine başvuran 7-11 yaş grubu çocuklarin beslenme durumlarinin saptanmasi

    OpenAIRE

    ÖZDEMİR BOZBULUT, Rukiye

    2010-01-01

        Bu çalışma; pediatrik endokrinoloji polikliniğine başvuran 7-11 yaş grubu çocukların beslenme alışkanlıklarının saptamak, antropometrik ölçümlerinin yapılarak büyüme gelişme geriliklerinin ve obezite durumlarını tespit etmek, çocukların beslenme alışkanlıklarının biyokimyasal parametreleri üzerine etkisini belirlemek ve  halk sağlığı hizmetlerine katkıda bulunmak amacıyla planlanmıştır. Bu araştırma Gazi Üniversitesi Tıp Fakültesi Pediatrik Endokrinoloj...

  9. Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

    Science.gov (United States)

    Boehm, Manja; Krause-Gruszczynska, Malgorzata; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Oyarzabal, Omar A.; Backert, Steffen

    2011-01-01

    Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin−/−, integrin beta1−/−, and focal adhesion kinase (FAK)−/− deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin−/−, integrin beta1−/−, and FAK−/− knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells. PMID:22919583

  10. Cyr61 induces the expression of monocyte chemoattractant protein-1 via the integrin ανβ3, FAK, PI3K/Akt, and NF-κB pathways in retinal vascular endothelial cells.

    Science.gov (United States)

    You, Jian-Jang; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2014-01-01

    Diabetes causes a number of metabolic and physiological abnormalities in the retina. Many of the molecular and physiological abnormalities that develop during diabetic retinopathy are due to inflammation. Monocyte chemoattractant protein-1 (MCP-1) is an important factor involved in diabetic retinopathy. In a previous study, we found that cysteine-rich 61 (Cyr61), an important angiogenic factor, also plays an important role in diabetic retinopathy. In addition to the direct effects of Cyr61, we observed that Cyr61 can induce the expression of MCP-1. However, the mechanism through which this occurs is not completely understood in chorioretinal vascular endothelial cells. We therefore investigated the effects of Cyr61 on MCP-1 expression in this cell type. Cyr61 stimulated the expression of MCP-1 at the mRNA, protein, and secreted protein levels in a dose-dependent and time-dependent manner. Both total MCP-1 levels and secreted MCP-1 levels were attenuated during the response to Cyr61 stimulation by pretreatment with integrin ανβ3-blocking antibodies, a FAK inhibitor (PF573228), a PI3K inhibitor (LY294002), and an Akt inhibitor (A6730). Electrophoretic mobility shift assays revealed that the above inhibitors suppressed the activation of NF-κB. Additionally, deletion of the NF-κB-binding element in the MCP-1 gene promoter led to a decrease in expression in luciferase reporter assays. These results show that the induction of MCP-1 by Cyr61 is mediated through the activation of the integrin ανβ3, FAK, PI3K/Akt, and IKK/NF-κB pathways in chorioretinal vascular endothelial cells.

  11. Ethanol-mediated regulation of cytochrome P450 2A6 expression in monocytes: role of oxidative stress-mediated PKC/MEK/Nrf2 pathway.

    Directory of Open Access Journals (Sweden)

    Mengyao Jin

    Full Text Available Cytochrome P450 2A6 (CYP2A6 is known to metabolize nicotine, the major constituent of tobacco, leading to the production of toxic metabolites and induction of oxidative stress that result in liver damage and lung cancer. Recently, we have shown that CYP2A6 is induced by ethanol and metabolizes nicotine into cotinine and other metabolites leading to generation of reactive oxygen species (ROS in U937 monocytes. However, the mechanism by which CYP2A6 is induced by ethanol is unknown. In this study, we have examined the role of the PKC/Nrf2 pathway (protein kinase C-mediated phosphorylation and translocation of nuclear erythroid 2-related factor 2 to the nucleus in ethanol-mediated CYP2A6 induction. Our results showed that 100 mM ethanol significantly induced CYP2A6 mRNA and protein (~150% and increased ROS formation, and induction of gene expression and ROS were both completely blocked by treatment with either a CYP2E1 inhibitor (diallyl sulfide or an antioxidant (vitamin C. The results suggest the role of oxidative stress in the regulation of CYP2A6 expression. Subsequently, we investigated the role of Nrf2 pathway in oxidative stress-mediated regulation of CYP2A6 expression in U937 monocytes. Our results showed that butylated hydroxyanisole, a stabilizer of nuclear Nrf2, increased CYP2A6 levels >200%. Staurosporine, an inhibitor of PKC, completely abolished ethanol-induced CYP2A6 expression. Furthermore, our results showed that a specific inhibitor of mitogen-activated protein kinase kinase (MEK (U0126 completely abolished ethanol-mediated CYP2A6 induction and Nrf2 translocation. Overall, these results suggest that CYP2E1-mediated oxidative stress produced as a result of ethanol metabolism translocates Nrf2 into the nucleus through PKC/MEK pathway, resulting in the induction of CYP2A6 in monocytes. An increased level of CYP2A6 in monocytes is expected to further increase oxidative stress in smokers through CYP2A6-mediated nicotine metabolism

  12. Estradiol-17beta-BSA stimulates Ca(2+) uptake through nongenomic pathways in primary rabbit kidney proximal tubule cells: involvement of cAMP and PKC.

    Science.gov (United States)

    Han, H J; Lee, Y H; Park, S H

    2000-04-01

    The effect of estradiol-17beta-BSA (E(2)-BSA) on Ca(2+) uptake and its related signal pathways were examined in the primary cultured rabbit kidney proximal tubule cells. E(2)-BSA (10(-9) M) significantly stimulated Ca(2+) uptake from 2 h by 13% and at 8 h by 35% as compared to control, respectively. This stimulatory effect of E(2)-BSA was not inhibited by tamoxifen (10(-8) M, an intracellular estrogen receptor antagonist), actinomycin D (10(-7) M, a transcription inhibitor), and cycloheximide (4 x 10(-5) M, a protein synthesis inhibitor). However, E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by methoxyverapamil (10(-6) M, an L-type calcium channel blocker) and 5-(N-ethyl-N-isopropyl)-amiloride (10(-5) M, a Na(+)/H(+) antiporter blocker). These results suggest that E(2)-BSA stimulates Ca(2+) uptake through nongenomic pathways. Thus, we investigated which signal pathways were related to E(2)-BSA-induced stimulation of Ca(2+) uptake. 8-Br-cAMP (10(-6) M) alone increased Ca(2+) uptake by 22% compared to control. When E(2)-BSA combined with 8-Br-cAMP, Ca(2+) uptake was not significantly stimulated compared to E(2)-BSA. SQ 22536 (10(-6) M, an adenylate cyclase inhibitor) and myristoylated protein kinase A inhibitor amide 14-22 (10(-6) M, a protein kinase A inhibitor) blocked E(2)-BSA-induced stimulation of Ca(2+) uptake and E(2)-BSA also increased cAMP generation by 26% of that of control. In addition, TPA (0.02 ng/ml, an artificial PKC promoter) stimulated the Ca(2+) uptake by 14%, and the cotreatment of TPA and E(2)-BSA did not significantly stimulate Ca(2+) uptake compared to E(2)-BSA. E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by U 73122 (10(-6) M, a phospholipase C inhibitor) or bisindolylmaleimide I (10(-6) M, a protein kinase C inhibitor). Indeed, E(2)-BSA stimulated PKC activity by 26%. In conclusion, E(2)-BSA (10(-9) M) stimulated Ca(2+) uptake by nongenomic action, which is mediated by cAMP and PKC pathways.

  13. Shigella type III secretion protein MxiI is recognized by Naip2 to induce Nlrc4 inflammasome activation independently of Pkcδ.

    Directory of Open Access Journals (Sweden)

    Shiho Suzuki

    2014-02-01

    Full Text Available Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1β and proIL-18 leading to the release of mature IL-1β and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkcδ was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1β release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1β maturation and Asc pyroptosome formation. Notably, the Pkcδ kinase was dispensable for caspase-1 activation and secretion of IL-1β induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.

  14. PARP-inhibitor treatment prevents hypertension induced cardiac remodeling by favorable modulation of heat shock proteins, Akt-1/GSK-3β and several PKC isoforms.

    Directory of Open Access Journals (Sweden)

    Laszlo Deres

    Full Text Available Spontaneously hypertensive rat (SHR is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose polymerase enzyme (PARP plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286 treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group or placebo (SHR-C group for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group. Echocardiography was performed, brain-derived natriuretic peptide (BNP activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps and the phosphorylation state of Akt-1(Ser473, glycogen synthase kinase (GSK-3β(Ser9, forkhead transcription factor (FKHR(Ser256, mitogen activated protein kinases (MAPKs, and protein kinase C (PKC isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK1/2(Thr183-Tyr185, Akt-1(Ser473, GSK-3β(Ser9, FKHR(Ser256, and PKC ε(Ser729 and the level of Hsp90 were increased, while the activity of PKC α/βII(Thr638/641, ζ/λ(410/403 were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling.

  15. 关于蛋白激酶C-γ及其在中枢中的作用的研究进展%cPKC-γand its role in central

    Institute of Scientific and Technical Information of China (English)

    郗平; 王静; 李俊发; 傅涛

    2016-01-01

    蛋白激酶C-γ是经典型蛋白激酶C家族中的一个亚型。作为蛋白激酶C家族中的一员,它具有蛋白激酶C的普遍作用,同时又具有其特异作用。在磷脂酰丝氨酸存在的情况下,蛋白激酶C-γ可被Ca2+、磷脂酰甘油等激活。蛋白激酶C-γ具有神经特异性,主要在大脑皮层和脊髓的神经元中表达。 cPKC-γ的缺失或突变是导致许多神经系统疾病的原因之一,可诱发原发性视网膜色素变性、白内障和脊髓小脑共济失调等疾病。异常视觉经验可通过神经发育可塑性影响视皮层发育最终导致弱视产生,作为一种神经特异性蛋白激酶,cPKC-γ是否在视觉发育可塑性中发挥作用,仍存在争议。本文中笔者主要从cPKC-γ的生物化学性质、分子作用机制和与其相关的疾病方面进行综述。%Protein kinase C gamma ( cPKC-γ) is a subtype of conventional PKC family .As a member of protein kinase C ,it has common effects of protein kinase C ,and special effect of itself .In the presence of phosphatidylserine , it can be activated by Ca 2+and phosphatidyl glycerol ( DAG ) .cPKC-γhas neuron specificity ,mainly in brain and spinal cord .Its absence or mutation will cause many nervous system diseases , such as retinitis pigmentosa ( RP) ,cataract and spinocerebellar ataxia .Abnormal visual experience can lead to amblyopia by affect neuroplasticity of visual cortex .As a neuron-specific protein kinase , its role in visual plasticity is still controversial .This article summarizes the biochemical properties ,molecular mechanism and related diseases of cPKC-γ.

  16. The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.

    Science.gov (United States)

    Du, Xiao-Yu; Huang, Jian; Xu, Liang-Quan; Tang, Dan-Feng; Wu, Lei; Zhang, Li-Xia; Pan, Xiao-Ling; Chen, Wei-Yun; Zheng, Li-Ping; Zheng, Yue-Hui

    2012-08-20

    C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c

  17. The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model.

    Directory of Open Access Journals (Sweden)

    Bo Sun

    Full Text Available Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC20 and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.

  18. The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model.

    Science.gov (United States)

    Sun, Bo; Hu, Chen; Fang, Huan; Zhu, Lina; Gao, Ning; Zhu, Jingci

    2015-01-01

    Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI) mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC) and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC20 and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.

  19. Niacin activates the PI3K/Akt cascade via PKC- and EGFR-transactivation-dependent pathways through hydroxyl-carboxylic acid receptor 2.

    Directory of Open Access Journals (Sweden)

    Huawang Sun

    Full Text Available Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.

  20. Shikonin inhibits TNF-α production through suppressing PKC-NF-κB-dependent decrease of IL-10 in rheumatoid arthritis-like cell model.

    Science.gov (United States)

    Sun, Wen-Xiao; Liu, Yan; Zhou, Wei; Li, He-Wei; Yang, Jian; Chen, Zhen-Bing

    2017-04-01

    Shikonin, a major effective component in the Chinese herbal medicine Lithospermum erythrorhizon Sieb., exhibits an anti-inflammatory property towards rheumatoid arthritis (RA), but the potential mechanism is unclear. Our aim was to investigate the mechanism of shikonin on the lipopolysaccharide (LPS)-induced fibroblast-like synoviocyte (LiFLS) inflammation model. Fibroblast-like synoviocytes (FLSs) were treated with 200 μg/ml of LPS for 24 h to establish the RA-like model, LiFLS. FLSs were pretreated with shikonin (0.1-1 μM) for 30 min in the treatment groups. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays were used to detect mRNA and protein levels of interleukin (IL)-10 and tumor necrosis factor (TNF)-α. Signal proteins involved in IL-10 production were analyzed by Western blotting. Shikonin significantly reversed the inhibitory effects of LPS on IL-10 expression in FLSs by inactivating the PKC-NF-κB pathway. In addition, shikonin inhibited LPS-induced TNF-α expression in FLSs, and this effect was markedly diminished by IL-10-neutralizing antibody. The IL-10-mediated suppression of TNF-α transcription was demonstrated by no response to the protein synthesis inhibitor cyclohexamide and no mRNA decay. Shikonin inhibits LPS-induced TNF-α production in FLSs through suppressing the PKC-NF-κB-dependent decrease in IL-10, and this study also highlights the potential application of shikonin in the treatment of RA.

