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. 2008 Aug;39(2):243-51.
doi: 10.1165/rcmb.2007-0378OC. Epub 2008 Feb 28.

Transcription factors sp1 and sp3 regulate expression of human extracellular superoxide dismutase in lung fibroblasts

Igor N Zelko  1 , Michael R Mueller, Rodney J Folz
Affiliations

Transcription factors sp1 and sp3 regulate expression of human extracellular superoxide dismutase in lung fibroblasts

Igor N Zelko et al. Am J Respir Cell Mol Biol. 2008 Aug.

Abstract

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.

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Figures

<b>Figure 1.</b>
Figure 1.
Extracellular superoxide dismutase (EC-SOD) expression, genomic sequence, and transcriptional start sites. (A) EC-SOD mRNA levels in different cells. Total RNA was purified, reverse transcribed, and amplified with primers specific for EC-SOD or GAPDH. Amplified products were separated on 1.2% agarose gel and visualized with ethidium bromide. (B) Nucleotide sequence of 5′-flanking region of EC-SOD gene. Transcriptional start sites are depicted as arrows, exon sequences are underlined, and the Sp1/Sp3 protected region is boxed. The previously identified transcriptional start site, depicted at position −620, was not confirmed in our experiments.
<b>Figure 2.</b>
Figure 2.
Functional properties of the EC-SOD promoter fragments in A549 and MRC5 cells. (A) 5′ regions of EC-SOD promoter were fused with the luciferase reporter gene in pGL3-Basic vector. Transcription start sites are depicted as arrows. A Dashed oval represents the putative Sp1/Sp3 binding site. (B) All constructs were transiently transfected into A549 or MRC5 cells, and luciferase activity was measured 24 hours later. Firefly luciferase activities are normalized to Renilla luciferase activity produced by co-transfecting the control plasmid pRL-CMV. Results shown are mean ± SD from at least two independent transfection experiments, each performed in quadruplicate.
<b>Figure 3.</b>
Figure 3.
Identification of trans-factors that bind to EC-SOD proximal promoter. (A) An end-labeled −263/+45 bp fragment of the human EC-SOD promoter was incubated with BSA (Lanes 2–3 and 7–8) or MRC5 nuclear extract (Lanes 4–5 and 9–10) and digested with increasing amount of DNase I. The single protected region (marked as black boxes on the right side of the gel) is identified at positions −198/−211. The nucleotide sequence of protected region shown at the bottom. Lanes 1 and 6, G+A, Maxam-Gilbert sequencing ladder of the probe DNA. (B) Binding of transcription factors from Sp-like family to the EC-SOD promoter. An end-labeled hSOD3 (−228/−178) oligonucleotide was incubated with A549 or MRC5 nuclear extract. Competition assays were performed with 50- and 200-fold excesses of unlabeled oligonucleotides to analyze the specificity of Sp1/Sp3 binding. The self and Sp1 consensus oligonucleotides eliminated shifted bands (Lanes 5–8). Oligonucleotides representing the consensus binding site for Egr-1 show some competition for DNA-protein complexes (Lanes 9 and 10), whereas the AP-1–specific probe shows no competition (Lanes 11 and 12). (C) For supershift experiments, antibodies specific for Sp1, Sp2, Sp3, or nonimmune (n.i.) IgG were added to the reaction mixture. DNA-protein complexes were separated on 5% nondenaturing polyacrylamide gel, dried, and exposed to X-ray film.
<b>Figure 4.</b>
Figure 4.
Activation of EC-SOD promoter by Sp1/Sp3 in vivo. Drosophila SL2 cells were transfected with 600 ng of promoter-reporter constructs depicted in the legend and 600 ng of pPac-O, pPac-Sp1, pPac-Sp2, or pPac-Sp3 plasmid as indicated. The luciferase activities were measured 24 hours later. Assays were repeated at least three times, and the mean ± SD for each value is indicated. Asterisks indicate the values of luciferase activity that are significantly different from those of control cells (P < 0.001).
<b>Figure 5.</b>
Figure 5.
Occupacy of EC-SOD promoter by Sp1/Sp3 and RNA polymerase II (Pol. II) in vivo. (A) Chromatin immunoprecipitation assay was used to analyze the interaction of RNA polymerase II, Sp3, Sp2, and Sp1 to the EC-SOD proximal promoter in A549 and MRC5 cells. Binding of RNA polymerase II to the GAPDH promoter was used as a positive control. Gel images are representative of at least three independent experiments. (B) Quantitative analysis of images presented in A. The intensity of fluorescence from amplified bands was compared and expressed as relative abundance of the protein at corresponding promoter. Asterisks indicate amplified bands whose intensity is significantly higher than the intensity of corresponding control IgG bands (P < 0.05). Bars represent ±SD.
<b>Figure 6.</b>
Figure 6.
Expression of Sp1 and Sp3 in A549 and MRC5 cells. (A) Relative mRNA levels of Sp1 and Sp3 in A549 and MRC5 cells. The mRNA levels were determined using quantitative RT-PCR and SYBR Green dye. Sp1 and Sp3 mRNA levels were normalized to GAPDH expression. Amplification was performed in triplicate, and SD is depicted as an error bar. Asterisks indicate the levels of mRNAs in MRC5 cells that are significantly different from those of A549 cells (P < 0.01). (B) Sp1 and Sp3 expression was assessed using Western blot. Nuclear extracts from A549 and MRC5 cells were separated on denaturing PAAG, transferred onto polyvinylidene difluoride membrane, and incubated with Sp1-, Sp3-, or GAPDH-specific IgG.
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