Abstract
Global DNA hydroxymethylation mediated by the TET (ten-eleven translocation) enzyme was induced in allergen-induced airway hyperresponsiveness in mouse lung tissues and specifically in isolated airway smooth muscle (ASM) cells. TET is an α-ketoglutarate (α-KG)–dependent enzyme, and the production of α-KG is catalyzed by IDH (isocitrate dehydrogenase). However, the role of IDH in the regulation of DNA hydroxymethylation in ASM cells is unknown. In comparison with nonasthmatic cells, asthmatic ASM cells exhibited higher TET activity and IDH2 (but not IDH-1 or IDH-3) gene expression levels. We modified the expression of IDH2 in ASM cells from humans with asthma by siRNA and examined the α-KG levels, TET activity, global DNA hydroxymethylation, cell proliferation, and expression of ASM phenotypic genes. Inhibition of IDH2 in asthmatic ASM cells decreased the α-KG levels, TET activity, and global DNA hydroxymethylation, and reversed the aberrant ASM phenotypes (including decreased cell proliferation and ASM phenotypic gene expression). Specifically, asthmatic cells transfected with siRNA against IDH2 showed decreased 5hmC (5-hydroxymethylcytosine) levels at the TGFB2 (transforming growth factor-β2) promoter determined by oxidative bisulfite sequencing. Taken together, our findings reveal that IDH2 plays an important role in the epigenetic regulation of ASM phenotypic changes in asthmatic ASM cells, suggesting that IDH2 is a potential therapeutic target for reversing the abnormal phenotypes seen in asthma.
Keywords: isocitrate dehydrogenase 2, ten-eleven translocation, DNA hydroxymethylation, α-ketoglutarate, airway smooth muscle cells
Clinical Relevance
We demonstrated a novel role of IDH2 (isocitrate dehydrogenase 2), which catalyzes the production of α-ketoglutarate and alters subsequent TET (ten-eleven translocation)-mediated DNA hydroxymethylation, in modulating the transcription activity of genes that regulate airway smooth muscle cell functions. Targeting IDH2 or α-ketoglutarate production may serve as a new approach to reverse the aberrant phenotypes seen in individuals with asthma.
Airway hyperresponsiveness (AHR) is a hallmark of asthma pathogenesis, which is caused by airway inflammation and airway remodeling (1). Numerous studies have revealed that airway remodeling caused by stimulants in patients with asthma might be independent, at least in part, of inflammatory responses (2–4). Airway smooth muscle (ASM) cells serve as the primary effectors of AHR by exaggerating the response to bronchoconstrictor stimuli and increasing ASM thickness by depositing the extracellular matrix and inducing inflammation (5, 6). Any dysregulation of ASM phenotypic genes could lead to the aberrant cell functions seen in humans with asthma. It has been revealed that epigenetic changes (DNA methylation, histone modifications, and noncoding RNAs) in ASM cells prime airway inflammation and remodeling in asthma pathogenesis (reviewed by Kaczmarek and colleagues [7]). We previously showed that decreased promoter methylation of PDE4D (phosphodiesterase 4D) contributes to the increased expression of PDE4D and the increased proliferation and migration of human asthmatic ASM cells (8). Clifford and colleagues reported that loss of repression of vascular endothelial growth factor via histone methylation at its promoter is found in human asthmatic ASM cells (9). Increased expression of long noncoding RNA TCF7 was shown to enhance the viability/proliferation and migration of human asthmatic ASM cells (10). Another study demonstrated that a set of noncoding microRNAs mediated airway inflammation and remodeling through the regulation of “asthma-related” genes (11). Given the fact that epigenetic changes are reversible, the identification of epigenetic mechanisms underlying ASM function may shed light on the reversal of aberrant ASM phenotypes seen in humans with asthma.
DNA methylation is one of the epigenetic mechanisms that regulate gene transcription. The TET (ten-eleven translocation) enzyme oxidizes 5mC (5-methylcytosine) into 5hmC (5-hydroxymethylcytosine), or subsequently to 5fC (5-formylcytosine) and 5caC (5-carboxylcytosine), which ultimately facilitates the removal of DNA methylation (12–14). Previously, we demonstrated that house dust mite–induced AHR in an experimental model was associated with a substantial induction of global DNA hydroxymethylation and differential gene-specific demethylation changes in both lung tissues and isolated tracheal ASM cells (15). In addition, we found increased global 5hmC and Tet1 mRNA expression levels in lung tissues from mice that showed increased airway remodeling (16). Taken together, these findings suggest a novel role of TET-mediated DNA hydroxymethylation in regulating airway remodeling and airway reactivity. Nevertheless, the regulation of TET in ASM cells is not fully understood.
