title: Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways.
abstract: Increased oxidative stress has been reported in vivo in the diabetic state via the production of reactive oxygen species (ROS). Such stress is bound to play a key role on activation of circulating monocytes, leading to the accelerated atherosclerosis observed in diabetics. However the exact molecular mechanisms of monocyte activation by high glucose is currently unclear. Here, we demonstrate that chronic high glucose (CHG) causes a dramatic increase in the release of the inflammatory cytokine tumor necrosis factor alpha (TNFalpha), at least in part through enhanced TNFalpha mRNA transcription, mediated by ROS via activation of transcription factors nuclear factor kappaB (NF-kappaB) and activating protein-1 (AP-1). TNFalpha accumulation in the conditioned media was increased 10-fold and mRNA levels were increased 11.5-fold by CHG. The following observations supported that both NF-kappaB and AP-1 mediated enhanced TNFalpha transcription by CHG: 1) A 295-base pair fragment of the proximal TNFalpha promoter containing NF-kappaB and AP-1 sites reproduced the effects of CHG on TNFalpha transcription in a luciferase reporter assay, 2) mutational analyses of both NF-kappaB and the AP-1 sites abrogated 90% of the luciferase activity, 3) gel-shift analysis using the binding sites showed activation of NF-kappaB and AP-1 in CHG nuclear extracts, and 4) Western blot analyses demonstrated elevated nuclear levels of p65 and p50 and decreased cytosolic levels of IkappaBalpha in CHG-treated monocytes. That ROS acted as a key intermediate in the CHG pathway was supported by the following evidence: 1) increased superoxide levels similar to those observed with PMA or TNFalpha, 2) increased phosphorylation of stress-responsive mitogen-activated protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNFalpha production, the 295TNFluc reporter activity, activation of NF-kappaB, and repression of IkappaBalpha by antioxidants and p38 mitogen-activated protein kinase inhibitors. The study suggests that ROS function as key components in the regulatory pathway progressing from elevated glucose to monocyte activation.
authors.affiliation: Department of Diabetes and Endocrinology and Graduate School of Biological Sciences, City of Hope National Medical Center, Duarte, California 91010, USA.
authors.forename: M
authors.initials: M
authors.lastname: Guha
authors.forename: W
authors.initials: W
authors.lastname: Bai
authors.forename: J L
authors.initials: JL
authors.lastname: Nadler
authors.forename: R
authors.initials: R
authors.lastname: Natarajan
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D006943:Hyperglycemia; D007093:Imidazoles / Q000494:pharmacology; D008163:Luminescent Measurements; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009000:Monocytes / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000502:physiology; D013859:Thiocarbamates / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000494:pharmacology; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D007093:Imidazoles; D016328:NF-kappa B; D011725:Pyridines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C066229:prolinedithiocarbamate; D011392:Proline; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole; D000111:Acetylcysteine