  1. PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 Cells.

    Science.gov (United States)

    Gonzalez, Alexis A; Salinas-Parra, Nicolas; Leach, Dan; Navar, L Gabriel; Prieto, Minolfa C

    2017-07-12

    During the early phase of angiotensin (ANG) II-dependent hypertension tubular prostaglandin E2 (PGE2) is increased. Renin synthesis and secretion in the collecting duct (CD) is upregulated by ANGII contributing to further intratubular ANGII formation. However, what happens first and whether the triggering mechanism is independent of tubular ANGII, remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular (JG) cells via E-prostanoid (EP) receptors through cAMP/CREB pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. M-1 CD cell line expressed EP1, EP3 and EP4 but not EP2. Dose response experiments in the presence of AT1 receptor blockade with candesartan demonstrated that 10-6 M PGE2 maximally increases renin mRNA (~4 fold) and prorenin/renin protein levels (~2 fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161,982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10 fold increase). To further evaluate the signaling pathway involved we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative (DN). Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation and augmentation of renin. Knockdown of EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through EP1 receptor via PKC/cAMP/CREB pathway. Therefore, we conclude that during early stages of ANGII-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANGII formation and further stimulation of renin. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  2. Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

    Science.gov (United States)

    Réus, Gislaine Z; Stringari, Roberto B; Ribeiro, Karine F; Ferraro, Ana K; Vitto, Marcelo F; Cesconetto, Patrícia; Souza, Claúdio T; Quevedo, João

    2011-08-01

    A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

  3. Physiological crosstalk between the AC/PKA and PLC/PKC pathways modulates melatonin-mediated, monochromatic-light-induced proliferation of T-lymphocytes in chickens.

    Science.gov (United States)

    Guo, Qingyun; Wang, Zixu; Dong, Yulan; Cao, Jing; Chen, Yaoxing

    2017-06-28

    Previous study has demonstrated that melatonin plays a critical role in monochromatic-light-induced lymphocyte proliferation in response to T cell mitogen concanavalin A (ConA). However, its intracellular mechanism is still unclear. In this study, we investigate the intracellular signal pathways of melatonin receptor-mediated T-lymphocyte proliferation in the spleens of chicks exposed to different light wavelengths. Results showed that green light enhanced T-lymphocyte proliferation by 2.46-6.83% and increased splenic mRNA and protein expressions of melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) by 16.05-40.43% compared with the white, red and blue light groups. However, pinealectomy resulted in a decrease in T-lymphocyte proliferation and melatonin receptor expression with no statistically significant differences between the different light groups. In vitro experiments showed that the Mel1b selective antagonist 4P-PDOT, the Mel1c selective antagonist prazosin and the mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 suppressed both melatonin-induced lymphocyte proliferation in response to ConA and melatonin- and ConA-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) activity but that the Mel1a/Mel1b non-selective antagonist luzindole did not. In addition, pretreatment with forskolin (FSK, the adenylyl cyclase activator), H89 (the PKA inhibitor), U73122 (the PLC inhibitor) or Go6983 (the broad spectrum PKC inhibitor) markedly attenuated melatonin- and ConA-stimulated T-lymphocyte proliferation and ERK1/2 activity. These results demonstrate that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways followed by ERK1/2 activation.

  4. Snail regulated by PKC/GSK-3β pathway is crucial for EGF-induced epithelial-mesenchymal transition (EMT) of cancer cells.

    Science.gov (United States)

    Liu, Zong-cai; Chen, Xiao-hui; Song, Hai-xing; Wang, Hong-sheng; Zhang, Ge; Wang, Hao; Chen, Dan-yang; Fang, Rui; Liu, Hao; Cai, Shao-hui; Du, Jun

    2014-11-01

    Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3β inhibition and (3) that GSK-3β inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3β signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

  5. Synergistic effect of high glucose and ANG II on proliferation of mouse embryonic stem cells: involvement of PKC and MAPKs as well as AT1 receptor.

    Science.gov (United States)

    Kim, Yun Hee; Han, Ho Jae

    2008-05-01

    This study examined the synergistic effect of high glucose levels and ANG II on proliferation and its related signal pathways using mouse embryonic stem (ES) cells. The combined use of a high glucose concentration (25 mM) and ANG II increased the level of [3H]thymidine/BrdU incorporation, and the number of cells compared with either treatment alone. Each treatment with high glucose or ANG II increased the cell population in the S phase compared with control, and the combined treatment of a high glucose concentration and ANG II significantly increased the number of cells in the S phase according to FACS analysis. Moreover, the high glucose-induced increase in [3H]thymidine incorporation was blocked by inhibiting the ANG II type 1 (AT1) receptor. The combined high glucose and ANG II significantly increased the STAT3 phosphorylation compared with high glucose or ANG II alone. ANG II stimulated the influx of Ca2+ in 25 mM glucose compared with 5 mM glucose. High glucose levels increase the level of PKC alpha, epsilon, and zeta translocation from the cytosol to the membrane fraction. In an examination of other signal pathways, the combined treatment significantly increased the level of p44/42, p38 MAPKs phosphorylation compared with either treatment alone. Indeed, the combined treatment increased the mRNA expression level of the protooncogenes and cell cycle regulatory proteins. In conclusion, the combined treatment of a high glucose concentration and ANG II had a synergistic effect in stimulating mouse ES cell proliferation through the Ca2+/PKC, MAPKs, and the AT1 receptor.

  6. Eğitim Fakültesi Öğrencilerinin Görüşlerine Göre Üniversitenin Örgütsel İmajının Değerlendirilmesi

    Directory of Open Access Journals (Sweden)

    Fatma KÖYBAŞI

    2016-12-01

    Full Text Available Öz: Bu araştırmanın amacı, eğitim fakültesi öğrencilerinin görüşlerine göre Cumhuriyet Üniversitesi’nin örgütsel imajını değerlendirmektir. Çalışma, betimsel tarama modelinde nicel bir çalışmadır. Araştırmanın örneklemi 2014-2015 akademik yılı Cumhuriyet Üniversitesi Eğitim Fakültesi’nde öğrenim gören 951 öğrenciden oluşmaktadır. Araştırmanın verileri, Kazoleas vd. (2001 tarafından geliştirilen ve Polat (2011 tarafından Türkçeye uyarlanan örgütsel imaj ölçeği ile toplanmıştır. Örgütsel imaj ölçeği 6 boyutlu olup toplam ölçeğin güvenirlik katsayısı .94’tür. Veriler, betimsel nicel analiz tekniği ile çözümlenmiş, ayrıca fark testleri (t-testi ve Anova kullanılmıştır. Araştırmanın bulgularına göre Cumhuriyet Üniversitesi Eğitim Fakültesi öğrencileri üniversitenin örgütsel imajını düşük düzeyde değerlendirmişlerdir. Kalite alt boyutu hariç (orta düzey diğer alt boyutlardaki imaj puanları düşük düzeyde olduğu ortaya çıkmıştır. Ayrıca üniversite öğrencilerinin görüşleri cinsiyet değişkenine göre anlamlı bir farklılık göstermemiştir. Diğer değişkenler olan memleket, öğrenim durumu, sınıf düzeyi ve yerleşim birimi değişkenlerine göre öğrencilerinin üniversitenin örgütsel imajına ilişkin görüşleri anlamlı bir farklılık göstermektedir. Memleket değişkenine göre öğrencilerin görüşlerindeki farklılık Sivaslı olan öğrenciler lehinedir. Öğrenim durumu değişkenine göre öğrencilerin görüşlerindeki farklılık 1.Öğretim öğrencileri lehinedir. 4. Sınıf öğrencileri 2. Sınıf öğrencilerine göre üniversitenin örgütsel imajını daha düşük düzeyde değerlendirdikleri ortaya çıkmıştır. Ayrıca, ilçede yaşayan öğrencilerin ilde yaşayan öğrencilere göre üniversitenin örgütsel imajını daha olumlu değerlendirdikleri bulunmuştur. Araştırma bulgular

  7. 大麻素受体1与FAK在血吸虫肝纤维化小鼠肝组织中的表达%Expression and significance of cannabinoid receptor 1 and FAK in hepatic tissues of schistosomal hepatic fibrosis mice

    Institute of Scientific and Technical Information of China (English)

    欧阳兵; 彭忠田; 王培

    2014-01-01

    目的:探讨大麻素受体1(CB1)与黏着斑激酶(FAK)在血吸虫肝纤维化小鼠肝组织中的表达。方法将32只昆明小鼠分为正常组(n=10)和模型组(n=22),两组动物均普通喂养,8周后处死取肝组织。模型组小鼠采用腹壁贴附法建立血吸虫肝纤维化小鼠模型。根据纤维化程度将模型组分为Ⅰ级肝纤维化组(n=4)、Ⅱ级肝纤维化组(n=8)和Ⅲ级肝纤维化组(n=10)。HE染色观察病理变化,Masson染色观察肝纤维化程度,免疫组织化学法检测不同纤维化组CB 1的表达,RT-PCR法检测各组CB 1 mRNA及FAK mRNA的表达。结果模型组可见明显虫卵结节及纤维化改变;不同纤维化组均可见CB 1、CB 1 mRNA及FAK mRNA表达,且随着肝脏纤维化程度的增加,CB1、CB1 mRNA及FAK mRNA的表达增加(P<0.05)。结论 CB1和FAK参与了血吸虫肝纤维化的发生与发展。%Objective To investigate the expression and significance of cannabinoid receptor 1 (CB1)and focal adhe-sion kinase (FAK)in hepatic tissues of schistosomal hepatic fibrosis mice.Methods A total of 32 Kunming mice were randomly divided into two groups:normal control group (n=10)and model group (n=22).The schistosome-induced liver fibrosis models were established by attaching cercaria to the skin on the ventral side of the mice and allo-wing infection to occur via direct penetration.All mice were raised normally,and at week 8 all mice were sacrificed to gain hepatic tissue samples.Mice in the model group were divided into 3 subgroups according to the severity of hepatic fibrosis.The pathological changes were determined by HE staining,the degrees of fibrosis were examined by Masson staining,the expression of CB1 was detected by immunohistochemical method,and the expressions of FAK mRNA and CB 1 mRNA were tested by reverse transcription polymerase chain reaction (RT-PCR).Results In the model group, significant schistosome egg

  8. Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-kappaB signal pathways in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Han, Ho Jae

    2008-03-01

    It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [(3)H]thymidine incorporation in a dose (> or =3 mg/ml)- and time (> or =3 h)-dependent manner, intracellular Ca(2+) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca(2+) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca(2+) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-kappaB (NF-kappaB) and inhibitor of NF-kappaB (IkappaB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca(2+), PKC, EGFR, p44/42 MAPK, or NF-kappaB signal pathways blocked the BSA-induced incorporation of [(3)H]thymidine. Consequently, the inhibition of Ca(2+), PKC, EGFR, p44/42 MAPKs, or NF-kappaB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21(WAF/Cip1) and p27(Kip1) expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca(2+)/PKC as well as the EGFR-dependent p44/42 MAPK and NF-kappaB signal pathways in PTCs.

  9. Ginsenosides-Rbl inhibits ET-1-induced cardiomyocyte hypertrophy via PKC pathway in neonatal rats%人参皂甙 Rbl 通过 PKC 途径抑制 ET-1诱发的乳鼠心肌肥大

    Institute of Scientific and Technical Information of China (English)

    孔宏亮; 黄带发; 王聿杰

    2015-01-01

    Objective:To explore whether ginsenosides-Rb1 (Gs-Rb1)can relieve cardiomyocyte hypertrophy induced by endothelin-1 (ET-1)via protein kinase C (PKC)system.Methods:Cardiomyocytes of neonatal rat were random-ly divided into blank control group,Gs-Rb1 group,ET-1 group,Gs-Rb1+ET-1 group,ET-1+CHE (chelerythrine, PKC blocker)group and Gs-Rb1 +ET-1 +CHE group.After 96h intervention,cardiomyocyte surface area,total protein content,PKC activity,c-fos and p-c-jun expressions were measured.Results: (1)Cardiomyocyte surface area and total protein content in Gs-Rb1+ET-1 group were significantly lower than those of ET-1 group (P <0.05~<0.001),but not significant different with those of Gs-Rb1+ET-1+CHE group,P =0.569;(2)PKC activity in Gs-Rb1+ET-1 group was significantly lower than that of ET-1 group [(9.3±0.6)pmol·min-1 ·mg-1 vs.(14.1± 0.9)pmol·min-1 ·mg-1 ],but significantly higher than that of Gs-Rb1+ET-1+CHE group [(2.7±0.2)pmol· min-1 ·mg-1 ],P <0.001 all;(3)Expressions of c-fos and p-c-jun gene and protein in ET-1 group were significant-ly higher than those of blank control group (P <0.001 all);compared with ET-1 group,there were significant re-ductions in expressions of c-fos [mRNA/protein:(0.53±0.05/0.39±0.02)vs.(0.43±0.03/0.31±0.03)]and p-c-jun [mRNA/protein:(0.64±0.04/0.44±0.02)vs.(0.33±0.05/0.37±0.03)]in Gs-Rb1+ET-1 group and ex-pressions of c-fos [mRNA/protein:0.41 ± 0.05/0.31 ± 0.02]and p-c-jun [mRNA/protein:0.31 ± 0.05/0.36 ±0.03]in ET-1+CHE group (P <0.05 or <0.001),expressions of c-fos and p-c-jun gene and protein in Gs-Rb1+ET-1+CHE group were significantly lower than those of Gs-Rb1+ET-1 group and ET-1+CHE group (P <0.05 or<0.001).Conclusion:Gs-Rb1 can significantly inhibit cardiomyocyte hypertrophy induced by ET-1 and PKC system is one of pathways mediating this biological effect.%目的:探讨人参皂甙 Rb1(Gs-Rb1)是否可通过蛋白激酶 C (PKC)系统减轻内皮素-1(ET-1)诱导的乳鼠心肌细胞肥大。方法:

  10. WJ9708012 exerts anticancer activity through PKC-α related crosstalk of mitochondrial and endoplasmic reticulum stresses in human hormone-refractory prostate cancer cells

    Institute of Scientific and Technical Information of China (English)

    Ting-chun KUO; Wei-jan HUANG; Jih-hwa GUH

    2011-01-01

    Aim: To investigate the anticancer mechanism of a methoxyflavanone derivative,WJ9708012,highlighting its role on a crosstalk between endoplasmic reticulum(ER)and mitochondrial stress.Methods: Cell proliferation was examined using sulforhodamine B assay.Cell-cycle progression,Ca2+mobilization and mitochondrial membrane potential(Δψm)were detected using flow cytometric analysis.Protein expression was detected using Western blot.Results: WJ9708012 displayed an antiproliferative and apoptotic activity in human hormone-refractory prostate cancer cells with IC50values of 6.4 and 5.3 μmol/L in PC-3 and DU-145 cells.WJ9708012 induced a prompt increase of cytosolic Ca2+level and activation of protein kinase C(PKC)-α.The cleavage of p-calpain was also induced by WJ9708012.Furthermore,WJ9708012 induced cell-cycle arrest at G1-phase associated with down-regulation of cyclin D1,cyclin E and cyclin-dependent kinase-4 expressions.It also caused a rapid and time-dependent decrease of phosphorylation level of mTOR(Ser2448),4E-BP1(Thr37/Thr46/Thr70)and p70S6K(Thr389),indicating the inhibition of mTOR-mediated translational pathways.The ER stress was activated by the identification of up-regulated GADD153 and glucose-regulated protein-78 protein levels.The subsequent mitochondrial stress was also identified by the observation of a decreased Bcl-2 and Bcl-xL expressions,an increased truncated Bid and Bad and a loss of Δψm.Conclusion: WJ9708012 induces an increase of cytosolic Ca2+concentration and activation of PKC-α.Subsequently,a crosstalk between ER stress and mitochondrial insult is induced,leading to the inhibition of mTOR pathways and arrest of the cell-cycle at G1phase.The apoptosis is ultimately induced by a severe damage of mitochondrial function.