TET is an α-ketoglutarate (α-KG)–dependent enzyme, and the production of α-KG is catalyzed by IDH (isocitrate dehydrogenase). IDHs are a key family of enzymes that are responsible for the conversion of glucose and fatty acid carbons in the citric acid cycle. They catalyze the decarboxylation of isocitrate to produce α-KG in parallel with the reduction of NAD(P) to NAD(P)H. Mammals express three different isoforms of IDHs: cytosolic NADP+-dependent IDH1, mitochondrial NADP+-dependent IDH2, and mitochondrial NAD+-dependent IDH3 (17). IDH1 and IDH2, the NADP+-dependent isoforms, produce NADPH, which is used to reduce glutathione, which participates in the defense against reactive oxygen species and oxidative damage. The inhibition of α-KG production via mutation of IDH1/2 could cause metabolic perturbations and epigenomic alterations (18). IDH1 and IDH2 mutants show inhibition of 5hmC production by TET1 and TET2 (19). In the present study, we found that asthmatic ASM cells exhibited higher IDH2 (but not IDH1 and IDH3) gene expression levels and TET activity. Therefore, we examined the role of IDH2 in the regulation of TET in ASM cells. We demonstrated that the upregulation of IDH2 contributes to increased TET activity and TET-mediated hydroxymethylation in human asthmatic ASM cells. Our results may provide new insights into the epigenetic modulation of the aberrant ASM functions seen in individuals with asthma.
Methods
Details regarding the methods used in this work are provided in the data supplement.
Human ASM Cell Culture
Human ASM cells were isolated from the bilateral lung and trachea of deceased lung donors with and without asthma as described previously (20). The characteristics of these subjects are shown in Table E3 of the data supplement. Subjects were selected by asthmatic status, and not by sex, age, cause of death, or medication history. All experiments were conducted in low-passage cell culture (passages 3–12).
Transfection with siRNA or DNA Plasmid of IDH2
Cells were transfected with 50 nM siRNA against IDH2 (4390825; Ambion) or a scramble sequence (AM4635; Ambion) for IDH2 silencing. Alternatively, the cells were transfected with 500 ng of IDH2 (NM_002168) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression. Functional assays were performed 2 days after the transfection.
mRNA Level by Quantitative PCR
Total RNA was reverse transcribed and then subjected to either TaqMan-based or SYBR Green–based quantitative PCR (qPCR). The 2-ΔΔCt method was used to calculate the relative expression ratio (RER) of transcripts normalized to the housekeeping gene RPL19. The primer sequences are listed in Table E1.
α-KG Assay
ASM cells were lysed in an ice-cold α-KG assay buffer using a Dounce homogenizer. The α-KG level was measured using the α-KG Assay Kit (Sigma Aldrich).
TET Activity Assay
Total nuclear protein (50 μg, isolated using the EpiQuick nuclear extraction kit; Epigentek) was subjected to an Epigenase 5mC hydroxylase TET activity assay (Epigentek).
Global 5hmC Contents in DNA
Genomic DNA (400 ng) was used to measure the levels of 5hmC in the DNA using the 5hmC ELISA kit (Zymo Research).
Oxidative Bisulfite Sequencing of Human TGFB2 Promoter
The hydroxymethylation and methylation status of the human TGFB2 promoter were assessed using the TrueMethyl Seq kit (CEGX). In brief, isolated genomic DNA was either oxidized (conversion of 5hmC into 5fC) or not oxidized (5hmC was maintained). Both oxidized and nonoxidized DNA was treated with bisulfite, followed by PCR using a primer flanking the promoter region. The PCR products were then subcloned and sequenced. The hydroxymethylation status at each CpG site in the human TGFB2 promoter was determined by subtraction from the nonoxidized (5mC + 5hmC) and oxidized (5mC) DNA.
5hmC Enrichment qPCR
Mse I–digested (NEB) DNA was immunoprecipitated with an antibody against 5hmC or IgG (mock control) (EpiQuik hMeDIP kit; Epigentek). DNA released from the reverse crosslink of a DNA–protein complex was purified before qPCR. One percent of the starting fragmented DNA was used as input. The enrichment of 5hmC at the promoter was quantified by SYBR Green–based qPCR. The primers are listed in Table E1.
Statistical Analysis
Technical triplicates were included in all of the assays. The results are expressed as mean ± SEM. Student’s t test was used to analyze the differences in characteristics between nonasthmatic and asthmatic ASM cells. One-way ANOVA with Tukey’s multiple comparison test was used to determine whether the differences between the treatment groups were statistically significant. All of the data were analyzed and plotted with Prism6 (GraphPad).
