Histone acetylation regulates the cell-specific and interferon-γ-inducible expression of extracellular superoxide dismutase in human pulmonary arteries

Abstract

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.

Figure 1.

Expression of extracellular superoxide dismutase (EC-SOD) in different tissues and cells. (A) Expression of EC-SOD in liver, lung, and pulmonary arteries (PA). (B) Analysis of EC-SOD expression in human pulmonary artery endothelial cells (HPAECs) and in human pulmonary artery smooth muscle cells (HPASMCs). Total RNA from liver, lung, and pulmonary artery cells was isolated and purified. Real-time PCR was used to determine relative concentrations of EC-SOD mRNA in those tissues and cells. Concentrations of EC-SOD mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At least four independent amplifications were performed for each analysis. The results shown represent the mean ± SD.

Figure 2.

Methylation status of CpG sites within 5′-flanking and coding regions of the EC-SOD gene. (A) Schematic representation of EC-SOD gene organization. The distribution of CpG sites within EC-SOD genomic DNA are shown as vertical gray bars. The ratio of observed versus expected CpG frequency is plotted, and the CpG island is marked as a pattern bar. (B) McrBC-based assay. The genomic DNA from HPAECs and HPASMCs was purified and digested with McrBC methylation–sensitive nuclease. The integrity of DNA was analyzed using primer pairs that amplify different region of the EC-SOD promoter. Amplified products were separated on 1.2% agarose gel and visualized using ethidium bromide under ultraviolet light. (C) Methylation status of CpG sites within the EC-SOD promoter, using bisulfite sequencing. At least 10 individual clones were sequenced for each cell type. (D and E) Methylation status of CpG sites within the EC-SOD coding region in HPASMCs (D) and HPAECs (E). From 9–12 clones were sequenced for each CpG site.

Figure 3.

Acetylation status of histones is important for cell-specific expression of EC-SOD. (A) HPAECs and HPASMCs were exposed to 5-aza-2′-deoxycytidine (5-aza-dC; 0.5 μM and 2 μM) or trichostatin A (TSA; 1.5 μM and 3 μM), or a combination of both substances for 48 hours. (B) Cells were exposed to DMSO or 2 μM of 5-aza-dC for 96 hours. Total RNA was extracted, and EC-SOD mRNA was measured by quantitative real-time RT-PCR. The data were normalized to concentrations of GAPDH mRNA. The results shown represent the mean ± SD from at least two independent experiments. *P < 0.01, significant difference, according to ANOVA/Bonferroni post hoc test.

Figure 4.

Regulation of EC-SOD and gp91^phox transcription by cytokines. MRC5 cells (A and B) and HPASMCs (C–F) were exposed to 5,000 U/ml of IFN-γ (A, C, and E) or 30 ng/ml of TNF-α (B, D, and F) for the times indicated. Total RNA was extracted, and EC-SOD and gp91^phox mRNA was measured by quantitative real-time RT-PCR. The data were normalized to concentrations of GAPDH mRNA. The results shown represent the mean ± SD from at least two independent experiments.

Figure 5.

Role of Janus tyrosine kinase (JAK)/signal transducers and activators of transcription proteins (STAT) signaling pathway in IFN-γ–dependent induction of EC-SOD. (A) Time course of inducible expression of interferon-responsive factor–1 (IRF-1) and EC-SOD in HPASMCs. Cells were incubated with 5,000 U/ml of IFN-γ for the indicated times. (B and C) Effects of JAK1, JAK2 (AG490), and STAT3 inhibitors on IFN-γ–induced EC-SOD (B) and IRF-1 (C) mRNA concentrations in HPASMCs. Cells were incubated with vehicle (Control) or IFN-γ (5,000 U/ml) in the presence or absence of the tested compound for 48 hours. Total RNA was isolated, and gene-specific mRNA concentrations were analyzed, using quantitative RT-PCR. Concentrations of EC-SOD and IRF-1 mRNA were normalized to the expression of GAPHD. The results shown represent the mean ± SD. *P < 0.001 and **P < 0.01, compared with cells treated with IFN-γ, and ^#P < 0.001, compared with cells treated with vehicle, according to ANOVA/Bonferroni post hoc test.

Figure 6.

Stability of EC-SOD mRNA after exposure to IFN-γ. (A) HPASMCs were exposed to IFN-γ (5,000 U/ml) or PBS for 72 hours, followed by treatment with actinomycin D (10 μg/ml). Total RNA was collected at the times indicated, and concentrations of EC-SOD mRNA were determined by quantitative RT-PCR. (B) Stability of glucose transporter–1 (GLUT1) mRNA under control conditions is shown for comparison with mRNA stability. The data shown represent at least two independent experiments.

Figure 7.

Role of histone acetylation in IFN-γ–inducible EC-SOD expression. HPAECs (A) and HPASMCs (B) were exposed to either IFN-γ (5,000 U/ml) or TSA (1.5 μM) or a combination of both substances for 48 hours. Total RNA was extracted, and EC-SOD mRNA was measured by quantitative real-time RT-PCR. The data were normalized to concentrations of GAPDH mRNA. The results shown represent the mean ± SD from at least two independent experiments. *P < 0.001, compared with nontreated cells, according to ANOVA/Bonferroni post hoc test. **P < 0.01, according to t test.