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• W2109971777 abstract "A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(−/−) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(−/−) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cδ, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity. A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(−/−) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(−/−) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cδ, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity. The free radical theory of aging predicts that oxygen-derived free radicals produced throughout life cause progressive damage and inflammation, ultimately leading to death (1Harman D. J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6606) Google Scholar). In the long-lived p66Shc-deficient mouse, embryonic fibroblasts produce less ROS and are more resistant to stressors, including hydrogen peroxide, and signal less through ROS-dependent pathways (2Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1479) Google Scholar, 3Berniakovich I. Trinei M. Stendardo M. Migliaccio E. Minucci S. Bernardi P. Pelicci P.G. Giorgio M. J. Biol. Chem. 2008; 283: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). p66Shc-deficient mice produce less mitochondrial ROS following CCl4 stimulation (4Giorgio M. Migliaccio E. Orsini F. Paolucci D. Moroni M. Contursi C. Pelliccia G. Luzi L. Minucci S. Marcaccio M. Pinton P. Rizzuto R. Bernardi P. Paolucci F. Pelicci P.G. Cell. 2005; 122: 221-233Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar). p66Shc KO mice also have reduced systemic and tissue oxidative stress (5Napoli C. Martin-Padura I. de Nigris F. Giorgio M. Mansueto G. Somma P. Condorelli M. Sica G. De Rosa G. Pelicci P. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2112-2116Crossref PubMed Scopus (332) Google Scholar); are resistant to atherosclerosis (6Martin-Padura I. de Nigris F. Migliaccio E. Mansueto G. Minardi S. Rienzo M. Lerman L.O. Stendardo M. Giorgio M. De Rosa G. Pelicci P.G. Napoli C. Endothelium. 2008; 15: 276-287Crossref PubMed Scopus (46) Google Scholar), oxidant-related endothelial dysfunction (7Camici G.G. Schiavoni M. Francia P. Bachschmid M. Martin-Padura I. Hersberger M. Tanner F.C. Pelicci P. Volpe M. Anversa P. Lüscher T.F. Cosentino F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5217-5222Crossref PubMed Scopus (204) Google Scholar), kidney oxidant injury (8Menini S. Iacobini C. Ricci C. Oddi G. Pesce C. Pugliese F. Block K. Abboud H.E. Giorgio M. Migliaccio E. Pelicci P.G. Pugliese G. Diabetologia. 2007; 50: 1997-2007Crossref PubMed Scopus (55) Google Scholar); and are protected from high fat diet-induced obesity (3Berniakovich I. Trinei M. Stendardo M. Migliaccio E. Minucci S. Bernardi P. Pelicci P.G. Giorgio M. J. Biol. Chem. 2008; 283: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We carried out a microarray study that indicated alterations in transcripts related to heme and NADPH oxidase superoxide production and thus investigated the impact of the p66Shc deficiency on ROS-generating activity of macrophages. p66Shc(−/−) mice have been described previously (2Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1479) Google Scholar). Mice were kept pathogen-free through the study at a barrier facility at the University of California (Davis, CA). All experimental procedures were approved by the Institutional Animal Care and Use Committee and were performed in compliance with local, state, and federal regulations. Mice used for this study were 2–6 months old and were age-matched for each experiment. Diphenyleneiodonium (DPI) 2The abbreviations used are: DPIdiphenyleneiodoniumMAPKmitogen-activated protein kinasePI3Kphosphoinositide 3-kinasePMAphorbol 12-myristate 13-acetatefMLPN-formyl-Met-Leu-PheAAarachidonic acidPMperitoneal macrophage(s)PBSphosphate-buffered salineFBSfetal bovine serumSODsuperoxide dismutasesiRNAsmall interfering RNACHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGTPγSguanosine 5′-O-(thiotriphosphate)PHOXNADPH oxidasePKCprotein kinase CERKextracellular signal-regulated kinaseKOknock-outPAKp21-activated kinaseGPCRG protein-coupled receptorPIP3phosphatidylinositol 3′,4′,5′-trisphosphateMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseH2FFdihydro2′4,5,6,7,7′hexafluorofluorosceinBSAbovine serum albuminIRinfrared. and gliotoxin were purchased from Axxora LLC (San