Results
Asthmatic ASM cells showed increased global DNA hydroxymethylation and TET activity compared with nonasthmatic ASM cells. The global 5hmC level was higher in asthmatic ASM cells than in nonasthmatic ASM cells (Figure 1A). In asthmatic ASM cells, the increased 5hmC level (150% that of nonasthmatic cells) was associated with the upregulation of TET activity (Figure 1B), which is consistent with our previous study in which we found that the global 5hmC level was increased in ASM cells isolated from mice that showed increased AHR (16). In addition, TET1 (but not TET2) mRNA was abundantly expressed in human asthmatic ASM cells at a twofold higher level than in nonasthmatic ASM cells (P < 0.001) (Figures E1A and E1B), suggesting that TET1 may contribute to the upregulation of TET enzymes (Figure 1B). TET3 had very low abundance in both asthmatic and nonasthmatic ASM cells (data not shown). We next examined whether TET upregulation is modulated by the availability of α-KG, which is one of the substrates for TET activity (12). Figure 1C shows that asthmatic ASM cells had a 30% higher α-KG level than nonasthmatic ASM cells. Furthermore, we determined the expression of the IDH gene in ASM cells, given the fact that IDH enzymes facilitate the conversion of isocitrate to α-KG, which may contribute to TET activity and increased global DNA hydroxymethylation (19). Asthmatic ASM cells showed a twofold higher mRNA level of IDH2 than nonasthmatic ASM cells (Figure 1D), whereas there was no difference in the mRNA level of IDH1 and IDH3A between asthmatic and nonasthmatic ASM cells (Figures E1C and E1D). These results suggested that the overexpression of IDH2 in asthmatic ASM cells is associated with increased TET activity and TET-mediated DNA hydroxymethylation.
Figure 1.
Differences in global DNA 5hmC (5-hydroxymethylcytosine) levels, TET (ten-eleven translocation) activity, α-ketoglutarate (α-KG) levels, and mRNA levels of IDH2 (isocitrate dehydrogenase 2) between nonasthmatic and asthmatic airway smooth muscle (ASM) cells. (A and B) Global DNA 5hmC levels (A) and TET activity (B) in asthmatic ASM cells relative to nonasthmatic cells were assayed by ELISA. (C) α-KG levels in cells were assayed by a coupled enzyme assay. (D) mRNA levels of IDH2 were assayed by quantitative PCR (qPCR). Each data point represents an individual lung donor. Values are mean ± SEM (six lung donors without asthma and six with asthma). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with nonasthmatic ASM cells.
To further examine the role of IDH2 in TET activity and TET-mediated DNA hydroxymethylation in asthmatic ASM cells, we introduced siRNA against IDH2 (siIDH2) to suppress expression of the IDH2 gene. Asthmatic ASM cells transfected with siIDH2 for 2 days showed a reduction in the mRNA level of IDH2 by 75% as compared with those transfected with scramble siRNA control (siCTL) (Figure 2A). This suggests that IDH2 was silenced successfully by siIDH2 in asthmatic ASM cells. In accordance with the reduction in IDH2 gene expression levels by siIDH2, there was a significant decrease in the α-KG levels by ∼25% (relative to siCTL; similar to the levels found in nonasthmatic ASM cells) in asthmatic ASM cells (Figure 2B). In addition, siIDH2 decreased the percentage of TET activity by 30% (relative to siCTL) (Figure 2C), and there was a corresponding 50% decrease in the global 5hmC levels (relative to siCTL) (Figure 2D). There were no changes in the mRNA levels of TET1 and TET2 upon siIDH2 treatment (Figure E2). This suggests that IDH2 modulates the activity of the TET enzymes, but not the transcription of TET1/2. Our findings show the role of IDH2 in the modulation of TET activity and global DNA hydroxymethylation in human asthmatic ASM cells, in part through the production of α-KG.
Figure 2.
Knockdown of IDH2 by siIDH2 (siRNA against IDH2) decreased gene expression of IDH2, α-KG levels, TET activity, and global 5hmC levels in human asthmatic ASM cells. (A) mRNA levels of IDH2 were assayed by qPCR. (B) α-KG levels in cells were assayed by a coupled enzyme assay. (C and D) TET activity (C) and global 5hmC levels (D) were examined by ELISA. Each data point represents an individual lung donor. (A–C) All data are represented as the percentage change relative to lipofectamine (LF) controls and shown as mean values ± SEM (six lung donors with asthma). (D) Absolute amount of 5hmC in genomic DNA are present. *P < 0.05 and ****P < 0.0001 in comparison with siCTL (scramble siRNA control)-treated asthmatic ASM cells.
