Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress

2003 ◽  
Vol 285 (2) ◽  
pp. C334-C342 ◽  
Author(s):  
Satoshi Numazawa ◽  
Makie Ishikawa ◽  
Aya Yoshida ◽  
Sachiko Tanaka ◽  
Takemi Yoshida
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Kinase Assay ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Kinase C

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769–42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12- O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCι activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCι, but not by a kinase inactive mutant aPKCι(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCι, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCι(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in the nuclear translocation and ARE transactivation of Nrf2 by oxidative stress.

scholarly journals The Skin-Whitening Effects of Ectoine via the Suppression of α-MSH-Stimulated Melanogenesis and the Activation of Antioxidant Nrf2 Pathways in UVA-Irradiated Keratinocytes

Antioxidants ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 63 ◽  
Author(s):  
You-Cheng Hseu ◽  
Xuan-Zao Chen ◽  
Yugandhar Vudhya Gowrisankar ◽  
Hung-Rong Yen ◽  
Jing-Yuan Chuang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Hacat Cells ◽  
Skin Whitening ◽  
B16f10 Cells ◽  
Kinase C ◽  
Protein Kinase Ckii

Ultraviolet A (UVA)-irradiation induced reactive oxygen species (ROS) production mediates excessive melanogenesis in skin cells leading to pigmentation. We demonstrated the depigmenting and anti-melanogenic effects of Ectoine, a natural bacterial osmolyte, in UVA-irradiated human (HaCaT) keratinocytes, and the underlying molecular mechanisms were elucidated. HaCaT cells were pre-treated with low concentrations of Ectoine (0.5–1.5 μM) and assayed for various depigmenting and anti-melanogenic parameters. This pre-treatment significantly downregulated ROS generation, α-melanocyte-stimulating hormone (α-MSH) production, and proopiomelanocortin (POMC) expression in UVA-irradiated HaCaT cells. Also, antioxidant heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase [quinone 1] (NQO-1), and γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) protein expressions were mediated via the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) whose knockdown indeed impaired this effect signifying the importance of the Nrf2 pathway. Ectoine was mediating the activation of Nrf2 via the p38, protein kinase B (also known as AKT), protein kinase C (PKC), and casein kinase II protein kinase (CKII) pathways. The conditioned medium obtained from the Ectoine pre-treated and UVA-irradiated HaCaT cells downregulated the tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1/-2), cyclic AMP (c-AMP) protein kinase, c-AMP response element-binding protein (CREB), and microphthalmia-associated transcription factor (MITF) expressions leading to melanoma B16F10 cells having inhibited melanin synthesis. Interestingly, this anti-melanogenic effect in α-MSH-stimulated B16F10 cells was observable only at 50–400 μM concentrations of Ectoine, signifying the key role played by Ectoine (0.5–1 μM)-treated keratinocytes in skin whitening effects. We concluded that Ectoine could be used as an effective topical natural cosmetic agent with depigmenting and anti-melanogenic efficacy.

scholarly journals Propofol Inhibits Ischemia/Reperfusion-Induced Cardiotoxicity Through the Protein Kinase C/Nuclear Factor Erythroid 2-Related Factor Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Shengqiang Li ◽  
Zhen Lei ◽  
Meng Zhao ◽  
Yonghao Hou ◽  
Di Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ischemia Reperfusion ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Nuclear Factor ◽  
H9c2 Cells ◽  
Nrf2 Pathway ◽  
Kinase C ◽  
Creatine Kinase Mb