Diego, CA), phorbol 12-myristate 13-acetate (PMA) was from Enzo Life Sciences International Inc. (Plymouth Meeting, PA); N-formyl-Met-Leu-Phe (fMLP) was from Tocris Bioscience (Ellisville, MO); arachidonic acid (AA) was from Acros Organics (Morris Plains, NJ); OxyBURST Green H2HFF-BSA dye was purchased from Molecular Probes, Inc. (Eugene, OR); goat anti-p22phox antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit anti-p40phox polyclonal and rabbit anti-p67phox polyclonal antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY); rabbit anti-phospho-p40phox polyclonal antibody was from Cell Signaling Technology Inc. (Danvers, MA); goat anti-p47phox polyclonal, goat anti-Rac2 polyclonal, mouse anti-PKCδ polyclonal, and rabbit anti-phospho-PKCδ monoclonal antibody were from Abcam Inc. (Cambridge, MA); rabbit anti-Shc polyclonal antibodies and mouse anti-gp91phox antibody were from BD Biosciences; Rac/Cdc42 assay reagent PAK-1 p21-binding domain-agarose conjugate and mouse monoclonal anti-Rac1 antibody were from Millipore Inc. (Temecula, CA); and goat anti-rabbit monoclonal antibody labeled with infrared (IR) dye 700CW, donkey anti-mouse monoclonal antibody labeled with IR dye 800CW, and donkey anti-goat polyclonal antibodies conjugated with IR dye 800CW were from Li-Cor Biosciences (Lincoln, NE). Primers for quantification of p66Shc forward (5′-gaaagttggggcggtgac-3′) and reverse (5′-gacccattctgcctcctc-3′), actin β forward (5′-tggaacggtgaaggcgacagcagttg-3′) and reverse (5′-gtggcttttgggagggtgagggactt-3′), and p66Shc-specific siRNA (9Kisielow M. Kleiner S. Nagasawa M. Faisal A. Nagamine Y. Biochem. J. 2002; 363: 1-5Crossref PubMed Scopus (75) Google Scholar) were synthesized by Integrated DNA Technology (Coralville, IA), and non-target control siRNA-AllStar was purchased from Qiagen (Valencia, CA). RAW264.7 cells were from ATCC (Manassas, VA), Bio-Lyte 5/8 and Bio-Lyte 3/10 ampholytes were from Bio-Rad. diphenyleneiodonium mitogen-activated protein kinase phosphoinositide 3-kinase phorbol 12-myristate 13-acetate N-formyl-Met-Leu-Phe arachidonic acid peritoneal macrophage(s) phosphate-buffered saline fetal bovine serum superoxide dismutase small interfering RNA 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid guanosine 5′-O-(thiotriphosphate) NADPH oxidase protein kinase C extracellular signal-regulated kinase knock-out p21-activated kinase G protein-coupled receptor phosphatidylinositol 3′,4′,5′-trisphosphate mitogen-activated protein kinase/extracellular signal-regulated kinase kinase dihydro2′4,5,6,7,7′hexafluorofluoroscein bovine serum albumin infrared. PM were harvested 4 days after thioglycollate injection of the peritoneal cavity. Cells were washed with chilled PBS, red blood cells were hypotonically lysed, and macrophages were resuspended in RPMI 1640 containing 15% FBS, 50 μg/ml penicillin, and 50 μg/ml streptomycin and plated on 100-mm round Petri dishes. After a 2-h incubation at 37 °C, 5% CO2 non-adherent cells were removed, and the remaining adherent cells were cultured in RPMI 1640 containing 15% FBS, 50 μg/ml penicillin, and 50 μg/ml streptomycin for no more than 48 h before functional assays. RAW264.7 cells were cultured in RPMI 1640 containing 15% FBS, 50 μg/ml penicillin, and 50 μg/ml streptomycin and subcultured twice a week. Total RNA was extracted by direct lysis of the cells on the tissue culture plate using an RNeasy minikit (Qiagen) according to the manufacturer's instructions. Equal RNA amounts were added to Superscript II First Strand reverse transcriptase reaction mixture (Invitrogen) with oligo(dT) primer. The resulting templates were subject to SYBR Green-based quantitative PCR using specific primers, listed above. The cycling conditions were 94 °C for 3 min as initial denaturation followed by 35 cycles of 94 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s and finished by melting curve by gradual heating until 95 °C. Only experiments with a single melting peak were considered for analysis. Reaction qualities have being verified by gel electrophoresis. PCR was carried out using a LightCycler480 real time PCR instrument and LightCycler480 analysis software (Roche Applied Science). Blood heme measurement was performed on heart blood samples. Blood was collected in capillary tubes containing electrolyte-balanced heparin at 70 IU/ml of blood. Following collection, blood samples were immediately analyzed for hemoglobin levels on