We next sought to determine whether IDH2 alters gene-specific DNA hydroxymethylation in asthmatic ASM cells. Previously, we showed that epigenetic changes in TGFB2 signaling were associated with increased allergen-induced AHR in mice, using a genome-wide DNA methylation profiling technique (MeDIP-seq [methylated DNA immunoprecipitation sequencing]) (16). Strikingly, we demonstrated a significant reduction in Tgfb2 promoter methylation (∼60%) and a corresponding induction in Tgfb2 gene expression (∼75%) in tracheal ASM cells isolated from mice that showed increased AHR (16). Upregulation of TGF-β signaling has been observed in humans with bronchial asthma (21). TGFβ1 is reported to induce ASM cell shortening and hyperresponsiveness (22), and decrease β2-agonist relaxation of human ASM cells (23). Genome-wide association studies revealed a strong association of the TGFB2 genetic variant with chronic obstructive pulmonary disease (24), suggesting that TGFB2 plays a role in lung disease pathogenesis. However, to date, no study has demonstrated the epigenetic regulation of TGFB2 in ASM cells. Here, we found that asthmatic ASM cells showed a twofold higher TGFB2 mRNA level (P < 0.05) than nonasthmatic ASM cells (Figure 3A). In silico analysis revealed high CG dinucleotide (CpG) contents (GC% > 60%) in the human TGFB2 promoter region, which covered a total of 17 CpG sites that encompassed transcription start site and 5′ untranslated region (Figure 3B), suggesting a possible role of promoter methylation in gene regulation. In accord with the increased TGFB2 expression, the percentage of methylation of TGFB2 was significantly lower in asthmatic ASM cells (Figure 3C). Hence, we hypothesized that IDH2 modulates the gene expression of TGFB2 through DNA hydroxymethylation of the TGFB2 promoter in asthmatic ASM cells. After siIDH2 knockdown, the mRNA level of TGFB2 was decreased by 40% in siIDH2-transfected cells compared with siCTL-transfected cells (Figure 3D). The percentages of DNA methylation and hydroxymethylation of each CpG site at the 5′ TGFB2 promoter were examined by oxidative bisulfite sequencing (oxBS-seq). Two of the 17 CpG sites (1, 9) showed a significant increase in the percentage of methylation (compared with siCTL) upon siIDH2 knockdown (Figure 3E). In contrast, suppression of IDH2 by siIDH2 decreased promoter hydroxymethylation at CpG sites 1 and 9 (Figure 3F). These results indicate that IDH2 could affect the CpG-site specific hydroxymethylation of the TGFB2 promoter and the correlated TGFB2 gene expression, which may lead to the modulation of TGFB2 signaling in asthmatic ASM cells. TGFB2 signaling contributes to airway remodeling by modulating cell proliferation (25). We demonstrated that siIDH2 knockdown could reduce cell proliferation (Figure E3), suggesting that IDH2 may modulate ASM cell functions, at least through the epigenetic regulation of ASM phenotypic genes such as TGFB2.
Figure 3.
Knockdown of IDH2 by siIDH2 reduced TGFB2 (transforming growth factor-β2) mRNA levels and hydroxymethylation of the TGFB2 promoter in human asthmatic ASM cells. (A) mRNA levels of TGFB2 in samples were assayed by qPCR. (B) Schematic diagram of CpG dinucleotide (GC) content (percentage) in the 5′ promoter region of TGFB2. In silico analysis identified the CpG islands (shaded in gray in the genomic DNA sequence) based on a GC content > 60% with an observed/expected ratio of 0.6 (MethPrimer). The PCR amplicon generated by PCR is indicated by the regions bounded by arrows. (C) Average of TGFB2 promoter methylation in nonasthmatic and asthmatic ASM cells was assayed by bisulfite sequencing. (D) mRNA levels of TGFB2 in asthmatic ASM cells after siRNA treatment were assayed by qPCR. (E and F) The average percentage of methylation (E) and hydroxymethylation (F) of each CpG site of the TGFB2 promoter in asthmatic ASM cells (six donors) was assayed by oxidative bisulfite sequencing. Four to six individual clones from each donor were picked for sequencing. Each data point represents the mean of six donors in LF controls (solid circles) and siCTL-treated (solid squares) and siIDH2-treated (open triangles) asthmatic ASM cells. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with LF controls. #P = 0.05 compared with siCTL-transfected cells. ATG = translation start site; Ave. = average; TSS = transcription start site; UTR = untranslated region.
