Activation of NF-kappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation.
OBJECTIVE
We investigated whether NADPH oxidase-dependent production of superoxide contributes to activation of NF-kappaB in endothelial cells by the saturated free fatty acid palmitate.


RESULTS
After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 mumol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a >2-fold increase in superoxide levels, an effect associated with activation of NF-kappaB signaling as measured by phospho-IkappaBalpha, NF-kappaB activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of 3 different interventions-pretreatment with superoxide dismutase (SOD), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA-attenuated palmitate-mediated NF-kappaB signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitate-mediated superoxide production was not affected by overexpression of a phosphorylation mutant IkappaBalpha (NF-kappaB super repressor) that blocks cellular inflammation downstream of IKKbeta/NF-kappaB. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4(-/-) mice, compared to low-fat fed controls.


CONCLUSIONS
These results suggest that NADPH oxidase-dependent superoxide production links palmitate-stimulated TLR4 activation to NF-kappaB signaling in endothelial cells.
Maloney
Ezekiel
E
Department of Medicine, University of Washington, Seattle, WA 98109, USA.
Sweet
Ian R
IR
Hockenbery
David M
DM
Pham
Matilda
M
Rizzo
Norma O
NO
Tateya
Sanshiro
S
Handa
Priya
P
Schwartz
Michael W
MW
Kim
Francis
F
D000818:Animals; D001013:Aorta, Thoracic / Q000201:enzymology / Q000276:immunology; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D002478:Cells, Cultured; D004041:Dietary Fats / Q000008:administration & dosage / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D004041:Dietary Fats; D020672:I-kappa B Proteins; C508600:IL6 protein, human; D015850:Interleukin-6; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D009861:Onium Compounds; D012333:RNA, Messenger; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D013481:Superoxides; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D019308:Palmitic Acid; C007517:diphenyleneiodonium; D013482:Superoxide Dismutase; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C422943:Nox4 protein, mouse; D053592:Interleukin-1 Receptor-Associated Kinases