a Radiometer OSM3 hemoximeter (Copenhagen, Denmark). Cells were cultivated and transfected on 6-well plates (Nunc) started at 200,000 cells/well. Transfection was performed as described, and after 48 h, cells were washed with PBS, pH 7.4, at 37 °C, and 3 ml of KRP, which contains PBS, pH 7.4, 1 mm CaCl2, 1.5 mm MgCl2, 5.5 mm glucose, and 10 μg/ml OxyBURST Green H2HFF-BSA dye, was added. Cells were incubated at 37 °C in the dark for 2 min, and readings of fluorescence at 530 nm excited at 480 nm were taken for 12 min using CytoFluor Multi-Well Plate Reader (PerSeptive Biosystems). Then PMA until 3 μg/ml was added to experimental wells, and readings were continued for the next 30 min, and then DMSO (solvent for PMA) was added to control cells, used as base line. After the assay, one-half of the cells from each well were taken to the cell count and trypan blue-based viability assay, and the number of cells in each well was calculated. Fluorescence readings were normalized to exact viable cell number in each well. Another half of the cells were used for either protein extraction for Western blots or total RNA extraction for semiquantitative reverse transcription-PCR. For the gliotoxin or DPI inhibition of NAD(P)H-oxidase, gliotoxin or DPI was directly added to the tissue culture medium until a final concentration of 10 μg/ml for gliotoxin or 10 μm for DPI, and cells were preincubated for 10 min at 37 °C prior to measurements of superoxide production. For the H2HFF-based assay, cells were cultivated at 37 °C and 5% CO2 on 100-mm tissue culture dishes for 24–48 h after harvesting. Medium was RPMI 1640 containing 15% FBS, 50 μg/ml penicillin, 50 μg/ml streptomycin. Cells were washed with ice-cold PBS, pH 7.4, and resuspended in ice-cold KRP. Aliquots were taken for the cell counting and viability assay, and the exact number of cells was calculated. The indicated number of cells was taken into a warm 96-well plate with fresh KRP supplemented with OxyBURST Green H2HFF-BSA dye until 10 μg/ml, and the final volume was 190 μl. After a 2-min incubation in the dark, readings were started at emission of 530 nm and excitation of 480 nm. As a stimulus, PMA at 3 μg/ml, fMLP at 3 μm, or AA at 15 μm was added at minute 12, and readings were continued during the next 30 min. A 10-s mixing step was used in each reading cycle of the instrument to prevent the cells from sedimentation. Gliotoxin or DPI inhibition was done similarly, as described above. For the SOD-inhibitable cytochrome c reduction-based assay, PM were harvested from the peritoneal cavity 4 days after thioglycollate injection by lavage with ice-cold PBS. Contaminating erythrocytes were hypotonically lysed, and PM were resuspended in KRP, pH 7.4, at 5 × 106 cells/ml and kept on ice until use. The reaction mixture was 250 μl and contained 50 μm cytochrome c and 6.25 × 105 PM in KRP. The superoxide release was induced with 4 μg of PMA, and readings of absorbance at 550 nm were taken for 10 min using a Tecan Spectra Rainbow 96-well plate spectrophotometer (Tecan). The same assay was carried out in the presence of SOD at 2.5 μg/reaction in order to evaluate the superoxide-independent change in absorbance. A sample without PMA was used as a negative control. siRNA Transfection of RAW264.7 cells was carried out on 6-well plates (Nunc). 200,000 cells/well were plated in antibiotic-free RPMI 1640, 15% FBS and after settlement washed with PBS. The transfection mixture was prepared using 70 nm siRNA specific for p66Shc or nonspecific AllStar siRNA (Qiagen), Opti-MEM medium (Invitrogen) and Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 6 h of transfection in 600 μl of transfection mixture/well, cells were fed with fresh RPMI 1640 supplemented with 15% FBS. After 48 h of cultivation, cells were used in NAD(P)H-oxidase measurement assays followed by cell counting and RNA or protein extraction as described above. Total RNA was extracted from the following tissues of individual age-matched mice: liver, spleen, lungs, epididymal fat, and PM at 3 months of age and liver, retroperitoneal fat, and spleen at 12 months of age. TRIzol reagent was used according to the manufacturer's instructions (Invitrogen), and then RNA was purified using RNeasy Mini Kit (Qiagen). Two mutant and two control mice were used for each experiment. In total, 16 samples from mutant mice and 16 samples from control mice tissues were used. First and second strand of cDNA were generated using the One-Cycle cDNA Synthesis Kit (Affymetrix), and labeled cRNAs were synthesized using the Gene Chip IVT Labeling System (Affymetrix), fragmented, and hybridized to the Mouse Genome 430 2.0 arrays (Affymetrix) according to the manufacturer's instructions. Resulting CEL files were analyzed for each group of tissue individually using dChip (DNA chip analyzer) software (10Schadt E.E. Li C. Ellis B. Wong W.H. J. Cell. Biochem. 