title: Cellular crosstalk between TNF-α, NADPH oxidase, PKCβ2, and C2GNT in human leukocytes.
abstract: Increasing evidence suggests that chronic, sub-clinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have established the potential role of the inflammatory enzyme, core 2 β-1, 6-N-acetylglucosaminyltransferase (C2GNT) in diabetic retinopathy. The present study was designed to explore the NADPH oxidase signaling pathway in the tumor necrosis factor-alpha (TNF-α)-induced activity of C2GNT in leukocytes. Human leukocytes (U937 cells) and an Epstein-Barr-transformed lymphoblastoid cell line deficient in p47phox (F10007 cells) were used for the study. Cells were exposed to TNF-α for 24h in the presence and absence of 1) NADPH oxidase inhibitors (apocynin and scrambled and unscrambled gp91ds-tat), 2) LY379196 (specific protein kinase C β1/2 (PKCβ1/2) inhibitor), and 3) the antioxidant tiron. Subsequent C2GNT and NADPH activity was measured and the adhesion of U937 and F10007 cells to endothelial cells was assessed. TNF-α-induced C2GNT activity (1813±326 pmol/h/mg protein) (mean±SEM) in human leukocytes was significantly reversed with apocynin (153±82 pmol/h/mg protein), unscrambled gp91ds-tat (244±122 pmol/h/mg protein) and tiron (756±87 pmol/h/mg protein). We further supported this C2GNT-NADPH oxidase link using p47phox-deficient leukocytes. The deficiency in p47phox prevented TNF-α-induced NADPH oxidase and C2GNT activity and adherence to endothelial cells. The response to TNF-α was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. Our results demonstrate for the first time a novel signaling crosstalk between TNF-α, NADPH oxidase, PKCβ1/2 and C2GNT in leukocytes.
authors.affiliation: Institute of Biomedical and Clinical Science, Peninsula College of Medicine and Dentistry, Universities of Exeter & Plymouth, UK. joanna.tarr@pms.ac.uk
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Tarr
authors.forename: Ning
authors.initials: N
authors.lastname: Ding
authors.forename: Kirti
authors.initials: K
authors.lastname: Kaul
authors.forename: Anna
authors.initials: A
authors.lastname: Antonell
authors.forename: Luis A
authors.initials: LA
authors.lastname: Pérez-Jurado
authors.forename: Rakesh
authors.initials: R
authors.lastname: Chibber
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics*; D010084:Oxidation-Reduction; D010957:Plasmids; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000098:Acetophenones; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D014409:Tumor Necrosis Factor-alpha; C473031:gp91ds-tat protein, chimeric; C056165:acetovanillone; D019255:NADPH Oxidases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; D011493:Protein Kinase C; D064546:Protein Kinase C beta