Our results demonstrate that the expression of EC-SOD can be induced by IFN-γ in both a dose-dependent and time-dependent manner via the activation of JAK/STAT signaling pathways. This induction is, at least in part, selective for HPASMCs, where the basal level of EC-SOD expression is already higher compared with that in endothelial cells. In addition, we show, for the first time to the best of our knowledge, that histone acetylation and DNA methylation are involved in the cell-specific expression of EC-SOD in pulmonary vascular cells. EC-SOD is well-documented to regulate vascular tone by controlling the concentrations of superoxide radicals within the vascular wall. Superoxide radicals interact with nitric oxide at very rapid, diffusion-limited rates to produce peroxynitrate. This reaction not only inactivates the vasodilating activity of nitric oxide, but can also modify vascular proteins, lipids, and nucleic acids. The nitration of vascular proteins in tyrosine residues and the oxidation of methionine and cysteine residues can produce profound effects on signal transduction pathways and vascular responsiveness (32
). In this regard, we decided to investigate the molecular mechanisms that govern the expression of EC-SOD in vascular cells, and to elucidate potential epigenetic approaches to modify the expression of EC-SOD, using pharmacologic agents.
Our previously published data indicate that DNA methylation is important in the repression of EC-SOD expression in pulmonary adenocarcinoma A549 cells (17
). We and others determined that EC-SOD is expressed at very high concentrations in pulmonary arteries, specifically in pulmonary artery smooth muscle cells. Therefore, we analyzed the role of DNA methylation in the suppression of EC-SOD production in endothelial cells. DNA methylation is an important regulator of gene expression. In mammals, the primary targets for methylation are cytosines, followed by guanosine bases. The majority of CpG dinucleotides are methylated and distributed throughout all the genome. However, the distribution pattern of CpG sites is not random throughout different genetic elements, including genes, noncoding sequences, and transposones. The promoters and 5′-flanking regions of many genes exhibit a high frequency of CpG sites (~1 CpG per 10 bp), compared with 1 CpG site per 100 bp on average for the rest of the genome. These high-density CpG regions are called “CpG islands” (CGIs), and tend to be hypomethylated. The EC-SOD promoter and 5′-flanking region do not contain any CGIs, but the coding region is characterized by very high frequencies of CpG sites. This feature of EC-SOD cDNA attracted our attention as a potential epigenetically driven regulatory element. An analysis of DNA methylation within the coding region of the EC-SOD gene in smooth muscle and endothelial cells showed substantial differences. In pulmonary smooth muscle cells, the percentage of methylated cytosines was considerably lower compared with that in endothelial cells. The functional significance of these differences is supported by the finding that cytosine demethylation agents were able to reactivate EC-SOD gene transcription only in endothelial cells. Thus, this differential pattern of DNA methylation may regulate the cell-specific expression of EC-SOD in pulmonary arteries. Recently, Maunakea and colleagues demonstrated that the majority of methylated CGIs are associated with intragenic and intergenic regions, but not with promoters (34
). They concluded that DNA methylation does not seem to play a major role in gene regulation by 5′ CGI promoters. These intergenic methylated CGIs can regulate the levels of alternative transcripts expressed in a cell-specific or tissue-specific fashion. The analysis of CpG sites within the EC-SOD promoter region found no differences in their methylation frequencies, and a very low percentage of them were methylated. The most striking difference in the methylation of cytosines was observed in the distal promoter, where CpG sites were mostly methylated in endothelial cells and completely unmethylated in smooth muscle cells. This pattern of methylation is in good agreement with current hypotheses stating that hypermethylated promoter regions are associated with repressive chromatin structures. It would be interesting to analyze the presence of cis-regulatory elements in this region of the distal promoter.