  11. Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

    Science.gov (United States)

    Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-12-01

    The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

  12. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

    Science.gov (United States)

    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  13. Hypoxia stimulates invasion and migration of human cervical cancer cell lines HeLa/SiHa through the Rab11 trafficking of integrin αvβ3/FAK/PI3K pathway-mediated Rac1 activation

    Indian Academy of Sciences (India)

    HAO XU; YUAN YUAN; WENQIAN WU; MIN ZHOU; QIAN JIANG; LINJUN NIU; JIAYIN JI; NIANLI LIU; LONGZHEN ZHANG; XIA WANG

    2017-09-01

    Hypoxia plays a key role in tumour cell survival, invasion, and metastasis. An increasing number of studies have attemptedto characterize the tumour response to hypoxia and to identify predictive markers of disease. Here we show that hypoxiaincreases tumour cell invasion and migration by the modulation of Rab11, an important molecule for vesicular trafficking.In our study, we found that Rab11, together with the activation of Rac1, could stimulate invasion and migration of cervicalcancer cell lines HeLa/SiHa in hypoxia. Activation of Rac1 activity by hypoxia seems to be central to carcinoma invasion.We also found that these effects could be related to the integrin αvβ3. In addition, we studied the molecular pathway for thisprocess. Our results showed that in cervical cancer cell lines HeLa/SiHa, Rac1 activation in hypoxia could stimulateinvasion and migration, and this process was mediated by integrin αvβ3-mediated FAK and PI3K phosphorylation.Furthermore, hypoxia induced a dramatic increase in αvβ3 integrin surface expression, and this increase is dependent onRab11. In conclusion, our study might provide a new mechanism for the effect of hypoxia on stimulating cervicalcarcinoma invasion.

  14. The roles of FAK and GP Ⅱ b/Ⅲ a in platelet signal transduction%GPⅡb/Ⅲa和FAK在血小板信号转导中的作用

    Institute of Scientific and Technical Information of China (English)

    胡惠静; 刘彦虹; 吴晓岩

    2010-01-01

    GPⅡb/Ⅲa是血小板膜上最重要的糖蛋白,不但参与血小板的黏附、聚集反应,同时介导血小板的信号转导,在维持血小板正常功能中起关键作用.近几年对GPⅡ b/Ⅲa介导的双向信号转导过程的研究有了较大进展,其中信号转导分子--黏着斑激酶在信号转导中所起的重要作用也日益引起人们的重视.%CP Ⅱ b/Ⅲ a is an important glycoprotein that presents on the surface of platelet and is critical for the maintenance of the normal function of platelets. GP Ⅱ b/Ⅲ a plays a key role in the processes of platelet adhesion and aggregation. It also mediates signal transduction. In recent years, great advance has been made in the inside-out and outside-in signal transduction mediated by GP Ⅱ b/Ⅲ a. Furthermore, focal adhesion kinase (FAK), a kind of the signal transduction molecule, is drawing increasing attention in signal transduction mediated by GP II b/Ⅲ a.

  15. Expression of FAK and FGF-2 in nasopharyngeal carcinoma and their clinical significance%FAK和FGF-2在鼻咽癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    张娟; 李文光; 陈永平

    2014-01-01

    目的:研究黏着斑激酶(FAK)及成纤维细胞生长因子-2(FGF-2)在鼻咽癌中的表达及其临床意义.方法:应用免疫组织化学法检测鼻咽癌及鼻咽炎组织中FAK及FGF-2蛋白表达情况,并分析其与临床病理参数的关系.结果:在鼻咽癌组织中FAK、FGF-2蛋白阳性表达率与鼻咽炎组织中的表达率比较差异均有统计学意义(均P<0.05);FAK、FGF-2蛋白的表达与淋巴结转移有关(分别P<0.01和P<0.05),与其他病理参数无关(P>0.05);FAK的表达与FGF-2的表达呈正相关(r=0.350,P<0.01).结论:FAK、FGF-2的高表达在鼻咽癌的发生、发展中具有一定的相关性.

  16. Factors of affecting students' career values: The example of faculty of sports scienceÖğrencilerin kariyer değerlerini etkileyen faktörler: Spor bilimleri fakültesi örneği

    Directory of Open Access Journals (Sweden)

    Adem Pala

    2016-04-01

    Full Text Available Purpose of this study was to investigation of factors of affecting students’ values faculty of sports science. The survey population was consisted of Sports Science Faculty students. The sample constituted a total of 400 students from Marmara University, Istanbul University, Mugla Sıtkı Kocman University and Tokat Gazi Osman Pasa University (220 male and 180 female third grade students and fourth grade students in 2015-2016 academic year. The survey “Career Anchor Scale” constituted by Eyuboglu (2006 was used as a data collection tool. Frequency and percentage of the data were calculated, independent t test, one-way ANOVA test and Tukey test was used for data analysis on SPSS 20.0 (Statistical package for social sciences package program. Findings; Participatied students whose 220 male and 180 female and mean of age was 20,9. Consequently, significant differences were found between genders in terms of Career Anchor  “career satisfaction”, “safety/steady” and “ self-commitment” sub dimension (p<0.05. Significant differences were found between university variable  in terms of Career Anchor “safety/steady” “pure defiance”, “life-style”,  and “entrepreneurship/creativity” sub dimension (p<0.05.   Özet Bu çalışma spor bilimleri fakültesi öğrencilerinin kariyer değer algılarının tespit etmek amacıyla yapılmıştır. Çalışmanın everenini Türkiye’deki Spor Bilimleri Fakültelerinde 2015-2016 eğitim-öğretim yılında 3.  ve 4. sınıflarda okuyan öğrenciler oluşturmaktadır. Örneklem grubunu ise Marmara Üniversitesi, İstanbul Üniversitesi, Tokat Gazi Osman Paşa Üniversitesi ve Muğla Sıtkı Koçman Üniversitesi Spor Bilimleri Fakültelerinde okuyan 400 öğrenci oluşturmaktadır. Örneklem grubunun görüşlerini tespit etmek amacıyla Eyüboğlu (2006 tarafından geliştirilen “Kariyer Değerleri Ölçeği” veri toplama aracı olarak kullanılmıştır. Elde edilen veriler

  17. Marine Bromophenol Bis (2,3-Dibromo-4,5-dihydroxy-phenyl-methane Inhibits the Proliferation, Migration, and Invasion of Hepatocellular Carcinoma Cells via Modulating β1-Integrin/FAK Signaling

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    Ning Wu

    2015-02-01

    Full Text Available Bis (2,3-dibromo-4,5-dihydroxy-phenyl-methane (BDDPM is a natural bromophenol compound derived from marine algae. Previous reports have shown that BDDPM possesses antimicrobial activity. In the present study, we found that BDDPM has cytotoxic activity on a wide range of tumor cells, including BEL-7402 cells (IC50 = 8.7 μg/mL. Further studies have shown that prior to the onset of apoptosis, the BDDPM induces BEL-7402 cell detachment by decreasing the adherence of cells to the extracellular matrix (ECM. Detachment experiments have shown that the treatment of BEL-7402 cells with low concentrations of BDDPM (5.0 μg/mL significantly inhibits cell adhesion to fibronectin and collagen IV as well as cell migration and invasion. High doses of BDDPM (10.0 μg/mL completely inhibit the migration of BEL-7402 cells, and the expression level of MMPs (MMP-2 and MMP-9 is significantly decreased. Moreover, the expression of β1-integrin and focal adhesion kinase (FAK is found to be down-regulated by BDDPM. This study suggests that BDDPM has a potential to be developed as a novel anticancer therapeutic agent due to its anti-metastatic activity and also indicates that BDDPM, which has a unique chemical structure, could serve as a lead compound for rational drug design and for future development of anticancer agents.

  18. Marine bromophenol bis (2,3-dibromo-4,5-dihydroxy-phenyl)-methane inhibits the proliferation, migration, and invasion of hepatocellular carcinoma cells via modulating β1-integrin/FAK signaling.

    Science.gov (United States)

    Wu, Ning; Luo, Jiao; Jiang, Bo; Wang, Lijun; Wang, Shuaiyu; Wang, Changhui; Fu, Changqing; Li, Jian; Shi, Dayong

    2015-02-13

    Bis (2,3-dibromo-4,5-dihydroxy-phenyl)-methane (BDDPM) is a natural bromophenol compound derived from marine algae. Previous reports have shown that BDDPM possesses antimicrobial activity. In the present study, we found that BDDPM has cytotoxic activity on a wide range of tumor cells, including BEL-7402 cells (IC50 = 8.7 μg/mL). Further studies have shown that prior to the onset of apoptosis, the BDDPM induces BEL-7402 cell detachment by decreasing the adherence of cells to the extracellular matrix (ECM). Detachment experiments have shown that the treatment of BEL-7402 cells with low concentrations of BDDPM (5.0 μg/mL) significantly inhibits cell adhesion to fibronectin and collagen IV as well as cell migration and invasion. High doses of BDDPM (10.0 μg/mL) completely inhibit the migration of BEL-7402 cells, and the expression level of MMPs (MMP-2 and MMP-9) is significantly decreased. Moreover, the expression of β1-integrin and focal adhesion kinase (FAK) is found to be down-regulated by BDDPM. This study suggests that BDDPM has a potential to be developed as a novel anticancer therapeutic agent due to its anti-metastatic activity and also indicates that BDDPM, which has a unique chemical structure, could serve as a lead compound for rational drug design and for future development of anticancer agents.

  19. Tıp Eğitiminde Değişim ve Trakya Üniversitesi Tıp Fakültesinde Değişim Süreci Üzerine Notlar

    OpenAIRE

    Eskiocak, Muzaffer; Sarıdoğan, Kenan; Tezel, Ahmet; Adalı, M. Kemal; Çağlar, Tuncay; Otkun, Metin; Özdemir, Ferda; Şahin, Melih; Mert, Selva; Kutlu, A. Kemal

    2005-01-01

    Tıp eğitiminde değişim günümüzün önemli tartışma ve eylem alanlarındadır. Bu yazıda tıp eğitiminde değişim süreci ve değişim dinamikleri konusunda Fakültemizden deneyim aktarmak amaçlanmıştır. Bu bağlamda tıp eğitiminin amacı, değişim dinamikleri ve değişimin yönü, eğitim ve süreç yönetimi irdelenmiştir. Yönetimi bir bilimsel eylem olarak düşünülmezse akademisyenlerin bilimsel özerklikleri hastane hizmeti yönetimi sürecinden olumsuz etkilenebilir sonucuna varılmıştır....

  20. Toxic effects of TiO2 nanoparticles in primary cultured rat sertoli cells are mediated via a dysregulated Ca(2+) /PKC/p38 MAPK/NF-κB cascade.

    Science.gov (United States)

    Ye, Lingqun; Hong, Fashui; Ze, Xiao; Li, Lingjuan; Zhou, Yaoming; Ze, Yuguan

    2017-05-01

    Although numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) can be accumulated in various animal organs and can cause toxicity, there is currently only limited data regarding reproductive toxicity especially on the toxic mechanisms of TiO2 NPs in Sertoli cells. In order to investigate the mechanism of reproductive toxicity, primary cultured rat Sertoli cells were exposed to 5, 15, or 30 μg/mL TiO2 NPs for 24 h, and TiO2 NPs internalization, expression of PKC (p-PKC) and p38 MAPK (p-p38 MAPK) as well as calcium homeostasis were examined. Our findings demonstrated that TiO2 NPs crossed the membrane into the cytoplasm or nucleus, and significantly suppressed cell viability of primary cultured rat Sertoli cells in a concentration-dependent manner. Furthermore, immunological dysfunction caused by TiO2 NPs was involved in the increased expression of NF-κB, TNF-α, and IL-1β, and decreased IκB expression. TiO2 NPs significantly decreased Ca(2+) -ATPase and Ca(2+) /Mg(2+) -ATPase activity and enhanced intracellular Ca(2+) levels, and up-regulated the expression of p-PKC and p-p38 MAPK in a dose-dependent manner in primary cultured rat Sertoli cells. Taken together, these findings indicate that TiO2 NPs may induce immunological dysfunction of primary cultured rat Sertoli cells by stimulating the Ca(2+) /PKC/p38 MAPK cascade, which triggers NF-κB activation and ultimately induces the expression of inflammatory cytokines in primary cultured rat Sertoli cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1374-1382, 2017.

