Activation of NF-kappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation

Arterioscler Thromb Vasc Biol. 2009 Sep;29(9):1370-5. doi: 10.1161/ATVBAHA.109.188813. Epub 2009 Jun 18.

Abstract

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.

Methods and 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Aorta, Thoracic / enzymology
  • Aorta, Thoracic / immunology
  • Bone Morphogenetic Protein 4 / metabolism
  • Cells, Cultured
  • Dietary Fats / administration & dosage
  • Dietary Fats / metabolism
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Endothelial Cells / immunology
  • Humans
  • I-kappa B Proteins / metabolism
  • Intercellular Adhesion Molecule-1 / metabolism
  • Interleukin-1 Receptor-Associated Kinases / metabolism
  • Interleukin-6 / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Myeloid Differentiation Factor 88 / metabolism
  • NADPH Oxidase 4
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism*
  • Onium Compounds / pharmacology
  • Palmitic Acid / metabolism*
  • Phosphorylation
  • RNA Interference
  • RNA, Messenger / metabolism
  • Signal Transduction
  • Superoxide Dismutase / metabolism
  • Superoxides / metabolism*
  • Time Factors
  • Toll-Like Receptor 4 / deficiency
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*

Substances

  • Bmp4 protein, mouse
  • Bone Morphogenetic Protein 4
  • Dietary Fats
  • I-kappa B Proteins
  • IL6 protein, human
  • Interleukin-6
  • MYD88 protein, human
  • Myeloid Differentiation Factor 88
  • NF-kappa B
  • NFKBIA protein, human
  • Nfkbia protein, mouse
  • Onium Compounds
  • RNA, Messenger
  • TLR4 protein, human
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • Superoxides
  • Intercellular Adhesion Molecule-1
  • NF-KappaB Inhibitor alpha
  • Palmitic Acid
  • diphenyleneiodonium
  • Superoxide Dismutase
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX4 protein, human
  • Nox4 protein, mouse
  • Interleukin-1 Receptor-Associated Kinases