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author:("elko, Igor N.")
1.  Histone Acetylation Regulates the Cell-Specific and Interferon-γ–Inducible Expression of Extracellular Superoxide Dismutase in Human Pulmonary Arteries 
Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle–specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ–inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ–dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ–inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation.
doi:10.1165/rcmb.2011-0012OC
PMCID: PMC3262691  PMID: 21493784
extracellular superoxide dismutase; promoter; epigenetic; transcription; pulmonary arteries; endothelial cells; smooth muscle cells
2.  Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure 
FEBS letters  2010;584(13):2947-2952.
The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knockout mice are more susceptible to bleomycin induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.
doi:10.1016/j.febslet.2010.05.025
PMCID: PMC2892677  PMID: 20493858
oxidative stress; reactive oxygen species; lung injury; pulmonary fibrosis
3.  CpG METHYLATION ATTENUATES SP1 AND SP3 BINDING TO HUMAN EXTRACELLULAR SUPEROXIDE DISMUTASE PROMOTER AND REGULATES ITS CELL-SPECIFIC EXPRESSION 
Free radical biology & medicine  2010;48(7):895-904.
Extracellular superoxide dismutase (EC-SOD) plays an important role in maintaining normal redox homeostasis in the lung. It is expressed at very high levels in pulmonary fibroblasts, alveolar type II epithelial cells and smooth muscle cells. The molecular mechanism(s) governing this cell-specific expression of EC-SOD are mostly unknown. In our previous studies we showed that EC-SOD cell specific expression was not attributed to differential transcriptional regulation, suggesting that other, possibly epigenetic, mechanisms are involved in regulation of its expression. In this paper, we found high levels of promoter methylation in A549 cells and correspondingly low levels of methylation in MRC5 cells. Inhibition of DNA methyltransferase activity by 5-azacytidine in A549 cells reactivated EC-SOD transcription (2.75±0.16 fold, p<0.001) demonstrating the importance of methylation in repression of EC-SOD expression. Furthermore, methylation of cytosines in the promoter markedly decreased Sp1/Sp3 driven promoter activity to 30.09±2.85% (p<0.001) compare to unmethylated promoter. This attenuation of transcription in the promoter-reporter construct was, at least in part, attributed to the binding of methyl-binding protein MeCP2 in the insect cells. However, no binding of MeCP2 or MBD2 proteins to EC-SOD promoter was detected in mammalian cells in vivo. We also found marked differences in the chromatin organization of the EC-SOD promoter between these two cell lines, further supporting the important role epigenetic modifications play in the regulation of EC-SOD expression.
doi:10.1016/j.freeradbiomed.2010.01.007
PMCID: PMC2838251  PMID: 20079429
reactive oxygen species; transcription; epigenetic regulation; DNA methylation; methyl-binding proteins; lung; chromatin organization
4.  Novel Mechanism for Regulation of Extracellular SOD Transcription and Activity by Copper: Role of Antioxidant-1 
Free radical biology & medicine  2008;46(1):95-104.
Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone Antioxidant-1 (Atox1) involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response element (RE) at the SOD3 promoter. Gel shift and ChIP assays reveal that Atox1 directly binds to the Atox1-RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated re-expression in Atox1-/- cells with nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, re-expression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1-/- cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through transcription factor function of Atox1, while full activity of SOD3 requires both copper chaperone and transcription factor function of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.
doi:10.1016/j.freeradbiomed.2008.09.039
PMCID: PMC2630370  PMID: 18977292
Antioxidant-1; SOD3; Copper; Transcription Factor; Copper Chaperone
5.  Transcription Factors Sp1 and Sp3 Regulate Expression of Human Extracellular Superoxide Dismutase in Lung Fibroblasts 
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.
doi:10.1165/rcmb.2007-0378OC
PMCID: PMC2542458  PMID: 18314536
extracellular superoxide dismutase; promoter; transcription; Sp1 gene family; antioxidant

Results 1-5 (5)