  1. DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412.

    Science.gov (United States)

    Seedhouse, C H; Hunter, H M; Lloyd-Lewis, B; Massip, A-M; Pallis, M; Carter, G I; Grundy, M; Shang, S; Russell, N H

    2006-12-01

    The presence of internal tandem duplications (ITD) mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor influences the risk of relapse in acute myeloid leukaemia (AML). We have investigated DNA repair in FLT3-ITD and wild-type (WT) cells. Using the comet assay, we have demonstrated that the FLT3 inhibitor PKC412 significantly inhibits repair of DNA damage in the MV4-11-FLT3-ITD cell line and FLT3-ITD patient samples but not in the HL-60-FLT3-WT cell line or FLT3-WT patient samples. Following the discovery that transcript levels of the DNA repair gene RAD51 are significantly correlated with FLT3 transcript levels in FLT3-ITD patients, we further investigated the role of RAD51 in FLT3-ITD-AML. The reduction in DNA repair in PKC412-treated FLT3-ITD cells was shown to be associated with downregulation of RAD51 mRNA and protein expression and correlates with the maintenance of phosphorylated H2AX levels, implying that PKC412 inhibits the homologous recombination double-strand break repair pathway in FLT3-ITD cells. Using FLT3-short interfering RNA (siRNA), we also demonstrated that genetic silencing of FLT3 results in RAD51 downregulation in FLT3-ITD cells but not in FLT3-WT cells. This work suggests that the use of FLT3 inhibitors such as PKC412 may reverse the drug-resistant phenotype of FLT3-ITD-AML cells by inhibiting repair of chemotherapy-induced genotoxic damage and thereby reduce the risk of disease relapse.

  2. E-cadherin、FAK、nm23-H1蛋白表达与声门上型喉癌颈淋巴结转移的相关因素研究%Expression of E-cadherin, FAK and nm23-H1 proteins in carcinoma of supraglottic larynx with lymph node metastasis

    Institute of Scientific and Technical Information of China (English)

    张靖华; 陈英武; 平金良; 徐炜; 顾栋烨

    2011-01-01

    Objective To investigate the expression ofE- cadherin, FAK (FocalAdhesion Kinase) and nm23- H1 proteins in carcinoma of supraglottic larynx with cervical lymph node metastasis. Methods Expression of E- cadherin, FAK and nm23- H1 proteins were detected by Envision two- step immunohistochemical method in 70 cases ofsupraglottic laryngeal carcinoma. The relationship ofE- cadherin, FAK and nm23- H1 expression with cervical lymph node metastasis was analyzed. Results E- cadherin and FAK were significantly correlated with cervical lymph node metastasis in supraglottic laryngeal cancer, but Nm23- Hl was not. Logistic regression showed that the diagnostic accuracy of combination of FAK expression, histological grade and T classification for cervical lymph node metastasis was 79.4%. Conclusions E- cadherin, FAK exprcssinn is significantly correlated with cerv ieal node metastasis of supraglottic carcinoma. Combination with tissue differentiation G, primary tumor staging T and FAK expression can predict cervical lymph node metastasis with high accuracy.%目的 探讨E-cadherin、FAK、nm23-H1蛋白表达与声门上型喉癌颈淋巴结转移的关系,以利于临床上预估颈淋巴结转移情况.方法 采用免疫组织化学Envision两步法检测70例声门上型喉癌中E-cadherin、FAK、nm23-H1的表达情况,结合患者临床资料分析3种免疫标志物(E-cadherin、FAK、nm23-H1蛋白)与颈淋巴结转移的相关性.结果 E-cadherin及FAK蛋白表达与颈淋巴结转移均明显相关(P<0.05),nm23-H1蛋白表达与颈淋巴结转移无明显相关(P >0.05).E-cadherin及FAK蛋白均阳性表达与颈淋巴结转移显著相关(P<0.01).肿瘤分期、远处转移、病理分化程度均与颈淋巴结转移相关(均P<0.01).原发肿瘤分期T、组织分化G、FAK被纳入Logistic回归方程,利用该方程可得颈淋巴结无转移的准确率为75.6%,颈淋巴结有转移的准确率为82.7%,总体准确率为79.4%.结论 声门上型喉癌

  3. The CCR5-antagonist Maraviroc reverses HIV-1 latency in vitro alone or in combination with the PKC-agonist Bryostatin-1.

    Science.gov (United States)

    López-Huertas, María Rosa; Jiménez-Tormo, Laura; Madrid-Elena, Nadia; Gutiérrez, Carolina; Rodríguez-Mora, Sara; Coiras, Mayte; Alcamí, José; Moreno, Santiago

    2017-05-24

    A potential strategy to cure HIV-1 infection is to use latency reversing agents (LRAs) to eliminate latent reservoirs established in resting CD4+ T (rCD4+) cells. As no drug has been shown to be completely effective, finding new drugs and combinations are of increasing importance. We studied the effect of Maraviroc (MVC), a CCR5 antagonist that activates NF-κB, on HIV-1 replication from latency. HIV-1-latency models based on CCL19 or IL7 treatment, before HIV-1 infection were used. Latently infected primary rCD4+ or central memory T cells were stimulated with MVC alone or in combination with Bryostatin-1, a PKC agonist known to reverse HIV-1 latency. MVC 5 μM and 0.31 μM were chosen for further studies although other concentrations of MVC also increased HIV-1 replication. MVC was as efficient as Bryostatin-1 in reactivating X4 and R5-tropic HIV-1. However, the combination of MVC and Bryostatin-1 was antagonistic, probably because Bryostatin-1 reduced CCR5 expression levels. Although HIV-1 reactivation had the same tendency in both latency models, statistical significance was only achieved in IL7-treated cells. These data suggest that MVC should be regarded as a new LRA with potency similar as Bryostatin-1. Further studies are required to describe the synergistic effect of MVC with other LRAs.

  4. The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis.

    Science.gov (United States)

    Abu-Siniyeh, Ahmed; Owen, Dylan M; Benzing, Carola; Rinkwitz, Silke; Becker, Thomas S; Majumdar, Arindam; Gaus, Katharina

    2016-01-01

    The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3-11 days post fertilization (dpf) with Laurdan 2-photon microscopy. We then applied a combination of Laurdan imaging, antisense knock-down and analysis of polarity markers to understand the relationship between membrane order and apical-basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.

  5. The inhibitory effect of pseudolaric acid B on gastric cancer and multidrug resistance via Cox-2/PKC-α/P-gp pathway.

    Directory of Open Access Journals (Sweden)

    Qian Sun

    Full Text Available AIM: To investigate the inhibitory effect of pseudolaric acid B on subcutaneous xenografts of human gastric adenocarcinoma and the underlying molecular mechanisms involved in its multidrug resistance. METHODS: Human gastric adenocarcinoma SGC7901 cells and drug-resistant SGC7901/ADR cells were injected into nude mice to establish a subcutaneous xenograft model. The effects of pseudolaric acid B with or without adriamycin treatment were compared by determining the tumor size and weight. Cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein expression levels were determined by immunohistochemistry and western blot. RESULTS: Pseudolaric acid B significantly suppressed the tumor growth induced by SGC7901 cells and SGC7901/ADR cells. The combination of pseudolaric acid B and the traditional chemotherapy drug adriamycin exhibited more potent inhibitory effects on the growth of gastric cancer in vivo than treatment with either pseudolaric acid B or adriamycin alone. Protein expression levels of cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein were inhibited by pseudolaric acid B alone or in combination with adriamycin in SGC7901/ADR cell xenografts. CONCLUSION: Pseudolaric acid B has a significant inhibitory effect and an additive inhibitory effect in combination with adriamycin on the growth of gastric cancer in vivo, which reverses the multidrug resistance of gastric neoplasm to chemotherapy drugs by downregulating the Cox-2/PKC-α/P-gp/mdr1 signaling pathway.

  6. Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

    Science.gov (United States)

    Worthmann, Kirstin; Leitges, Michael; Teng, Beina; Sestu, Marcello; Tossidou, Irini; Samson, Thomas; Haller, Hermann; Huber, Tobias B; Schiffer, Mario

    2013-12-01

    The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes.

  7. An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

    Directory of Open Access Journals (Sweden)

    Vanessa Moreira

    2014-01-01

    Full Text Available Phospholipases A2 (PLA2 are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PGE2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2. Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

  8. Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization

    Science.gov (United States)

    Shi, Ying; Lai, Xiangru; Ye, Lingyan; Chen, Keqiang; Cao, Zheng; Gong, Wanghua; Jin, Lili; Wang, Chunyan; Liu, Mingyong; Liao, Yuan; Wang, Ji Ming; Zhou, Naiming

    2017-01-01

    The niacin receptor HCA2 is implicated in controlling inflammatory host responses with yet poorly understood mechanistic basis. We previously reported that HCA2 in A431 epithelial cells transduced Gβγ-protein kinase C- and Gβγ-metalloproteinase/EGFR-dependent MAPK/ERK signaling cascades. Here, we investigated the role of HCA2 in macrophage-mediated inflammation and the underlying mechanisms. We found that proinflammatory stimulants LPS, IL-6 and IL-1β up-regulated the expression of HCA2 on macrophages. Niacin significantly inhibited macrophage chemotaxis in response to chemoattractants fMLF and CCL2 by disrupting polarized distribution of F-actin and Gβ protein. Niacin showed a selected additive effect on chemoattractant-induced activation of ERK1/2, JNK and PI3K pathways, but only the MEK inhibitor UO126 reduced niacin-mediated inhibition of macrophage chemotaxis, while activation of ERK1/2 by EGF alone did not inhibit fMLF-mediated migration of HEK293T cells co-expressing HCA2 and fMLF receptor FPR1. In addition, niacin induced heterologous desensitization and internalization of FPR1. Furthermore, niacin rescued mice from septic shock by diminishing inflammatory symptoms and the effect was abrogated in HCA2−/− mice. These results suggest that Gβγ/PKC-dependent ERK1/2 activation and heterologous desensitization of chemoattractant receptors are involved in the inhibition of chemoattractant-induced migration of macrophages by niacin. Thus, HCA2 plays a critical role in host protection against pro-inflammatory insults. PMID:28186140

  9. Thrombin/Matrix Metalloproteinase-9-Dependent SK-N-SH Cell Migration is Mediated Through a PLC/PKC/MAPKs/NF-κB Cascade.

    Science.gov (United States)

    Yang, Chien-Chung; Lin, Chih-Chung; Chien, Peter Tzu-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-11-01

    Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH3), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH3, Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

  10. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2.

    Science.gov (United States)

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-03-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.

  11. PAR2-PKC 通路调控 TRPV1介导内脏高敏感性参与肠易激综合征%PAR2-PKC pathway regulated TRPV1 mediates visceral hyperalgesia to participate in the irritable bowel syndrome

    Institute of Scientific and Technical Information of China (English)

    黄斌; 黄适; 张涛; 陈远能

    2015-01-01

    Visceral hyperalgesia is one of characteristic pathophysiological mechanisms of irritable bowel syndrome ( IBS) .The mechanism of visceral hyperalgesia is unclear and thought to involve a variety of enteric nervous secreted neurotransmitters , cytokines and receptors .The exact mechanism of visceral hyperalgesia at present has not yet fully un-derstood , but TRPV1 upregulation and phosphorylation mediated pain sensitization was considered to be one of the key events.Excessive activation of PAR-PKC pathway phosphorylate the TRPV 1 and lower its threshold , eventually leading to the occurring of visceral hyperalgesia .This article reviewed the recent advance about the visceral hyperalgesia led by the upregulation and phosphorylation of TRPV 1 which was regulated by PAR-PKC pathway .%内脏高敏感性是肠易激综合征( irritable bowel syndrome ,IBS)特征性病理生理机制之一,其发生涉及多种肠神经分泌的递质、细胞因子和受体,内脏高敏感性的发生机制目前尚未明确,但是辣椒素受体(transient receptor potential vanilloid 1,TRPV1)上调及磷酸化介导痛觉敏化被认为是内脏高敏感性发生的关键性事件之一。 PAR2-PKC通路过度活化,可介导TRPV1磷酸化,TRPV1开放阈值下降,内脏疼痛感知异常,最终导致内脏高敏感性的发生。本文就近年来关于PAR2-PKC通路调控TRPV1磷酸化及上调介导IBS内脏高敏感性的相关研究作一概述。

  12. Induction of TRIM22 by IFN-γ Involves JAK and PC-PLC/PKC, but Not MAPKs and pI3K/Akt/mTOR Pathways.

    Science.gov (United States)

    Gao, Bo; Xu, Wei; Wang, Yaxin; Zhong, Linmao; Xiong, Sidong

    2013-10-01

    Tripartite motif (TRIM) 22 plays an important role in interferons (IFNs)-mediated antiviral activity. We previously demonstrated that interferon regulatory factor-1 (IRF-1) played a central role in IFN-γ-induced TRIM22 expression via binding to a special cis-element named 5' extended IFN-stimulating response element (5'eISRE). In this study, we sought to identify the signaling pathways involved in TRIM22 induction by IFN-γ. By using various pharmacological inhibitors, it was found that the activity of tyrosine kinase and phosphatidylcholine-phospholipase C (PC-PLC), but not phosphatidylinositol-phospholipase C (PI-PLC) and phospholipase D (PLD), was required for IFN-γ-induced TRIM22 expression in HepG2 cells. Tyrosine kinase Janus kinase (JAK), not SRC and PYK2, played an indispensable role in TRIM22 induction. Inhibition of protein kinase C (PKC) activity also significantly attenuated IFN-γ induction of TRIM22. Although treatment with IFN-γ resulted in the stimulation of mitogen-activated protein kinases (MAPKs) (p38, ERK, and JNK) and pI3K/Akt/mTOR pathways in HepG2 cells, the inhibition of their activity did not affect IFN-γ-stimulated TRIM22 expression. Further studies showed that overexpression of JAK1 and PKCα activated TRIM22 promoter activity in a 5'eISRE-dependent manner, and inhibition of not only JAK but also PC-PLC/PKC pathways significantly attenuated IFN-γ-induced IRF-1 expression in HepG2 cells. Taken together, these data indicated that IFN-γ induced TRIM22 expression via activation of JAK and PC-PLC/PKC signaling pathways, which involved the cis-element 5'eISRE and the transactivator IRF-1.