Both hydrogen peroxide (H2O2, H) and ischemia/reperfusion (I/R) can damage cardiomyocytes, which was inhibited by propofol (P). The present research was designed to examine whether propofol can reduce myocardial I/R injury by activating protein kinase C (PKC)/nuclear factor erythroid-2-related factor 2 (NRF2) pathway in H9C2 cells and rat Langendorff models. H9C2 cells were disposed of no reagents (C), H2O2 for 24 h (H), propofol for 1 h before H2O2 (H+P), and chelerythrine (CHE, PKC inhibitor) for 1 h before propofol and H2O2 (H+P+CHE). N = 3. The PKC gene of H9C2 was knocked down by siRNA and overexpressed by phorbol 12-myristate 13-acetate (PMA, PKC agonist). The cell viability and the expressions of PKC, NRF2, or heme oxygenase-1(HO-1) were evaluated. Propofol significantly reduced H9C2 cell mortality induced by H2O2, and significantly increased NRF2 nuclear location and HO-1 expression, which were restrained by siRNA knockout of PKC and promoted by PMA. Rat hearts were treated with KrebsHenseleit solution for 120 min (C), with (I/R+P) or without (I/R) propofol for 20 min before stopping perfusion for 30 min and reperfusion for 60 min, and CHE for 10 min before treated with propofol. N = 6. The levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and creatine kinase-MB (CK-MB) in perfusion fluid and antioxidant enzymes in the myocardium were assessed. I/R, which increased LDH and CK-MB expression and reduced SOD expression, boosted the pathological damage and infarcts of the myocardium after reperfusion. However, propofol restrained all these effects, an activity that was antagonized by CHE. The results suggest that propofol pretreatment protects against I/R injury by activating of PKC/NRF2 pathway.

scholarly journals Sanghuangporus sanghuang Mycelium Prevents Paracetamol-Induced Hepatotoxicity through Regulating the MAPK/NF-κB, Keap1/Nrf2/HO-1, TLR4/PI3K/Akt and CaMKKβ/LKB1/AMPK Pathways and Suppressing Oxidative Stress and Inflammation

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 897
Author(s):  
Wen-Ping Jiang ◽  
Jeng-Shyan Deng ◽  
Shyh-Shyun Huang ◽  
Sheng-Hua Wu ◽  
Chin-Chu Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Liver Failure ◽  
Acute Liver Failure ◽  
Mitogen Activated Protein Kinase ◽  
Heme Oxygenase 1 ◽  
Molecular Evidence ◽  
Related Factor ◽  
Serum Aminotransferase ◽  
Compound C

Liver damage induced by paracetamol overdose is the main cause of acute liver failure worldwide. In order to study the hepatoprotective effect of Sanghuangporus sanghuang mycelium (SS) on paracetamol-induced liver injury, SS was administered orally every day for 6 days in mice before paracetamol treatment. SS decreased serum aminotransferase activities and the lipid profiles, protecting against paracetamol hepatotoxicity in mice. Furthermore, SS inhibited the lipid peroxidation marker malondialdehyde (MDA), hepatic cytochrome P450 2E1 (CYP2E1), and the histopathological changes in the liver and decreased inflammatory activity by inhibiting the production of proinflammatory cytokines in paracetamol-induced acute liver failure. Moreover, SS improved the levels of glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase in the liver. Significantly, SS diminished mitogen-activated protein kinase (MAPK), Toll-like receptor 4 (TLR4), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and the nuclear factor-kappa B (NF-κB) axis, as well as upregulated the Kelch-like ECH-associated protein 1 (Keap1)/erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, in paracetamol-induced mice. SS mainly inhibited the phosphorylation of the liver kinase B1 (LKB1), Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ), and AMP-activated protein kinase (AMPK) protein expression. Furthermore, the protective effects of SS on paracetamol-induced hepatotoxicity were abolished by compound C, an AMPK inhibitor. In summary, we provide novel molecular evidence that SS protects liver cells from paracetamol-induced hepatotoxicity by inhibiting oxidative stress and inflammation.

