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J. Biol. Chem., Vol. 275, Issue 23, 17728-17739, June 9, 2000
Gene
Expression in Monocytic Cells via Hyperglycemia-induced Oxidant
Stress-dependent and -independent Pathways*
§,
, and
From the 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 Department of Diabetes and Endocrinology and
Graduate School of Biological Sciences, City of Hope National
Medical Center, Duarte, California 91010, ¶ Genetics Institute,
Pasadena, California 91105, and Department of Internal
Medicine, University of Virginia, Charlottesville, Virginia 22908
(TNF), at least in part through enhanced TNF mRNA
transcription, mediated by ROS via activation of transcription factors
nuclear factor 
B (NF-B) and activating protein-1 (AP-1). TNF
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-B and AP-1 mediated enhanced
TNF transcription by CHG: 1) A 295-base pair fragment of the
proximal TNF promoter containing NF-B and AP-1 sites reproduced
the effects of CHG on TNF transcription in a luciferase reporter
assay, 2) mutational analyses of both NF-B and the AP-1 sites
abrogated 90% of the luciferase activity, 3) gel-shift analysis using
the binding sites showed activation of NF-B 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 IB 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 TNF, 2)
increased phosphorylation of stress-responsive mitogen-activated
protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNF production, the 295TNFluc reporter activity, activation of
NF-B, and repression of IB 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.
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