  13. Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong J.; Galoforo, S.S.; Berns, C.M. [William Beaumont Hospital, Royal Oak, MI (United States). Dept. of Radiation Oncology] [and others

    1997-08-01

    We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatment with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.

  14. Spinal D-Serine Increases PKC-Dependent GluN1 Phosphorylation Contributing to the Sigma-1 Receptor-Induced Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

    Science.gov (United States)

    Choi, Sheu-Ran; Moon, Ji-Young; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Hoon-Seong; Kang, Suk-Yun; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2017-04-01

    We have recently shown that spinal sigma-1 receptor (Sig-1R) activation facilitates nociception via an increase in phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). The present study was designed to examine whether the Sig-1R-induced facilitative effect on NMDA-induced nociception is mediated by D-serine, and whether D-serine modulates spinal pGluN1 expression and the development of neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the D-serine degrading enzyme, D-amino acid oxidase attenuated the facilitation of NMDA-induced nociception induced by the Sig-1R agonist, 2-(4-morpholinethyl)1-phenylcyclohexane carboxylate. Exogenous D-serine increased protein kinase C (PKC)-dependent (Ser896) pGluN1 expression and facilitated NMDA-induced nociception, which was attenuated by preteatment with the PKC inhibitor, chelerythrine. In CCI mice, administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt or D-amino acid oxidase on postoperative days 0 to 3 suppressed CCI-induced mechanical allodynia (MA) and pGluN1 expression on day 3 after CCI surgery. Intrathecal administration of D-serine restored MA as well as the GluN1 phosphorylation on day 3 after surgery that was suppressed by the Sig-1R antagonist, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide or the astrocyte inhibitor, fluorocitrate. In contrast, D-serine had no effect on CCI-induced thermal hyperalgesia or GluN1 expression. These results indicate that spinal D-serine: 1) mediates the facilitative effect of Sig-1R on NMDA-induced nociception, 2) modulates PKC-dependent pGluN1 expression, and 3) ultimately contributes to the induction of MA after peripheral nerve injury.

  15. Programming of neurotoxic cofactor CXCL-10 in HIV-1-associated dementia: abrogation of CXCL-10-induced neuro-glial toxicity in vitro by PKC activator

    Science.gov (United States)

    2012-01-01

    Background More than 50% of patients undergoing lifelong suppressive antiviral treatment for HIV-1 infection develop minor HIV-1-associated neurocognitive disorders. Neurological complications during HIV-1 infection are the result of direct neuronal damage by proinflammatory products released from HIV-1-infected or -uninfected activated lymphocytes, monocytes, macrophages, microglia and astrocytes. The specific pro-inflammatory products and their roles in neurotoxicity are far from clear. We investigated proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) of HIV-demented (HIV-D) and HIV-nondemented (HIV-ND) patients and studied their affect on neuroglial toxicity. Methods and results Bioplex array showed elevated levels of signatory chemokines or cytokines (IL-6, IFN-γ, CXCL10, MCP-1 and PDGF) in the CSF of HIV-D patients (n = 7) but not in that of HIV-ND patients (n = 7). Among the signatory cytokines and chemokines, CXCL10 was distinctly upregulated in-vitro in HIV-1 (NLENG1)-activated human fetal astrocytes, HIV-1 (Ba-L)-infected macrophages, and HIV-1 (NLENG1)-infected lymphocytes. Virus-infected macrophages also had increased levels of TNF-α. Consistently, human fetal astrocytes treated with HIV-1 and TNF-α induced the signatory molecules. CXCL10 in combination with HIV-1 synergistically enhanced neuronal toxicity and showed chemotactic activity (~ 40 fold) for activated peripheral blood mononuclear cells (PBMC), suggesting the intersection of signaling events imparted by HIV-1 and CXCL10 after binding to their respective surface receptors, CXCR4 and CXCR3, on neurons. Blocking CXCR3 and its downstream MAP kinase (MAPK) signaling pathway suppressed combined CXCL10 and HIV-1-induced neurotoxicity. Bryostatin, a PKC modulator and suppressor of CXCR4, conferred neuroprotection against combined insult with HIV-1 and CXCL10. Bryostatin also suppressed HIV-1 and CXCL10-induced PBMC chemotaxis. Although, therapeutic targeting of chemokines in

  16. Programming of neurotoxic cofactor CXCL-10 in HIV-1-associated dementia: abrogation of CXCL-10-induced neuro-glial toxicity in vitro by PKC activator

    Directory of Open Access Journals (Sweden)

    Mehla Rajeev

    2012-10-01

    Full Text Available Abstract Background More than 50% of patients undergoing lifelong suppressive antiviral treatment for HIV-1 infection develop minor HIV-1-associated neurocognitive disorders. Neurological complications during HIV-1 infection are the result of direct neuronal damage by proinflammatory products released from HIV-1-infected or -uninfected activated lymphocytes, monocytes, macrophages, microglia and astrocytes. The specific pro-inflammatory products and their roles in neurotoxicity are far from clear. We investigated proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF of HIV-demented (HIV-D and HIV-nondemented (HIV-ND patients and studied their affect on neuroglial toxicity. Methods and results Bioplex array showed elevated levels of signatory chemokines or cytokines (IL-6, IFN-γ, CXCL10, MCP-1 and PDGF in the CSF of HIV-D patients (n = 7 but not in that of HIV-ND patients (n = 7. Among the signatory cytokines and chemokines, CXCL10 was distinctly upregulated in-vitro in HIV-1 (NLENG1-activated human fetal astrocytes, HIV-1 (Ba-L-infected macrophages, and HIV-1 (NLENG1-infected lymphocytes. Virus-infected macrophages also had increased levels of TNF-α. Consistently, human fetal astrocytes treated with HIV-1 and TNF-α induced the signatory molecules. CXCL10 in combination with HIV-1 synergistically enhanced neuronal toxicity and showed chemotactic activity (~ 40 fold for activated peripheral blood mononuclear cells (PBMC, suggesting the intersection of signaling events imparted by HIV-1 and CXCL10 after binding to their respective surface receptors, CXCR4 and CXCR3, on neurons. Blocking CXCR3 and its downstream MAP kinase (MAPK signaling pathway suppressed combined CXCL10 and HIV-1-induced neurotoxicity. Bryostatin, a PKC modulator and suppressor of CXCR4, conferred neuroprotection against combined insult with HIV-1 and CXCL10. Bryostatin also suppressed HIV-1 and CXCL10-induced PBMC chemotaxis. Although, therapeutic targeting

  17. Gemiadamlarının Sağlık ve Emniyet Koşullarının Değerlendirilmesi: DEÜ Denizcilik Fakültesi Örneği

    Directory of Open Access Journals (Sweden)

    Barış KULEYİN

    2014-06-01

    Full Text Available Dünya ticaretinin % 90’lık kısmı denizyolu ticaretiyle gerçekleştirilmekte olup yaklaşık 50.000 gemi bu amaca hizmet etmektedir. Denizyolu ticaretinin lokomotifi olan gemilerde farklı milliyette 1.187.000 gemi adamı çalışmaktadır. Diğer bir deyişle bir milyondan fazla gemi adamı, dünya nüfusunun kalan kısmının yararı için çalışmaktadır. Denizcilerin ve sektörün önemi “Denizcilik olmadan dünya nüfusunun yarısı açlıktan yarısı da soğuktan yok olur.” ifadesi ile belirtilmektedir. Denizcilik mesleği, diğer mesleklere göre önemli farklılıklara sahiptir. Gemi adamlığı ve denizcilik mesleği emek yoğun bir yapıya sahip olduğundan emniyet açısından azami ölçüde dikkat isteyen bir meslektir. Çalışma esnasında yaşanacak küçük bir dikkatsizlik bile ciddi yaralanmalara ve hatta ölümlere yol açabilir. Bu çalışmayla, Dokuz Eylül Üniversitesi (DEÜ Denizcilik Fakültesi öğrencilerinin gemilerde emniyet kapsamında yaşadıkları problemlerin değerlendirilmesi amaçlanmış olup aynı zamanda açık deniz stajına gidecek öğrencilere gemilerde emniyet hakkında bir rehber kaynak oluşturulması amaçlanmıştır. Çalışmada kullanılan “Emniyet ve Yaralanma Bilgi Formu” verileri SPSS 20 istatistik programı aracılığıyla analiz edilmiştir.

  18. Bryostatin 1 inhibits phorbol ester-induced apoptosis in prostate cancer cells by differentially modulating protein kinase C (PKC) delta translocation and preventing PKCdelta-mediated release of tumor necrosis factor-alpha.

    Science.gov (United States)

    von Burstin, Vivian A; Xiao, Liqing; Kazanietz, Marcelo G

    2010-09-01

    Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCdelta-mediated release of TNFalpha, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFalpha release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCdelta to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCdelta peripheral translocation. Studies using a membrane-targeted PKCdelta construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCdelta may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy.

  19. Bryostatin 1 Inhibits Phorbol Ester-Induced Apoptosis in Prostate Cancer Cells by Differentially Modulating Protein Kinase C (PKC) δ Translocation and Preventing PKCδ-Mediated Release of Tumor Necrosis Factor-α

    Science.gov (United States)

    von Burstin, Vivian A.; Xiao, Liqing

    2010-01-01

    Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCδ-mediated release of TNFα, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFα release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCδ to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCδ peripheral translocation. Studies using a membrane-targeted PKCδ construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCδ may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy. PMID:20516369

  20. Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    ZENG QingHua; LI XingTing; ZHONG GuoGan; ZHANG WenJie; SUN ChengWen

    2009-01-01

    Using fura-2-acetoxymethyl eater (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]1) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats.The effect of ET-1 on [Ca2+]1 elevation was abolished in the presence of the ETA receptor blocker BQ123,but was not affected by the ETa receptor blocker BQ788. ET-1-induced an increase in [Ca2+]1, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibltors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an Increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETa receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.

  1. Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca2+]i elevation was abolished in the presence of the ETA receptor blocker BQ123, but was not affected by the ETB receptor blocker BQ788. ET-1-induced an increase in [Ca2+]i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.

  2. Hemşirelik Fakültesi Öğrencilerinin Sosyal Ağ Sitelerini Kullanma Amacı ile İletişim Becerileri Arasındaki İlişkinin İncelenmesi

    Directory of Open Access Journals (Sweden)

    Hatice Kaya

    2015-08-01

    Full Text Available Amaç: Araştırma, hemşirelik öğrencilerinin sosyal ağ sitelerini kullanma amacı ile iletişim becerileri arasındaki ilişkiyi belirlemeye yönelik tanımlayıcı ve ilişki arayıcı türde planlandı.Yöntem: Araştırmanın evrenini, bir Hemşirelik Fakültesi’nin 2011-2012 eğitim-öğretim yılında öğrenim gören tüm öğrenciler, örneklemini ise tabakalı rastgele örnekleme yöntemi ile belirlenen 238 öğrenci oluşturdu. Kurumdan yazılı, öğrencilerden sözlü bilgilendirilmiş onam alındı. Veriler Yapılandırılmış Soru Formu, Sosyal Ağ Siteleri Kullanım Amacı Ölçeği ve İletişim Becerileri Ölçeği (İBÖ ile toplandı. Verilerin analizinde; frekans, yüzde, aritmetik ortalama, standart sapma, Independent t testi, Spearman’s rho korelasyon testleri kullanıldı.Bulgular: Yaş ortalamasının 20.67±2.67 %78.2’sinin kız, %59.7’sinin bilgisayar kullandığı, %98.3’ünün internet, %86.1’inin sosyal ağ kullandığı, sosyal ağ sitelerini kullanan öğrencilerin %47.9’unun myspace, %26.1’inin twitter, %18’inin ise facebook’u tercih ettikleri saptandı. Öğrencilerin Sosyal Ağ Siteleri Kullanım Amacı Ölçeği toplam puan ortalamasının 38.69±17.49, İBÖ toplam puan ortalamasının 104.00±19.26 olduğu görüldü. Sosyal ağ sitelerini kullanan öğrencilerin İBÖ toplam puanı arasında anlamlı bir fark bulunmazken (p>0.05, İBÖ davranışsal alt boyutunda istatistiksel açıdan anlamlı bir fark saptandı (p<0.001.Sonuçlar: Öğrencilerin iletişim becerileri orta düzeydedir. Sosyal ağ sitelerini hem iletişim, hem kendini tanıtma, hem de eğitim amaçlı kullanmaktadırlar. Sosyal ağ sitelerini etkin kullanan öğrenciler, iletişim becerilerini davranışlarına yansıtabilmektedirler. 

  3. Mecanismos moleculares de respuesta al estrés oxidativo mediados por la ruta de integridad celular (vía PKC1-MAP quinasa)de Saccharonmyces cerevisiae

    OpenAIRE

    Vilella Mitjana, Felipe

    2006-01-01

    La via d'integritat cel·lular o via Pkc1-MAP quinasa de Saccharomyces cerevisiae té un paper central en els mecanismes de resposta cel·lular enfront de diferents estressos ambientals, com són: l'estrès tèrmic, l'estrès hipoosmòtic o qualsevol estrès que afecti a la paret cel·lular. En aquest treball de tesi doctoral es demostra que aquesta via de transducció de senyal també està implicada en la supervivència iadaptació enfront els efectes de l'estrès oxidatiu.Hem observat que les proteïnes Pk...

  4. Involvement of TRPV2 and SOCE in calcium influx disorder in DMD primary human myotubes with a specific contribution of α1-syntrophin and PLC/PKC in SOCE regulation.

