Activation of Manganese-containing superoxide dismutase (MnSOD) gene expression in adult rat lungs during exposure to a sublethal concentration of oxygen (85%) may play a critical role in allowing animals to survive a subsequent exposure to lethal concentrations of oxygen. The overall goal of this research is to define the molecular mechanisms for regulation of MnSOD gene in rat lungs in response to oxidant stress. Previous studies have shown that activation of transcription of MnSOD gene occurs only after three days of exposure to 85 % oxygen and at that time inflammation of the lungs is also evident. These observations suggest that induction of MnSOD gene transcription during hyperoxia may result from the response of lung cells to various cytokines generated during lung inflammation including tumor necrosis factor (TNF), interleukin-1 and interferon-gamma which are known to be capable of inducing MnSOD gene expression, rather than from a direct effect of oxygen tension. Expression/induction studies on transgenic mice carrying various rat MnSOD-I promoter deletion/chloramphenicol acetyltransferase (CAT) fusion genes have allowed us to map one of the hyperoxic injury responsive elements to between positions -507 and -405 and one of the TNF-alpha responsive elements to between -405 and -289 of the rat MnSOD-I promoter. In this application, the cis-acting regulatory DNA elements responsible for hyperoxia and cytokines will be mapped to the nucleotide level by initially constructing various CAT reporter genes under the transcriptional control of (1) promoter internal deletion mutants, (2) promoter base substitution mutants and (3) fusion promoters consisting of herpes thymidine kinase promoter and various regions of MnSOD-I promoter. Transgenic mice and tissue culture cells harboring these fusion genes will be generated, and the inducibility of expression of the reporter genes by hyperoxia and cytokines will be assessed. The similarity between the hyperoxic injury responsive element(s) and cytokine responsive elements will then be determined. The sites of interactions between promoter sequences and nuclear protein factors in lungs of rats and transgenic mice with or without exposure to hyperoxia or cytokine treatments will also be studied by in vivo footprinting. Finally, comparison will be made between the sites of DNA-protein interactions and sequences of hyperoxia and cytokines responsive elements. Accomplishment of the proposed research should provide valuable basic information toward understanding the nature of the regulatory elements which control the expression of this gene, as well as uncovering the molecular signal driven in vivo in the lungs by hyperoxia and cytokines. This knowledge should then direct development of strategies for augmenting MnSOD expression as a part of antioxidant defense against oxidant-induced lung diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL039585-08
Application #
2219278
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1987-09-30
Project End
1998-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
8
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Organized Research Units
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Darville, M I; Ho, Y S; Eizirik, D L (2000) NF-kappaB is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells. Endocrinology 141:153-62
Ho, Y S; Vincent, R; Dey, M S et al. (1998) Transgenic models for the study of lung antioxidant defense: enhanced manganese-containing superoxide dismutase activity gives partial protection to B6C3 hybrid mice exposed to hyperoxia. Am J Respir Cell Mol Biol 18:538-47
Ho, Y S; Dey, M S; Crapo, J D (1996) Antioxidant enzyme expression in rat lungs during hyperoxia. Am J Physiol 270:L810-8
Jackson, R M; Parish, G; Ho, Y S (1996) Effects of hypoxia on expression of superoxide dismutases in cultured ATII cells and lung fibroblasts. Am J Physiol 271:L955-62
Russell, W J; Ho, Y S; Parish, G et al. (1995) Effects of hypoxia on MnSOD expression in mouse lung. Am J Physiol 269:L221-6
Ho, Y S (1994) Transgenic models for the study of lung biology and disease. Am J Physiol 266:L319-53
Oberley, T D; Coursin, D B; Cihla, H P et al. (1993) Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme. Histochem J 25:267-79
Subramanian, R; Volovsek, A; Ho, Y S (1993) Lack of change in MnSOD during ischemia/reperfusion of isolated rat heart. J Mol Cell Cardiol 25:1179-86
Ho, Y S; Howard, A J (1992) Cloning and characterization of the rat glutathione peroxidase gene. FEBS Lett 301:5-9
Ho, Y S; Howard, A J; Crapo, J D (1991) Molecular structure of a functional rat gene for manganese-containing superoxide dismutase. Am J Respir Cell Mol Biol 4:278-86

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