2001; 37 (Suppl.): 120-125Crossref Scopus (282) Google Scholar). Updated annotations were obtained from the NetAffx data base, and multisample analysis was performed by combining the dChip lists using Excel. Probesets with a pCall of >20% and p < 0.05 were considered significantly altered. An additional set of CEL files was obtained. Liver, spleen, and lungs samples from 3-month-old p66Shc(−/−) and 3-month-old control mice were hybridized in a similar way to the Affymetrix Mouse Genome U74Av2 chips. RNA samples of each tissue from three animals were pulled together. Two chips for each RNA sample of control and two chips for each RNA sample of mutant mice were used. The CEL files were analyzed with dChip in our laboratory and incorporated into our expression data table using a >90% probeset match between two different chip formats, Mouse Genome 430 2.0 and U74Av2. Probesets were then resorted by number of times they were significantly changed through different experiments. Lists of the top 250 up- and down-regulated genes were categorized using the Onto-Express Pathway analysis tool, and lists of the 100 top up- and down-regulated genes were categorized using the Onto-Express bioprocess analysis tool (11Khatri P. Bhavsar P. Bawa G. Draghici S. Nucleic Acids Res. 2004; 32: W449-W456Crossref PubMed Scopus (124) Google Scholar). For filtering OntoExpress results, we used a cut-off value for p of 0.05. Lists of significantly altered pathways (which included 27 pathways) and significantly altered bioprocesses (18 bioprocesses) were sorted by impact factor and p value, respectively, and the four top up- and down-regulated pathways and bioprocesses were tabulated. Total protein was isolated by direct lysis of adherent cells or isolated from cell pellets washed with ice-cold PBS, pH 7.4, using cell lysis buffer (Cell Signaling Technologies), containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 mm phenylmethanesulfonyl fluoride and supplemented with Complete miniprotease inhibitor mixture and PhosStop phosphatase inhibitor mixture (Roche Applied Science). 40 μg of protein/line, as determined by a Bradford assay (Bio-Rad), were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, blocked with Odyssey Blocking Buffer (Li-Cor Biosciences), and hybridized with the indicated primary antibodies, followed by development with infrared IR dye 700CW- and/or 800CW-labeled secondary antibodies (Li-Cor Corp.). Blots were scanned on an Odyssey infrared imaging instrument and quantified using Odyssey 2.1 software. Use of different IR dye-labeled secondary antibodies allowed us to measure the level of housekeeping proteins at the same time as the proteins of interest on the same membrane and improved the accuracy of quantification and normalization. Macrophages were induced with DMSO (mock treatment) or PMA for 5 min, as indicated; reactions were stopped with 5 volumes of ice-cold PBS; cells were collected by quick centrifugation at 0 °C and lysed by sonication with First Dimension Buffer, containing 8.0 m urea, 2.0% Triton X-100, 5% β-mercaptoethanol, 1.6% Bio-Lyte 5/7 and 0.4% Bio-Lyte 3/10 ampholytes, and 1.5% CHAPS. Samples were isoelectrofocused on 4% PAGE (T = 25%, C = 3%), supplemented with 8 m urea, 1.5% CHAPS at a pH range of 4–9. Isoelectrofocusing gels were than transferred on top of 4–15% gradient SDS-PAGE for the second dimension. Blots were probed with goat anti-p47phox antibody. For the second hybridization, blots were probed with donkey anti-goat monoclonal antibodies (Li-Cor), labeled with IR dye 800CW. The membrane was scanned with an Odyssey infrared scanner and analyzed with Odyssey 2.1 software. Total protein was isolated from control and p66Shc(−/−) PM induced with PMA for 5 min or mock-treated as described above. Cells were collected and lysed by 20 strokes in CHAPS lysis buffer containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10% glycerol, 0.5% CHAPS and supplemented with Complete Mini protease inhibitor mixture and PhosStop phosphatase inhibitor mixture (Roche Applied Science). Unbroken cells were collected by brief centrifugation at 400 × g, and protein concentrations were determined. 