    Science.gov (United States)

    Harisseh, Rania; Chatelier, Aurélien; Magaud, Christophe; Déliot, Nadine; Constantin, Bruno

    2013-05-01

    Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca(2+)/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.

  5. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    Science.gov (United States)

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  6. Eğitim Fakültesi Öğrencilerinin Bilgisayar Destekli Uzaktan Eğitim Sistemi (Moodle Memnuniyet Düzeyleri. Education Faculty Students’ Levels of Satisfaction with the Computer-Assisted Distance Education System (Moodle

    Directory of Open Access Journals (Sweden)

    Murat YALMAN

    2013-09-01

    rneklemi Eğitim Fakültesinde 2011-2012 ve 2012-2013 öğretim yıllarında öğrenim gören 1050 öğrenciden oluşmaktadır. Bu çalışma sonucunda öğrencilerin uzaktan eğitim memnuniyet düzeylerine ilişkin güvenilirlik kat sayısı Cronbach Alpha değerleri 0,94 olarak hesaplanmıştır. Öğrencilerin büyük oranı (% 76,57 uzaktan eğitim yönetim sistemiyle aldıkları eğitimden sonra eğitim gördükleri bölüme ait uzaktan eğitim seçenekleri bile olsa yine de yüz yüze eğitimi seçecekleri belirlenmiştir. Bununla birlikte öğrencilerin % 58,29’u eğitim gördükleri bölüme ait dersleri hem örgün eğitimle yüz yüze, hem de uzaktan eğitim sistemi ile birlikte (harmanlanmış eğitim olarak almayı tercih etmektedir. Öğrencilerin cinsiyet değişkenine göre uzaktan eğitim memnuniyet düzeyleri arasında anlamlı farklılık bulunamazken, gördükleri derslerin yürütülmesine ilişkin seçimleri ve bilgisayar kullanabilme bilgi düzeyleri arasında p <0,05 düzeyinde anlamlı farklılığın olduğu belirlenmiştir. Bununla birlikte öğrencilerin % 40,57’si şu an gördükleri eğitim programlarının uzaktan eğitim yönetim sistemiyle verilmesi için uygun olduğunu ifade ederken sadece % 18,29’u uygun olmadığı konusunda fikir beyan etmişlerdir.

  7. PKC-β抑制剂LY333531对糖尿病大鼠心肌功能及Caveolins蛋白的影响%Study on the effect of LY333531 on myocardiac function and expression of myocardial protein Caveolins in diabetic rats

    Institute of Scientific and Technical Information of China (English)

    雷少青; 苏娃婷; 徐金金; 刘慧敏; 夏中元; 夏正远; 徐波

    2014-01-01

    Objective To study the effect of LY333531(LY)on myocardiac function and expression of myocardial protein Caveolins in diabetic rats. Methods A total of 24 male SD rats were randomly divided into control group(C),diabetic group(D)and diabetic group with LY treatment for 4 weeks. The plasma levels of triglyceride(TG),inflammatory factor TNF - α and IL -6 were detected by biochemical analysis. The cardiac function was evaluated by echocardiography. The expression of Cav -1,Cav -3 and PKC - β2 were detected by Western blot. Results In comparison with group C,the plasma levels of TG,TNF - α and IL -6 in group D were significantly increased and accompanied with insulted cardiac diastolic function. The protein expression of PKC - β2 and Cav -1 in diabetic rats was up - regulated,but the protein expression of Cav - 3 was down - regulated as compared with that of control rats. The treatment with LY prevented or reversed all these changes without impacting plasma levels of TNF - α and IL -6. Conclusion Selective inhibition of PKC - β with LY can significantly improve diabetic cardiac diastolic function, it may be related to the inhibition of excessive activation of PKC - β2 and restore the expression of Caveolins.%目的观察PKC-β抑制剂LY333531(LY)对糖尿病大鼠心肌功能及Caveolins蛋白的影响。方法24只SD雄性大鼠随机分为正常对照组( C)、糖尿病组( D)及糖尿病LY治疗组。4周后生化检测血浆甘油三酯( TG)、炎症因子TNF-α与IL-6水平,心脏超声评价心脏功能,Western Blot分析心肌组织Cav-1、Cav-3,PKC-β2蛋白表达水平。结果与C组比较,D组大鼠血浆TG、TNF-α与IL-6水平均增高,心脏舒张功能降低,心肌组织p-PKC-β2、Cav-1表达水平增加而Cav-3表达水平降低。与D组比较,LY除了不能降低糖尿病大鼠血浆TNF-α与IL-6水平外,均可明显抑制上述变化。结论 PKC-β抑制剂LY明显改善糖尿病心肌舒张功能,其机制可能与抑制PKC

  8. STATUS POPULASI IKAN NAPOLEON DI WILAYAH TAMAN NASIONAL BUNAKEN DAN KABUPATEN KARAS FAK-FAK

    Directory of Open Access Journals (Sweden)

    Isa Nagib Edrus

    2016-03-01

    Full Text Available Penelitian ikan napoleon di perairan Bunaken pada Oktober 2012 dan Kabupaten Karas pada Nopember 2010 bertujuan untuk mengetahui status populasi ikan tersebut di bawah usaha perlindungan otoritas Taman Nasional Bunaken dan pasca penutupan penangkapannya di Karas.  Metode Underwater Visual Census digunakan untuk mendapatkan data jumlah individu dan ukuran tubuh ikan napoleon.  Hasil penelitian menunjukkan bahwa rata-rata kepadatan ikan napoleon dari luas wilayah sensus 19,26 hektar di Bunaken adalah 1,71 individu per hektar dan dari luas sensus 44,61 hektar di perairan Karas adalah 1,41 individu per hektar. Distribusi panjang frekuensi populasi ikan ini di kedua wilayah tersebut menunjukkan adanya dua kelompok umur, dimana interval ukuran panjang adalah antara 15 cm sampai 100 cm.  Perkembangan populasi ikan tersebut di Bunaken selama 7 tahun menunjukkan peningkatan sebesar 427,5%, sedang di Karas selama kurun waktu 5 tahun sebesar 298%. Namun pertumbuhan populasi sebesar ini ternyata belum masuk pada kategori status kelimpahan yang membaik. Jadi perlindungan ikan ini disarankan untuk lebih diperketat dengan cara melakukan moratorium.   Study on humphead wrasse fish in Bunaken and Karas district waters recpectively in October 2012 and November 2010 were aimed to determine population status of the fish under protecting regulations of the Bunaken National Park Authority and fishing closed session in Karas.  A method used to get population sizes and body sizes was the Napoleon Underwater Visual Census. The results showed that an average of fish density was 1,71 individual/ha of 19.26 ha in area census at Bunaken and it was 1,41 individual/ha of 44.61ha in area census of Karas waters. Distribution frequencies of the fish population were under two cohorts  with body size interval ranged from 15cm to 100 cm. Population trend during seven  years in Bunaken have increased in 427.5 % and those during five years in Karas have increase in 298%; however, such population growing has not be categorized yet as a good level of abundance status. Thus, the study suggested to strictly protect the humphead warsse by using a moratorium approach.

  9. D-Saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Semantee [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Manna, Prasenjit [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India); Gachhui, Ratan [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Sil, Parames C., E-mail: parames@bosemain.boseinst.ac.in [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India)

    2013-02-15

    Increasing evidence suggests that oxidative stress is involved in the pathogenesis of diabetic nephropathy (DN) and this can be attenuated by antioxidants. D-Saccharic acid 1,4-lactone (DSL) is known for its detoxifying and antioxidant properties. Our early investigation showed that DSL can ameliorate alloxan (ALX) induced diabetes mellitus and oxidative stress in rats by inhibiting pancreatic β-cell apoptosis. In the present study we, therefore, investigated the protective role of DSL against renal injury in ALX induced diabetic rats. ALX exposure (at a dose of 120 mg/kg body weight, i. p., once) elevated the blood glucose level, serum markers related to renal injury, the production of reactive oxygen species (ROS), and disturbed the intra-cellular antioxidant machineries. Oral administration of DSL (80 mg/kg body weight) restored all these alterations close to normal. In addition, DSL could also normalize the aldose reductase activity which was found to increase in the diabetic rats. Investigating the mechanism of its protective activity, we observed the activation of different isoforms of PKC along with the accumulation of matrix proteins like collagen and fibronectin. The diabetic rats also showed nuclear translocation of NF-κB and increase in the concentration of inflammatory cytokines in the renal tissue. The activation of mitochondria dependent apoptotic pathway was observed in the diabetic rat kidneys. However, treatment of diabetic rats with DSL counteracted all these changes. These findings, for the first time, demonstrated that DSL could ameliorate renal dysfunction in diabetic rats by suppressing the oxidative stress related signalling pathways. - Highlights: ► Sustained hyperglycemia and oxidative stress lead to diabetic renal injury. ► D-saccharic acid 1,4-lactone prevents renal damage in alloxan-induced diabetes. ► It restores intra-cellular antioxidant machineries and kidney apoptosis. ► DSL reduces hyperglycemia-mediated oxidative stress

  10. Human Peritoneal Mesothelial Cell Death Induced by High-Glucose Hypertonic Solution Involves Ca2+ and Na+ Ions and Oxidative Stress with the Participation of PKC/NOX2 and PI3K/Akt Pathways

    Directory of Open Access Journals (Sweden)

    Felipe Simon

    2017-06-01

    Full Text Available Chronic peritoneal dialysis (PD therapy is equally efficient as hemodialysis while providing greater patient comfort and mobility. Therefore, PD is the treatment of choice for several types of renal patients. During PD, a high-glucose hyperosmotic (HGH solution is administered into the peritoneal cavity to generate an osmotic gradient that promotes water and solutes transport from peritoneal blood to the dialysis solution. Unfortunately, PD has been associated with a loss of peritoneal viability and function through the generation of a severe inflammatory state that induces human peritoneal mesothelial cell (HPMC death. Despite this deleterious effect, the precise molecular mechanism of HPMC death as induced by HGH solutions is far from being understood. Therefore, the aim of this study was to explore the pathways involved in HGH solution-induced HPMC death. HGH-induced HPMC death included influxes of intracellular Ca2+ and Na+. Furthermore, HGH-induced HPMC death was inhibited by antioxidant and reducing agents. In line with this, HPMC death was induced solely by increased oxidative stress. In addition to this, the cPKC/NOX2 and PI3K/Akt intracellular signaling pathways also participated in HGH-induced HPMC death. The participation of PI3K/Akt intracellular is in agreement with previously shown in rat PMC apoptosis. These findings contribute toward fully elucidating the underlying molecular mechanism mediating peritoneal mesothelial cell death induced by high-glucose solutions during peritoneal dialysis.

  11. İ.Ü. Orman Fakültesi Araştırma Ormanı’nda Eylül 2007-Ağustos 2009 Dönemi Güncel Polen Dağılımı

    OpenAIRE

    Karlıoğlu, Nurgül; Akkemik, Ünal

    2012-01-01

    ÖzetÇalışma, İ.Ü. Orman Fakültesi Araştırma Ormanı’nda vejetasyonun çeşitlilik gösterdiği 3 örnek alanda (orman içi, orman kenarı ve açık alan) 2007-2009 yılları arasında toprağa düşen odunsu ve otsu bitki polen yoğunluğunu (cm2/ay) incelemek ve polen yoğunluğu değerlerinin önemli meteorolojik parametrelerle olan ilişkisini ortaya koymak amacıyla gerçekleştirilmiştir. Üç ayrı örnek alana Tauber tipi polen tuzakları yerleştirilmiş ve aylık olarak değiştirilmiştir. 2007-2008 yılına ait odunsu b...

  12. Phorbol Esters from Jatropha Meal Triggered Apoptosis, Activated PKC-δ, Caspase-3 Proteins and Down-Regulated the Proto-Oncogenes in MCF-7 and HeLa Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Syahida Ahmad

    2012-09-01

    Full Text Available Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs. The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7 and cervical (HeLa cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC50 of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC50 concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun. These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

  13. Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-δ, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines.

    Science.gov (United States)

    Oskoueian, Ehsan; Abdullah, Norhani; Ahmad, Syahida

    2012-09-10

    Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs). The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7) and cervical (HeLa) cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC₅₀ of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA) as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC₅₀ concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun). These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

  14. Key distribution in PKC through Quantas

    CERN Document Server

    Goel, Aditya

    2010-01-01

    Cryptography literally means "The art & science of secret writing & sending a message between two parties in such a way that its contents cannot be understood by someone other than the intended recipient". and Quantum word is related with "Light". Thus, Quantum Cryptography is a way of descripting any information in the form of quantum particles. There are no classical cryptographic systems which are perfectly secure. In contrast to Classical cryptography which depends upon Mathematics, Quantum Cryptography utilizes the concepts of Quantum Physics which provides us the security against the cleverest marauders of the present age. In the view of increasing need of Network and Information Security, we do require methods to overcome the Molecular Computing technologies (A future technology) and other techniques of the various codebrakers. Both the parts i.e. Quantum Key distribution and Information transference from Sender to Receiver are much efficient and secure. It is based upon BB84 protocol. It can b...

