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Transcription Factors Sp1 and Sp3 Regulate Expression of Human Extracellular Superoxide Dismutase in Lung Fibroblasts

¹ Departments of Medicine and Biochemistry and Molecular Biology University of Louisville, Louisville, Kentucky

Correspondence and requests for reprints should be addressed to Rodney J. Folz, M.D., Ph.D., 550 S. Jackson Street/ACB – A3R43, University of Louisville, Louisville, KY. E-mail: rodney.folz{at}louisville.edu

The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5'-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter–luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.

Key Words: extracellular superoxide dismutase • promoter • transcription • Sp1 gene family • antioxidant

CLINICAL RELEVANCE We have identified key regulatory elements for human extracellular superoxide dismutase (EC-SOD), the major antioxidant enzyme in the lung. Since oxidative stress contributes to the development of lung disease, understanding factors that regulate EC-SOD may provide novel therapeutic strategies.

 

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