100 μg of total protein were adjusted to 60 μl with CHAPS lysis buffer and centrifuged at 100,000 × g for 30 min at +4 °C. Supernatant was called the cytosolic fraction, and pellet was washed with CHAPS lysis buffer and contained the membrane fraction of proteins. The cytosolic fraction was supplemented until 1× with Laemmli sample buffer, and the membrane fraction was resuspended in 1× Laemmli sample buffer. Fractions were boiled until they dissolved completely and were loaded on SDS-PAGE. Western blots with the indicated antibody were performed as described above. Macrophages were isolated from 3-month-old mice as described above, and 7 × 106 cells were resuspended in 6 ml of KRP medium. 3 ml of cell suspension were induced with stimulus: 3 μg/ml PMA for 5 min or 5 μm fMLP for 60 s, as indicated; the other half of the cell suspension was treated with an equal amount of DMSO (mock treatment). For fMLP induction, cells were pretreated with 10 μm cytochalasin b for 10 min. Reactions were stopped by a 5-volume dilution of ice-cold PBS, and cells were collected by rapid centrifugation and lysed in ice-cold lysis buffer: 25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Igepal CA 630, 10% glycerol, 25 mm NaF, 10 mm MgCl2, 1 mm EDTA, 1 mm sodium orthovanadate, and Complete Mini proteinase inhibitor mixture (Roche Applied Science). Lysates were rapidly clarified by low speed centrifugation at +4 °C and immediately frozen in liquid nitrogen until use. Aliquots of protein lysates were left for protein quantification. GTP-Rac1/2 was precipitated using Rac/Cdc41 assay reagent PAK-1 PBD-agarose conjugate (Millipore Inc.) from equal amounts of protein, as recommended by the manufacturer. For the positive controls, one-third aliquots of each experimental sample were loaded with 100 μm GTPγS for 10 min at 30 °C and than used for precipitation with PAK-1 p21-binding domain-agarose. Positive control samples were used to estimate total Rac available for GTP binding in a sample and to ensure that affinity reagent was not saturated. Precipitates were resolved on 15% SDS-PAGE, and blots were probed with mouse anti-Rac1 monoclonal (Millipore Inc.) and goat anti-Rac2 polyclonal antibody (Abcam Inc., Cambridge, MA). Blots were developed and quantified using infrared-labeled secondary antibodies as described above. Fluorescent arbitrary units from Rac bands were normalized to a 50 kDa constitutive band of constant intensity between all samples regardless of GTPγS loading of lysates. To identify transcripts altered in long-lived p66Shc knock-out mice, we microarrayed RNA from liver, spleen, lungs, fat, and peritoneal macrophages of 3-month-old males and 12-month-old males and females of p66Shc(−/−) and control mice. A total of 19 mutants and 19 age/sex-matched control animals were used for the hybridization with Affymetrix oligo-microarray chips, followed by analysis of gene expression data using dChip (10Schadt E.E. Li C. Ellis B. Wong W.H. J. Cell. Biochem. 2001; 37 (Suppl.): 120-125Crossref Scopus (282) Google Scholar). Each data set was analyzed individually, transcripts of p < 0.05 were organized in a megatable, and the top 250 up- and down-regulated genes were entered into OntoExpress (12Draghici S. Khatri P. Tarca A.L. Amin K. Done A. Voichita C. Georgescu C. Romero R. Genome Res. 2007; 17: 1537-1545Crossref PubMed Scopus (897) Google Scholar) as described under “Experimental Procedures.” The top four significantly altered pathways are presented. Also, lists of top 100 up- and down-regulated genes were analyzed with OntoExpress for biological process (11Khatri P. Bhavsar P. Bawa G. Draghici S. Nucleic Acids Res. 2004; 32: W449-W456Crossref PubMed Scopus (124) Google Scholar). The top four regulated bioprocesses are shown in Table 1.TABLE 1Genomic effects of p66Shc knock-outPathwaysBioprocessesDown-regulatedImpact factorp valueDown-regulatedNo. of altered genesp valuePhosphatidylinositol signaling system353.2E-14Negative regulation of programmed cell death63.3E-03Antigen processing and presentation281.4E-11Anti-apoptosis51.2E-03Chronic myeloid leukemia106.7E-04Heme biosynthetic process31.7E-04Adipocytokine signaling pathway82.4E-03Porphyrin biosynthetic process33.8E-04Up-regulatedImpact