Abstract
We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.
Highlights
Effect of H20,on AOEmRNA Levels-The addition of H202 to hamstertrachealepithelial cells (HTE) cells caused a dose-dependent increase in the levels of steady state message for catalase (Fig. 1).This response was observed in exponentially growing cells as well as incells which had reachedconfluence prior toexposure to HzOz.The greatest induction was observed with 100 and 250 p M H202 which resulted in increasesof 2.5- and 3-fold, respectively, in the levels of catalase mRNA
The message levels of glutathione peroxidase (GPX), which catalyzes the reductionof H202,were less responsive to H202C. ells growing exponentially did not demonstrate any change in thleevels of GPX mRNA inresponse to H202, but at concentrations of 100 and 250 p~ H202the levels of GPX mRNA increasedby as much as 50% in confluentcells
MnSOD mRNA increased3-fold as HTEcells advanced from log phase to confluence
Summary
Dept. of Biochemistry, Medical School, University of Vermont, Burlington, VT05405. The abbreviations used are: ROS, reactive oxygen species; SOD, occurred in HTE cells after exposure todifferentoxidant stresses. Preliminary experiments showed that the induction of MnSOD mRNA using the xanthine/xanthine oxidase system fluctuated depending on the sources of the enzyme and fetal bovine serum. Western Blot Analysis-Confluent epithelial cells were exposed to 50 p M xanthine (control cultures) and 0.05 or 0.2 unit/ml xanthine oxidase in serum containing medium for 24 or 48 h. Aliquots saved prior to lyophilization were used for protein determination [19] using bovine serum albumin, Fraction V (Sigma), as a standard.Samples including prestained molecular weight standards (GIBCO-BRL) were electrophoresed in a 15% SDS-polyacrylamide gel using a Mini-protean I1 Cell apparatus (Bio-Rad) and transferred to nitrocellulose (Schleicher and Schuell) as described previously [20].The nitrocellulose blot was stained with anti-MnSOD antibody diluted in 0.05% Tween-20 (Sigma) in CMF-PBS.
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