  15. IL-1通过PKC/MAPK激活蛋白激酶通路上调泡沫细胞ACAT-1的表达及活性%IL-1 increases expression and activity of ACAT-1 in foam cells via protein kinase C/mitogen activated protein kinase pathway

    Institute of Scientific and Technical Information of China (English)

    王庸晋; 王治平; 魏武; 周胜华

    2012-01-01

    Objective To investigate potential effect of interleukin-1 ( IL-1) of increasing Acyl-CoA; on cholesterol acyltransferase-1 (ACAT-1) expression and activity in foam cells by protein kinase C ( PKC )/mitogen-activated protein kinase (MAPK) signaling pathway. Methods Human monocytic leukemia cell line/(THP-l) was co-cultured with phorbol myristate acetate (PMA) and induced to differentiation into macrophages. The macrophages were cultured with oxidized low density lipoprotein (Ox-LDL) and induced to differentiate into foam cells. Oil red O staining was employed to examine cytoplasm lipid deposition. Activity of PKC was detected by Pep Tag assay for non-radioactive detection and MAPK activity was detected by Western blot in three different groups (the foam cells group, the foam cells IL-1 group, the foam cells IL-1/IL-1 monoclonal antibody group). PKC and MAPK inhibitors were separately added to the three different groups. Western blot and liquid phase scintillation counting were employed to examine the expression of ACAT-1 protein and ACAT-1 activity in different groups, respectively. Results THP-1 cells presented a suspension growth pattern, and showed significant changes in adhensive growth and oval shape. The PKC and MAPK activities in the the foam cells IL-1 group were higher than those in the foam cells group (P < 0. 05 ). The expression and activity of ACAT-1 in the the foam cells IL-1 group were higher than those in the foam cells group (P <0. 05). After added PKC and MAPK inhibitors, the up-regulation effects of IL-1 on the expression and activity of ACAT-1 were remarkably decreased (P <0. 05 ). Conclusion PKC/MAPK signaling pathway is involved in the IL-1 up-regulation effects on the ACAT-1 expression and activity in the the foam cells.%目的 研究白细胞介素1(IL-1)是否通过蛋白激酶C/丝裂原激活蛋白激酶(PKC/MAPK)信号通路上调泡沫细胞中脂酰CoA胆固醇酯酰转移酶-1(ACAT-1)的表达及活性.方法 复苏并培养人单

  16. 抗抑郁药物对慢性应激抑郁模型大鼠海马蛋白激酶C表达的干预作用%Effects of Fluoxetine and Tianeptine on the Expression of PKC in Hippocampus of Chronic Stress Depression Rats

    Institute of Scientific and Technical Information of China (English)

    吴枫; 孔令韬; 汤艳清

    2012-01-01

    Objective To investigate the effects of fluoxetine and tianeptine on the expression of PKC in hippocampus of chronic stress depression rats. Methods 60 male Wistar rats were randomly divided into depression group, fluoxetine group, tianeptine group and control group with each group 15 rats. The rats in depression group, fluoxetine group and tianeptine group were given stress stimulation for 21 days, and meanwhile rats in control group were kept normally. The rats in fluoxetine group were given fluoxetine by intragastric administration ( 10 mg/kg ), rats in tianeptine group were given tianeptine by intragastric administration ( 50 mg/kg ) . While the depression group and control group were given same volume of 0. 9% sodium chloride injection. The ethology examination was performed by using method of open - field and experiment of fluid consumption. The expression of PKC was detected by Western - blotting method. Results After the stress stimulation, horizontal crossing numbers, erection times, modification times and percentage of sacchar - consumption of the rats in depression group were significantly less than those of the control group ( P < 0. 01 ) . The horizontal crossing numbers, the erection times, the modification times and the percentage of sacchar - consumption of the rats in fluoxetine group were significantly less than those of the depression group ( P < 0. 05 ) . The percentage of sacchar - consumption of the rats in tianeptine group was significantly more than that of the depression group ( P < 0. 05 ) .In the test with Western - blotting method, the expression level of PKC in the hippocampus in depression group was significantly lower than that of the control group ( P <0. 01 ) . The expression level of PKC in the hippocampus in fluoxetine group was significantly higher than that of the depression group ( P < 0. 01 ), but no statistically significant difference showed between fluoxetine group and control group. The expression level of PKC in the

  17. Tumor-secreted LOXL2 Activates Fibroblasts Through FAK Signaling

    OpenAIRE

    Barker, Holly E.; Bird, Demelza; Lang, Georgina; Janine T. Erler

    2013-01-01

    Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown...

  18. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular...... fibroblasts, it was determined that expression of α-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared with control tumors which exhibited matrices with dense, parallel alignments....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α-SMA...

  19. Effect of changes of PKC activity on iNOS in posterior horn neurons of spinal cord in rats after formalin-induced inflammatory pain and hyperalgesia%甲醛足底致痛大鼠脊髓后角神经元内蛋白激酶C活性改变对一氧化氮合酶的影响

    Institute of Scientific and Technical Information of China (English)

    吕兴业

    2011-01-01

    目的 通过改变蛋白激酶C(protein kinase C,PKC)的活性观察其对甲醛炎性痛及痛觉过敏时大鼠脊髓后角一氧化氮合酶(nitric oxide synthase,NOS)尤其是对诱生型NOS(induciable NOS,iNOS)的影响,以探讨在该过程中PKC的作用机制.方法 将实验动物54只分为9组,每组6只,分男q为正常组、甲醛12 h组、甲醛24 h组、甲醛12 h加0.9%氯化钠溶液组、甲醛24 h加0.9%氯化钠溶液组、甲醛12 h加佛波醇脂(PMA)组、甲醛24 h加PMA组、甲醛12 h加灯盏花素乙(CH)组和甲醛24 h加CH组.先进行疼痛行为学检测,然后分别于注射甲醛后12、24 h将大鼠麻醉后取材,采用免疫组织化学方法观察脊髓后角iNOS阳性神经元数目及染色深度,以探讨PKC时NOS的影响.结果 与甲醛12 h组比较,甲醛12 h加PMA组iNOS阳性细胞数明显增加(P<0.01),神经细胞及神经纤维染色也明显加深(P<0.01);甲醛12 h加CH组iNOS阳性细胞数明显减少(P<0.01),神经细胞及神经纤维染色明显变浅(P<0.01).同时产生相应的行为痛觉过敏变化.结论 鞘内注射PKC兴奋荆PMA能显著增加L5脊髓后角神经元NOS的活性;鞘内注射PKC抑制剂CH能显著抑制L5脊髓后角神经元iNOS的活性.在甲醛炎性痛及痛觉过敏中,脊髓后角iNOS活性在一定程度上受PKC调控.%Objective To observe the effect of changes of protein kinase C (PKC) activity on nitric oxide synthase (NOS) and inducible NOS (iNOS) in posterior horn neurons of spinal cord in rats after formalin-induced inflammatory pain and hyperalgesia in order to explore the action mechanism of PKC during the process. Methods Fifty-four SD rats were randomly divided into 9 groups:control group, formalin 12h, formalin 24h, formalin 12h +NS,formalin 24 h + N, formalin 12h + PMA, formalin 24h + PMA, formalin 12h + CH and formalin 24h + CH groups. The mechanical and thermal pain threshold was measured by paw withdrawal latencies to yon Frey hair and radiant heat

  20. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  1. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  2. Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü Öğrencilerinin Eleştirel Düşünme Eğilimlerinin Çeşitli Değişkenlerle İlişkisinin İncelenmesi Investigation Of The Relationship Between Critical Thinking Dispositions And Miscellaneous Variables Of The Students Of The Faculty Of Education The Department Of Fine Arts Education

    Directory of Open Access Journals (Sweden)

    Onur TOPOĞLU

    2013-09-01

    students who are teacher candidates have lack ofcritical thinking abilities. One of the primary purposes of universityeducation is to educate the students to make them gained inquisitiveand analytically thinking abilities. Therefore, it is crucial that theteachers of tomorrow have to furnished with all those abilities to be ableto teach and pass the critical thinking abilities to their students. Bu çalışmada, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimleri ile cinsiyetleri, alan başarıları ve akademik başarıları arasındaki ilişkinin ortaya konması amaçlanmıştır. Çalışma, ilişkisel tarama modeline dayalı, betimsel bir araştırmadır. Çalışmada veri toplama aracı olarak kişisel bilgi formu ve Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimlerini ortaya koymak amacıyla ‘California Eleştirel Düşünme Eğilimleri Ölçeği’ (CCTDI kullanılmıştır. Çalışmanın evrenini, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümlerinde öğrenim görmekte olan öğrenciler oluştururken örneklemini 2011-2012 bahar yarıyılında Adnan Menderes Üniversitesi Eğitim Fakültesi Müzik Eğitimi Anabilim Dalı ve Resim-iş Anabilim Dalında öğrenim görmekte olan 204 öğrenci oluşturmaktadır. Araştırmanın sonucunda, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimlerinin, alanlarına ve cinsiyetlerine göre anlamlı farklılık göstermediği; ancak müzik bölümünde okuyan kız öğrencilerin açık fikirlilik ve doğruyu arama düzeylerinin erkeklere oranla önemli derecede yüksek olduğu, müzik öğrencilerinin eleştirel düşünme eğilimleri ile akademik başarıları ve alan başarıları arasında önemli ilişki olmadığı belirlenmiştir. Resim öğrencilerinin eleştirel düşünme eğilimleri ile akademik başarıları arasında pozitif yönde yüksek anlamlı ilişki, alan ba

  3. Akademisyenlerin Meslek Ahlakına Aykırı Olan Davranışlara İlişkin Algıları (Çomü Eğitim Fakültesi Örneği Academicians’ Perceptions Of Behaviours Against Occupational Ethics (A Case In Comu, Faculty Of Education

    Directory of Open Access Journals (Sweden)

    İlknur MAYA

    2013-07-01

    aninstitutional infrastructure, ethic values of an institution, questioningone’s conscience, values education and character education, andregular supervision. It was found that academicians experienced ethicaldilemmas between educational activities and research activities,between their work at university and willingness to work in privatesector, and between administrators’ pressures and their personal valuesin fulfilling their duties; respectively. Araştırmada, akademisyenlerin gerçekleştirdikleri eğitim-öğretim, araştırma ve topluma hizmet gibi etkinliklerde meslek ahlakına aykırı olarak algıladıkları davranışların neler olduğunun, nedenlerinin ve nasıl önlenebileceğinin belirlenmesi amaçlanmaktadır. Ayrıca çalışmada, akademisyenlerin yaşadıkları etik ikilemlerin neler olduğunun saptanmasına çalışılmaktadır. Araştırmada nitel araştırma yöntemlerinden yarı yapılandırılmış görüşme tekniği kullanılmıştır. Çalışma grubunu, Çanakkale Onsekiz Mart Üniversitesi Eğitim Fakültesinde görev yapmakta olan yüz akademisyenden kartopu örnekleme yoluyla seçilmiş elli kişi oluşturmaktadır. Verilerin analizinde, içerik analizi türlerinden tümevarımcı analiz kullanılmıştır. Akademisyenlerin meslek ahlakına aykırı olarak algıladıkları davranışlar, en çok eğitim-öğretim işleri daha sonra araştırma işlerinde görülmektedir. Akademisyenler, eğitim-öğretim işleri bakımından en çok mazeretsiz ders yapmamak, mazeretsiz derse geç girmek / dersten erken çıkmak, dersi etkili ve nitelikli olarak yürütmemek, üniversite dışında çalışma nedeniyle üniversitedeki sorumluluklarını yerine getirmemek ve öğrencilere eşit / adil davranmamak davranışlarını meslek ahlakına aykırı bulmaktadır. Akademisyenler, araştırma işleri bakımından en çok araştırmada alıntı ve kaynakları doğru vermemek, nitelikli ve özgün araştırmalar yapmamak ve araştırma verileri üzerinde oynamak

  4. SPAK deficiency corrects pseudohypoaldosteronism II caused by WNK4 mutation

    National Research Council Canada - National Science Library

    Chu, Pei-Yi; Cheng, Chih-Jen; Wu, Yi-Chang; Fang, Yu-Wei; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Yang, Sung-Sen; Lin, Shih-Hua

    2013-01-01

    .../+) mice were crossed with kidney tubule-specific (KSP) Osr1 knockout (KSP-Osr1 (-/-)) and Spak knockout (Spak (-/-)) mice. Blood pressure, plasma and urine biochemistries, and the relevant protein expression in the kidneys were examined...

  5. Contribution of new technologies to university education: Opinions of communication faculty students on augmented reality applicationsYeni teknolojilerin üniversite eğitimine katkısı: İletişim fakültesi öğrencilerinin artırılmış gerçeklik uygulamalarına ilişkin görüşleri

    Directory of Open Access Journals (Sweden)

    İdil Sayımer

    2015-12-01

    ğitim ve öğrenme üzerindeki olumlu etkileri nedeniyle eğitim süreçlerinde de yaygınlaşması kaçınılmaz olacaktır. AG’nin gerçek olanla sanal olanı bütünleştirme özelliği taşımasına bağlı olarak eğitim sürecinde görselliği ve yaratıcılığı temel alarak teori ile pratik arasındaki boşluğu doldurduğu, dolayısıyla öğrenimi kolaylaştırıp derinleştirdiği yönünde yeni çalışmalar mevcuttur. Bu çalışmada, Türkiye’de iletişim fakültesi öğrencilerinin AG uygulamaları hakkındaki farkındalık ve ilgi düzeyleri ile uygulama becerilerini keşfetmek amaçlanmıştır. Eğitim öğretim sürecinin taraflarından biri olan öğrencilere yönelik öğrenci merkezli yeni öğretim planlamaları yapabilmek için bu grubun düşünceleri ve teknoloji kullanma becerilerini saptamak önemlidir. Buradan hareketle görsel teknolojileri kullanan ve yaratıcılığın temel alındığı disiplinlerin yer aldığı iletişim fakültesi lisans öğrencileri üzerine bir araştırma yapılmıştır. Araştırmanın örneklemi Kocaeli Üniversitesi İletişim Fakültesi'nde bulunan beş bölümün öğrencilerinden oluşmaktadır. Veriler öğrenciler tarafından 2015 Güz dönemi başlangıcında doldurulan çevrim içi anket aracılığıyla toplanmıştır. Araştırmanın bulguları farklı bölümlerde öğrenim gören öğrencilerin konuya yaklaşımları arasındaki farklılıkları da ortaya koyan karşılaştırmalı bir çalışma olarak değerlendirilmektedir. Çalışmada bulgular çerçevesinde yapılan değerlendirme ile birlikte AG’nin öğrenim sürecine katkıları konusunda da öneriler yer almaktadır.

