Abstract
Mitochondrial levels of the anti-oxidant enzyme, manganese superoxide dismutase (MnSOD), are dramatically elevated in response to stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and lipopolysaccharide (LPS). However, the precise intracellular signaling pathways responsible for this inducible expression are poorly understood. MnSOD expression in pulmonary epithelial and endothelial cells, treated with inflammatory mediators and various inhibitors, was studied by Northern analysis. The mitochondrial electron transport chain inhibitors, antimycin A and myxothiazol, selectively blocked TNF-alpha-inducible expression of MnSOD but not that of IL-1beta or LPS, indicating different signaling pathways. N-Acetylcysteine could reliably decrease inducible MnSOD expression by TNF-alpha, but not IL-1, linking reactive oxygen species (ROS) to the TNF-alpha signaling pathway. Elevated levels of arachidonic acid have been demonstrated previously to generate mitochondrial ROS. A specific cytoplasmic phospholipase A(2) inhibitor reduced stimulated MnSOD expression by TNF-alpha, but not by IL-1beta, further supporting the role of ROS. Other investigators have shown that MnSOD expression may be regulated by NF-kappaB. Our results with a specific inhibitory kappa-kinase inhibitor indicate that NF-kappaB modulates IL-1beta signaling but not the TNF-alpha pathway. Thus, we have demonstrated that although inducible MnSOD transcription may appear similar at the messenger RNA level, the intracellular signaling pathways are differentially regulated.
Highlights
Manganese superoxide dismutase (MnSOD),1 a vital antioxidant enzyme localized to the mitochondrial matrix, catalyzes the dismutation of superoxide anions (O2.) to hydrogen peroxide (H2O2). transport chain is
Based on earlier experiments demonstrating stimulated expression of manganese superoxide dismutase (MnSOD) mRNA following treatment of a rat pulmonary epithelial-like cell line (L2 cells) with inflammatory mediators [7, 9], we initiated studies to evaluate the effect of TNF-␣ on MnSOD expression in cells treated with mitochondrial respiratory chain inhibitors
To evaluate whether signaling pathways for all three inflammatory mediators were similar when mitochondrial respiration is inhibited with antimycin A, we examined the effect that increasing concentrations of antimycin A had on the LPS- and IL-1-stimulated expression of MnSOD in VA cells (Fig. 3, A, C and D)
Summary
Materials—Recombinant human TNF-␣ (a gift from Genentech), IL-1 (a gift from NCI, National Institutes of Health), antimycin A, N-acetylcysteine, AACOCF3, SB 203580, PD 98059 (Calbiochem), I. Cell Culture—L2 cells, a rat pulmonary epithelial-like line (ATCC CCL 149), were grown in Ham’s F12K media (Life Technologies, Inc.) with 10% fetal bovine serum (Flow Laboratories, McLean, VA), 10 mM L-glutamine, and antibiotic/antimycotic solution (ABAM, Sigma) at 37 °C in room air, 5% CO2. VA cells, a rat pulmonary artery endothelial cell line, isolated from segments of pulmonary artery by mechanical methods [11], were grown in Medium 199 with Earle’s salts (Sigma) with sodium bicarbonate to pH 7.4, 10% fetal bovine serum, 10 mM L-glutamine, and antibiotic/antimycotic solution at 37 °C in room air, 5% CO2. LA9 cells, a mouse fibroblast antimycin-resistant mutant cell line (generously provided by Dr Neil Howell, see Ref. 31), were grown in Ham’s F12K media (Life Technologies, Inc.) with 10% fetal bovine serum (Flow Laboratories, McLean, VA), 10 mM L-glutamine, and antibiotic/antimycotic solution (ABAM, Sigma) at 37 °C in room air, 5% CO2. The unpaired Student’s t test was used to evaluate the significance of differences between groups, accepting p Ͻ 0.05 as the level of significance
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