Molecular Mechanisms of Tumor Necrosis Factor α Gene Expression in Monocytic Cells via Hyperglycemia-induced Oxidant Stress-dependent and -independent Pathways

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 α (TNFα), at least in part through enhanced TNFα mRNA transcription, mediated by ROS via activation of transcription factors nuclear factor κB (NF-κB) and activating protein-1 (AP-1). TNFα 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-κB and AP-1 mediated enhanced TNFα transcription by CHG: 1) A 295-base pair fragment of the proximal TNFα promoter containing NF-κB and AP-1 sites reproduced the effects of CHG on TNFα transcription in a luciferase reporter assay, 2) mutational analyses of both NF-κB and the AP-1 sites abrogated 90% of the luciferase activity, 3) gel-shift analysis using the binding sites showed activation of NF-κB 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 IκBα 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 TNFα, 2) increased phosphorylation of stress-responsive mitogen-activated protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNFα production, the 295TNFluc reporter activity, activation of NF-κB, and repression of IκBα 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.