  6. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    Science.gov (United States)

    2015-11-01

    interesting fact is that up-regulation of PKC occurs in human cancer, as extensively demonstrated in prostate, breast, lung , ovarian, and head and...neck cancer [14, 19, 22-27, 33]. PKC is essentially undetectable in normal prostate epithelium or benign prostatic epithelium , however it is highly...and lung cancer[19, 23, 26, 32-33]. Regardless, it is not yet clear if PKC overexpression is causally related to the initiation and progression of

  7. PKC-δ activation in neutrophils promotes fungal clearance.

    Science.gov (United States)

    Li, Xun; Cullere, Xavier; Nishi, Hiroshi; Saggu, Gurpanna; Durand, Enrique; Mansour, Michael K; Tam, Jenny M; Song, Xiu-Yu; Lin, Xin; Vyas, Jatin M; Mayadas, Tanya

    2016-09-01

    The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ-deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

  8. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    Science.gov (United States)

    2014-09-01

    Malik, A., Zaman, N., Sarfaraz, S., Siddiqui, I. A., Syed, D. N. et al (2007). Combined inhibitory effects of green tea polyphenols and selective...not only in prostate cancer but also in several other epithelial cancers including lung , breast, and thyroid cancer8, 13, 20-26. Studies from our...Cvarepsilon is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes. Oncogene 31: 2593-2600. 23 Hafeez, B. B

  9. Yeditepe Üniversitesi Diş Hekimliği Fakültesi öğrencilerinin, kan ve vücut sıvılarıyla bulaşan enfeksiyonlar, enfeksiyon kontrolü ve hepatit B enfeksiyonu ile ilgili bilgi düzeylerinin, tutumlarının ve hepatit B aşılanma ve serolojik durumlarının değerlen

    Directory of Open Access Journals (Sweden)

    Kemal Şençift

    2011-05-01

    Full Text Available

     

     

    Amaç: Bu çalışmanın amacı Yeditepe Üniversitesi Diş Hekimliği Fakültesi öğrencilerinin kan ve vücut sıvılarıyla bulaşan enfeksiyonlar, enfeksiyon kontrol yöntemleri ve hepatit B virüsü (HBV enfeksiyonu ile ilgili bilgi düzeylerini ve tutumlarını değerlendirmek, ve HBV aşılanma ve serolojik durumlarını incelemektir.

    Gereç ve Yöntem: Yeditepe Üniversitesi Diş Hekimliği Fakültesi’nde, 2010-2011 yılında eğitim gören 261 öğrenciye, HBV bilgi düzeyleri, aşılanma durumları ve enfeksiyon kontrolü konusundaki 52 sorudan oluşan anket formları dağıtıldı. Sonuçlar, tanımlayıcı istatistiksel metodlar, Student t testi,  Ki-Kare testi ve Fisher’s Exact Ki-Kare testi kullanılarak değerlendirildi. Veriler p<0,05 anlamlılık düzeyinde değerlendirildi.

    Bulgular: Yaş ortalaması 21,29 olan öğrencilerin 123’ü (%47,1 kendilerini HBV açısından risk grubunda görmekteydi. Öğrencilerin 207’sinin (%79,3 HBV aşısı yapt

  10. 电针"内关"穴对心肌缺血大鼠心肌氯离子通道相关基因表达的影响%Effect of Electroacupuncture Stimulation of "Neiguan" (PC 6) on Expression of Myocardial Chloride Channel-related Genes, PKC and PKG Proteins in Myocardial Ischemia Rats

    Institute of Scientific and Technical Information of China (English)

    白增华; 吴兆利; 苏妆; 丛培玮; 陈以国; 李春日; 武林

    2015-01-01

    目的:观察电针"内关"穴对心肌缺血大鼠心肌氯离子通道调控基因表达的影响,探讨经穴效应特异性的作用机制.方法.SD大鼠随机分为正常对照组10只,模型组、内关穴组、列缺穴组、非经非穴组各15只.皮下注射异丙肾上腺素建立心肌缺血模型.内关穴组取"内关"穴,列缺穴组取"列缺"穴、非经非穴组取"天枢"与"神阙"连线中点进行电针治疗,每日治疗1次,治疗7d.Western blot法和Real time-PCR法分别检测心肌组织蛋白激酶C(PKC)和氯离子通道基因囊性纤维化跨膜传导调节因子(CFTR)及钙激活氯通道(CLCa 1)的基因表达.结果:与正常对照组比较,模型组大鼠心肌PKC、CLCa l、CFTR表达升高(P<0.05).与模型组比较,内关穴组、列缺穴组及非经非穴组大鼠心肌PKC表达明显下降(P<0.05),内关穴组和列缺穴组与非经非穴组比较PKC表达下降(P<0.05).与模型组比较,内关穴组、列缺穴组及非经非穴组心肌CLCa l基因表达显著降低(P<0.05),列缺穴组、非经非穴组与内关穴组比较显著升高(P<0.05),列缺穴组与非经非穴组比较显著下降(P<0.05).与模型组比较,内关穴组、列缺穴组CFTR基因表达显著下降(P<0.05),列缺穴组、非经非穴组与内关穴组比较显著升高(P<0.05),而列缺穴组与非经非穴组比较显著下降(P<0.05).结论:电针不同穴位对心肌缺血大鼠心肌PKC、CFTR、CLCa 1的表达有不同的影响,"内关"穴具有特异性效应.

  11. Gazi Üniversitesi Gazi Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü Müzik Eğitimi Anabilim Dalı Keman Öğrencilerinin Aldıkları Keman Eğitiminde Karşılaştıkları Sorunlar ve Sorunları Çözmede İzledikleri Yollar Problems Encountered by Violin Students From Gazi University Faculty of Education Department of Education of Fine Arts Department of Music Education During Violin Education and Methods to be Followed to Solve These Problems

    Directory of Open Access Journals (Sweden)

    Gamze Elif TANINMIŞ

    2013-07-01

    Full Text Available This research, aims to determine problems encountered by violinstudents from Gazi University Faculty of Education Department ofEducation of Fine Arts Department of Music Education in the course ofviolin education and methods to be followed to solve these problems.This research is a descriptive work in terms of its aim and quality of thedata collected in accordance with this aim. Resources that are relevantto this topic have been reviewed and 87 undergraduate violin studentspursuing their education in Gazi University Faculty of EducationDepartment of Fine Arts Department of Music Education in academicyear 2011-12 have been surveyed in an effort to designate what kind ofproblems they encounter during their violin education and methodsthey follow to cope with them. The data acquired as results of thesurvey are presented and contrued in the form of charts. Inconsideration of the data, not to begin to start playing violin in an earlyage, not to allocate enough time for daily individual violin practice andinadequacy of course hours are seen as three most important problems.Three most important problems they experience while learning teachingmethods such as practices, etudes, works of art, etc. are the onesassociated with the use of bow, bowing techniques and double stop. It ispointed out that a clear majority of students play the musical pieces inparts, not throughly and receive help from their instructors in order tosolve the problems they encounter when they study teaching methodssuch as practices, etudes, works of art, etc. and instructor/s theyreceive help from find/s solution to their problems at the rate of 68.96. Bu araştırma, Gazi Üniversitesi Gazi Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü Müzik Eğitimi Anabilim Dalı keman öğrencilerinin aldıkları keman eğitiminde karşılaştıkları sorunları ve sorunları çözmede izledikleri yolları tespit etmeyi amaçlamaktadır. Araştırma, taşıdığı amaç ve bu amaca uygun

  12. Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK

    DEFF Research Database (Denmark)

    Baker, A-M; Bird, D; Lang, G;

    2013-01-01

    activity is required for LOX-mediated effects on proliferation and invasion in both in vitro and in vivo models of CRC. Furthermore, we use rheology to measure the relative stiffness of modified collagen matrices and subcutaneous tumors, and show that LOX-induced collagen cross-linking results...

  13. Blocking FAK Signaling in the Tumor or Stroma and Effects on Metastasis

    Science.gov (United States)

    2010-04-01

    PEG400, polyethylene glycol 400; PI, propidium iodide; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis...directional cell movement.2,3 To assess if PND-1186 would inhibit 4T1 cell migration, time lapse wound healing assays were performed in the presence...using vernier calipers and determined by length x width2/2) were grouped (n = 8 per group) and PND-1186 solubilized in polyeth- ylene glycol 400 (PEG400

  14. BALKAN DEVLETLERİNİN İTTİFAK ARAYIŞI VE OSMANLI DEVLETİ

    OpenAIRE

    ZEYREK, Suat

    2013-01-01

    IN SEARCH OF ALLIANCE OF BALKAN STATES AND OTTOMAN EMPIRE In the 19th century European politics was shaped by “balance policy” and European Powers, led by England, was keen to protect the territorial integrity of the Ottoman Empire. As Germany emerged to be a new challenging power in Europe, the demarcation of power blocs in the continent became more obvious. In the aftermath of the Treaty of Berlin, Western Powers, including Germany, encouraged minorities in the Ottoman Empire to form their ...

  15. The influence of extra framework aluminium on H-fak catalyzed cracking of light alkanes

    NARCIS (Netherlands)

    Narbeshuber, T.; Narbeshuber, T.F.; Brait, A.; Brait, A.; Seshan, Kulathuiyer; Lercher, J.A.

    1996-01-01

    The conversion of light linear and branched alkanes on two faujasite samples containing different concentrations of free Brønsted acid sites and extraframework alumina (EFAL) was studied between 733 K and 813 K. Protolytic cracking and bimolecular hydride transfer proceeded solely on Brønsted acid

  16. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    Science.gov (United States)

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  17. SPAK deficiency corrects pseudohypoaldosteronism II caused by WNK4 mutation.

    Science.gov (United States)

    Chu, Pei-Yi; Cheng, Chih-Jen; Wu, Yi-Chang; Fang, Yu-Wei; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Yang, Sung-Sen; Lin, Shih-Hua

    2013-01-01

    Stimulation of the OSR1 (Oxidative stress-responsive kinase-1)/SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase]-NCC (Na(+)-Cl(-) cotransporter) signaling cascade plays an important role in the WNK [With-No-Lysine (K)] kinase 4 D561A knock-in mouse model of pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. The aim of this study was to investigate the respective roles of Osr1 and Spak in the pathogenesis of PHA II in vivo. Wnk4 (D561A/+) mice were crossed with kidney tubule-specific (KSP) Osr1 knockout (KSP-Osr1 (-/-)) and Spak knockout (Spak (-/-)) mice. Blood pressure, plasma and urine biochemistries, and the relevant protein expression in the kidneys were examined. Wnk4 (D561A/+), KSP-Osr1 (-/-), and Spak (-/-) mice recapitulated the phenotypes of PHA II, Bartter-like syndrome, and Gitelman syndrome, respectively. Wnk4 (D561A/+).KSP-Osr1 (-/-) remained phenotypically PHA II while Wnk4 (D561A/+).Spak (-/-) mice became normotensive and lacked the PHA II phenotype. Phosphorylated Spak and Ncc were similarly increased in both Wnk4 (D561A/+) and Wnk4 (D561A/+).KSP-Osr1 (-/-) mice while phosphorylated Ncc normalized in Wnk4 (D561A/+).Spak (-/-) mice. Furthermore, Wnk4 (D561A/+).KSP-Osr1 (-/-) mice exhibited exaggerated salt excretion in response to thiazide diuretics while Wnk4 (D561A/+).Spak (-/-) mice exhibited normal responses. Wnk4(D561A/+).Spak (-/-).KSP-Osr1 (-/-) triple mutant mice had low blood pressure and diminished phosphorylated Ncc. Both SPAK and OSR1 are important in the maintenance of blood pressure but activation of SPAK-NCC plays the dominant role in PHA II. SPAK may be a therapeutic target for disorders with salt-sensitive hypertension related to WNK4 activation.

  18. SPAK deficiency corrects pseudohypoaldosteronism II caused by WNK4 mutation.

    Directory of Open Access Journals (Sweden)

    Pei-Yi Chu

    Full Text Available Stimulation of the OSR1 (Oxidative stress-responsive kinase-1/SPAK [STE20 (sterile 20/SPS1-related proline/alanine-rich kinase]-NCC (Na(+-Cl(- cotransporter signaling cascade plays an important role in the WNK [With-No-Lysine (K] kinase 4 D561A knock-in mouse model of pseudohypoaldosteronism type II (PHA II characterized by salt-sensitive hypertension and hyperkalemia. The aim of this study was to investigate the respective roles of Osr1 and Spak in the pathogenesis of PHA II in vivo. Wnk4 (D561A/+ mice were crossed with kidney tubule-specific (KSP Osr1 knockout (KSP-Osr1 (-/- and Spak knockout (Spak (-/- mice. Blood pressure, plasma and urine biochemistries, and the relevant protein expression in the kidneys were examined. Wnk4 (D561A/+, KSP-Osr1 (-/-, and Spak (-/- mice recapitulated the phenotypes of PHA II, Bartter-like syndrome, and Gitelman syndrome, respectively. Wnk4 (D561A/+.KSP-Osr1 (-/- remained phenotypically PHA II while Wnk4 (D561A/+.Spak (-/- mice became normotensive and lacked the PHA II phenotype. Phosphorylated Spak and Ncc were similarly increased in both Wnk4 (D561A/+ and Wnk4 (D561A/+.KSP-Osr1 (-/- mice while phosphorylated Ncc normalized in Wnk4 (D561A/+.Spak (-/- mice. Furthermore, Wnk4 (D561A/+.KSP-Osr1 (-/- mice exhibited exaggerated salt excretion in response to thiazide diuretics while Wnk4 (D561A/+.Spak (-/- mice exhibited normal responses. Wnk4(D561A/+.Spak (-/-.KSP-Osr1 (-/- triple mutant mice had low blood pressure and diminished phosphorylated Ncc. Both SPAK and OSR1 are important in the maintenance of blood pressure but activation of SPAK-NCC plays the dominant role in PHA II. SPAK may be a therapeutic target for disorders with salt-sensitive hypertension related to WNK4 activation.