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C

2003 ◽  
Vol 35 (6) ◽  
pp. 683-694 ◽  
Author(s):  
Flavia Pricci ◽  
Gaetano Leto ◽  
Lorena Amadio ◽  
Carla Iacobini ◽  
Samantha Cordone ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Endothelial Dysfunction ◽  
Kinase C

scholarly journals Ca2+-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2+-induced secretory response is accompanied by loss of Ca2+-induced protein phosphorylation

1992 ◽  
Vol 285 (3) ◽  
pp. 973-978 ◽  
Author(s):  
P M Jones ◽  
S J Persaud ◽  
S L Howell
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Prolonged Exposure ◽  
Kinase C ◽  
Dependent Protein ◽  
32P Incorporation ◽  
Rat Islets Of Langerhans

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide

2010 ◽  
Vol 299 (2) ◽  
pp. G320-G328 ◽  
Author(s):  
Claudia Stross ◽  
Angelika Helmer ◽  
Katrin Weissenberger ◽  
Boris Görg ◽  
Verena Keitel ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bile Salt ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Sodium Taurocholate ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Salt Uptake ◽  
Kinase C

Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca2+-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na+-K+-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 ± 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca2+-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

Protein kinase C-ζ modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt

2002 ◽  
Vol 282 (1) ◽  
pp. H320-H327 ◽  
Author(s):  
Yukitaka Shizukuda ◽  
Peter M. Buttrick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adult Rat ◽  
Kinase C ◽  
Adult Cardiac Myocytes ◽  
Pkc Ζ

We hypothesized that thromboxane A2 (TxA2) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a TxA2 mimetic [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a TxA2 receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. TxA2 stimulation resulted in membrane translocation of PKC-ζ but not PKC-α, -βII, -δ, and -ε at 3 min and 1 h. The activation of PKC-ζ by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-ζ pseudosubstrate peptide (ζ-PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with ζ-PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of ζ-PS. Therefore, our results suggest that the activation of PKC-ζ modulates TxA2 receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes.

Mechanism of Hepatic Heme Oxygenase-1 Induction by Isoflurane

2006 ◽  
Vol 104 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Alexander Hoetzel ◽  
Daniel Leitz ◽  
Rene Schmidt ◽  
Eva Tritschler ◽  
Inge Bauer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Heme Oxygenase ◽  
Kupffer Cell ◽  
Cell Function ◽  
Heme Oxygenase 1 ◽  
Gadolinium Chloride ◽  
Kinase C

Background The heme oxygenase pathway represents a major cell and organ protective system in the liver. The authors recently showed that isoflurane and sevoflurane up-regulate the inducible isoform heme oxygenase 1 (HO-1). Because the activating cascade remained unclear, it was the aim of this study to identify the underlying mechanism of this effect. Methods Rats were anesthetized with pentobarbital intravenously or with isoflurane per inhalation (2.3 vol%). Kupffer cell function was inhibited by dexamethasone or gadolinium chloride. Nitric oxide synthases were inhibited by either N(omega)-nitro-L-arginine methyl ester or S-methyl thiourea. N-acetyl-cysteine served as an antioxidant, and diethyldithiocarbamate served as an inhibitor of cytochrome P450 2E1. Protein kinase C and phospholipase A2 were inhibited by chelerythrine or quinacrine, respectively. HO-1 was analyzed in liver tissue by Northern blot, Western blot, immunostaining, and enzymatic activity assay. Results In contrast to pentobarbital, isoflurane induced HO-1 after 4-6 h in hepatocytes in the pericentral region of the liver. The induction was prevented in the presence of dexamethasone (P < 0.05) and gadolinium chloride (P < 0.05). Inhibition of nitric oxide synthases or reactive oxygen intermediates did not affect isoflurane-mediated HO-1 up-regulation. In contrast, chelerythrine (P < 0.05) and quinacrine (P < 0.05) resulted in a blockade of HO-1 induction. Conclusion The up-regulation of HO-1 by isoflurane in the liver is restricted to parenchymal cells and depends on Kupffer cell function. The induction is independent of nitric oxide or reactive oxygen species but does involve protein kinase C and phospholipase A2.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

2011 ◽  
Vol 22 (24) ◽  
pp. 4908-4917 ◽  
Author(s):  
Deepti Gadi ◽  
Alice Wagenknecht-Wiesner ◽  
David Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Fluorescent Protein ◽  
Immunoglobulin E ◽  
Dependent Manner ◽  
Effector Domain ◽  
Granule Exocytosis ◽  
Kinase C

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

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