IFN-γ is capable of activating a number of genes through the JAK and STAT pathways, and also of binding transcription factors to upstream regions or enhancers of inducible genes in specific DNA regulatory elements, known as the interferon-stimulated response element (ISRE) and the γ-activated sequence (GAS). IFN-γ was shown to induce the expression of EC-SOD in human vascular smooth muscle cells (35
). A simultaneous instillation of IFN-γ and TNF-α into the lungs of rats induced the expression of EC-SOD through, at least in part, an NF-κB–dependent mechanism (36
). Our data indicate a robust induction of EC-SOD in pulmonary fibroblasts and in smooth muscle cells of human pulmonary arteries. Thus, we analyzed the presence and location of ISRE with the consensus sequence 5′-A/GNGAANNGAAACT-3′, and of GAS with the consensus sequence 5′-TTCN(2
)GAA-3′, in close proximity to the EC-SOD gene. We found several GAS sequences located within the 5′-flanking region and near transcription start sites. However, cloning 1,000 bp of the EC-SOD 5′-flanking region in front of the luciferase reporter gene did not produce an IFN-γ–responsive construct (data not shown). Multiple GAS sequences were found within the intronic region and the 3′-flanking region of the EC-SOD gene. In addition, we found sequence 5′-AGG AAA AGG AAt CT-3′, located in close proximity to transcription start sites, and with a very close resemblance to ISRE apart from only one mismatch (T instead of A). However, analysis of the functional importance of these cis-elements in the IFN-γ–dependent induction of EC-SOD requires further investigation.
How histone modifications and chromatin organization regulate the IFN-γ–inducible expression of EC-SOD remain unclear at present. In one putative mechanism, the exposure of cells to IFN-γ can change the nucleosomal organization within the EC-SOD promoter region. We showed that the Sp1 and Sp3 transcription factors bind to the proximal promoter and facilitate transcription of the EC-SOD gene in pulmonary cells. The relaxation of nucleosomes in this region may expose Sp1/Sp3 binding sites, and allow interactions with their trans-factors. A good example of nucleosomal reorganization within the Sp1 binding site of the IL-12 promoter after exposure to LPS and IFN-γ was described elsewhere (37
). However, our data (not shown) indicate that the Sp1/Sp3–specific cis-element located in the proximal promoter is not likely to participate in the IFN-γ–mediated induction of the EC-SOD gene in vascular cells.
Another model suggests that exposure of endothelial cells to TSA increases the accessibility of some unidentified IFN–responsive elements to IFN-γ–inducible transcription factors, which can facilitate the transcription of EC-SOD. Exposure to cytokines was shown to induce a notable perturbation in the acetylation and methylation state of histones in specific regions of the genome. Similar synergistic effects of histone deacetylase inhibitors were described for a number of IFN-γ–inducible genes in murine trophoblast cells (38
) and for the reactivation of the caspase-8 gene in cancer cells (39
In our study, we observed a strikingly similar regulation of gp91phox
expression in response to IFN-γ and TNF-α exposure. The main catalytic, membrane-bound gp91phox
subunit (renamed Nox2) of the NADPH oxidase complex is mostly expressed in neutrophils, and is involved in the production of superoxide fluxes designed to kill invading pathogens. Recently, vascular smooth muscle cells were also shown to produce superoxide, mostly through NADPH oxidase activity. The production of superoxide radicals in the vascular wall at low concentrations is beneficial for vascular physiology because of their role in the regulation of multiple signaling pathways. However, very high concentrations of ROS can be detrimental for vascular cells. The IFN-γ–induced production of NO, combined with a burst in O2−
concentrations, can lead to the formation of peroxynitrite, a strong oxidizing molecule that can induce an inflammatory response in surrounding cells and tissues (40
). Hence, the orchestrated production of superoxide radicals and their dismutation may be a well-designed mechanism to maintain appropriate amounts of reactive oxygen and nitrogen species generated during an inflammatory response. Superoxide concentrations should be sufficiently high to keep pathogens from replication, but low enough not to cause any excessive damage to the vascular tissue of the host.
In conclusion, our results indicate that DNA methylation and histone deacetylation in human pulmonary endothelial cells are associated with the decreased expression of EC-SOD. In addition, we determined that the induction of EC-SOD by IFN-γ is regulated via JAK/STAT signaling pathways in smooth muscle cells and can be reactivated, at least in part, by exposure to the histone deacetylase inhibitor in endothelial cells.