factorp valueUp-regulatedNo. of altered genesp valueGlioma107.6E-04Actin cytoskeleton organization and biogenesis83.8E-03Natural killer cell-mediated cytotoxicity98.4E-04Actin filament-based process84.4E-03ECM-receptor interaction91.1E-03Cell-matrix adhesion43.1E-03Insulin signaling pathway61.2E-02Cell-substrate adhesion43.7E-03 Open table in a new tab PI3K and antigen processing were significantly altered, and heme transcripts were preferentially affected. Some of the genes underlying these down-regulated processes were related to NADPH oxidase activity. Given the involvement of heme in NADPH oxidase-dependent ROS production, this activity was specifically measured. NADPH oxidase (PHOX) is the basis of the respiratory burst. We measured the respiratory burst of p66Shc(−/−) peritoneal macrophages by two different methods: the conventional SOD-inhibitable reduction of cytochrome c assay and a more sensitive, fluorescence-based H2HFF-oxidation assay (Fig. 1). Both H2HFF dye oxidation and cytochrome c reduction were inhibitable with DPI or gliotoxin, specific PHOX inhibitors (13Hancock J.T. Jones O.T. Biochem. J. 1987; 242: 103-107Crossref PubMed Scopus (194) Google Scholar, 14Nishida S. Yoshida L.S. Shimoyama T. Nunoi H. Kobayashi T. Tsunawaki S. Infect. Immun. 2005; 73: 235-244Crossref PubMed Scopus (44) Google Scholar, 15Tsunawaki S. Yoshida L.S. Nishida S. Kobayashi T. Shimoyama T. Infect. Immun. 2004; 72: 3373-3382Crossref PubMed Scopus (121) Google Scholar, 16Yoshida L.S. Abe S. Tsunawaki S. Biochem. Biophys. Res. Commun. 2000; 268: 716-723Crossref PubMed Scopus (49) Google Scholar). As determined by the cytochrome c assay, the mean value of SOD-inhibitable superoxide production by 600,000 PMA-induced control macrophages was 151 pmol of superoxide/min, whereas p66Shc(−/−) macrophages produce 104 pmol of superoxide/min. Thus, mutant macrophages have 69% of the NAD(P)H-oxidase activity of control. The difference in means was significant, p < 0.000003 (Fig. 2A). Similarly, the H2HFF oxidation method demonstrated an about 40% defect in superoxide production by mutant macrophages, p = 0.01084 (Fig. 2B). Similar results were obtained for fMLP- and AA-stimulated macrophages (Fig. 2, C and D). Similar results were also obtained for p66Shc(−/−) mice in the 129 genetic background using the DCFDA assay.FIGURE 2NAD(P)H oxidase activity in peritoneal macrophages from control and p66Shc(−/−) mice. A, superoxide generation by PMA-induced peritoneal macrophages from p66Shc(−/−) and control mice, as indicated, was assayed using the SOD-inhibitable cytochrome c reduction method. Bars, mean amount of superoxide (pmol)/min/600,000 cells. Error bars, S.D. (n = 12). B, superoxide generation by PMA-induced peritoneal macrophages from p66Shc(−/−) and control mice, as indicated, was assayed using the H2HFF fluorescent dye oxidation method. Bars, means of slopes of fluorescence increase (excitation 480 nm, emission 530 nm) after PMA induction of macrophages. Non-induced cells were used as the base line. C, superoxide generation by fMLP-induced peritoneal macrophages from p66Shc(−/−) and control mice, as indicated, was assayed using the H2HFF fluorescent dye oxidation method. D, superoxide generation by AA-induced peritoneal macrophages from p66Shc(−/−) and control mice, as indicated, was assayed using the H2HFF fluorescent dye oxidation method. Error bars, S.D. For the H2HFF dye oxidation method, n = 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There are at least three explanations for a p66Shc-dependent defect in PMA-stimulated NAD(P)H-oxidase activity (i.e. a defect in expression in a PHOX component, in the heme necessary for superoxide production, or the rate of activation of the complex). First, we investigated the expression of the components of the NOX2 complex (i.e. Rac1/2, p40phox, p47phox, p67phox, p22phox, and gp91phox), using Western blots of protein extracts from peritoneal macrophages of p66Shc(−/−) and control mice (Fig. 3) (data not shown). There was no significant decrease in the protein levels of any tested NOX2 subunit" @default.
• W2109971777 created "2016-06-24" @default.
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• W2109971777 date "2010-01-01" @default.
• W2109971777 modified "2023-10-18" @default.
• W2109971777 title "Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice" @default.
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