To further investigate the effect of siIDH2 knockdown on ASM phenotypic properties, we measured the mRNA levels of ASM phenotypic genes (Table 1) in addition to TGFB2. ASM phenotypic genes function in cell hyperplasia (CCND1 [cyclin D1], PCNA [proliferating cell nuclear antigen], and SMA [smooth muscle actin]), collagen synthesis (COL1A [collagen type Iα] and COL3A [collagen type IIIα]), cell contraction (CAMK2D [calcium/calmodulin-dependent protein kinase II δ] and MLCK [myosin light chain kinase]), and growth factors (SMAD3 and TGFB1 [transforming growth factor β-1]). There were no significant differences in the mRNA levels of CCND1, PCNA, and SMA between siCTL-transfected and siIDH2-transfected cells. siIDH2 knockdown decreased the mRNA level of COL3A (but not COL1A) by 40%. We did not observe any change in the mRNA levels of genes involved in cell contraction (MLCK and CAMK2D). As shown in Figure 3A, siIDH2 knockdown led to a decreased mRNA level of TGFB2. The mRNA level of SMAD3, which is the downstream partner for TGFB2 signaling, was also reduced by 40% upon siIDH2 knockdown, although this was not statistically significant (P = 0.06).
Table 1.
siIDH2 Knockdown Altered Airway Smooth Muscle Phenotypic Gene Expression in Human Asthmatic Airway Smooth Muscle Cells
| Gene |
siCTL |
siIDH2 |
Versus siCTL | ||
|---|---|---|---|---|---|
| RER*, Mean | SEM | RER*, Mean | SEM | P Value† | |
| Cell hyperplasia |
|||||
| CCND1 | 0.95 | 0.12 | 1.12 | 0.27 | 0.65 |
| PCNA | 1.19 | 0.04 | 1.09 | 0.03 | 0.17 |
| SMA | 1.31 | 0.13 | 1.06 | 0.13 | 0.13 |
| Collagen synthesis |
|||||
| COL1A | 1.20 | 0.07 | 1.05 | 0.14 | 0.31 |
| COL3A | 1.41 | 0.19 | 0.88 | 0.12 | 0.01 |
| Cell contraction |
|||||
| CAMK2D | 1.03 | 0.08 | 0.99 | 0.06 | 0.91 |
| MLCK | 1.09 | 0.10 | 1.08 | 0.13 | >0.99 |
| Growth factors/mediators |
|||||
| SMAD3 | 1.03 | 0.10 | 0.60 | 0.11 | 0.06 |
| TGFB1 | 1.03 | 0.13 | 1.34 | 0.28 | 0.27 |
Definition of abbreviations: CAMK2D = calcium/calmodulin-dependent protein kinase IIδ; CCND1 = cyclin D1; COL1A = collagen type Iα; COL3A = collagen type IIIα; IDH2 = isocitrate dehydrogenase 2; MLCK = myosin light chain kinase; PCNA = proliferating cell nuclear antigen; RER = relative expression ratio; siCTL = scramble siRNA control; siIDH2 = siRNA against IDH2; SMA = smooth muscle actin; SMAD3 = SMAD family member 3; TGFB1 = transforming growth factor-β1.
The mRNA level of airway smooth muscle phenotypic genes was examined by quantitative PCR and normalized to that of the housekeeping gene RPL19. RER values were calculated by a 2−ΔΔCt method, and RER was assigned a value of 1.00 for lipofectamine controls. All data were expressed as mean values ± SEM. Data were obtained from six lung donors with asthma.
One-way ANOVA with Tukey’s multiple comparison test was used to determine statistical significance between siCTL- and siIDH2-treated cells.
Next, we aimed to test whether IDH2 modulates gene-specific transcription through DNA hydroxymethylation. We used 5hmC-enrichment qPCR to demonstrate 5hmC binding at the COL3A, SMAD3, and TGFB2 promoter regions (Table 2). 5hmC-enrichment qPCR is commonly used to detect binding of 5hmC at a specific region of the promoter (∼80–200 bp) in a relatively high-throughput manner as compared with oxBS-seq (which requires chemical treatment, PCR, cloning, and sequencing). For example, by using oxBS-seq, we identified a region at the TGFB2 promoter encompassing CpG sites 1 and 9, which was associated with the differential DNA hydroxymethylation changes observed upon siIDH2 treatment (Figure 3F). We next used 5hmC-enrichment qPCR instead of oxBS-seq to examine the changes in DNA hydroxymethylation at that region. There was a significant reduction in 5hmC enrichment at the COL3A and TGFB2 promoters upon siIDH2 knockdown. The SMAD3 promoter showed a trend in decreased 5hmC enrichment, but this was not statistically significant. Taken together, these findings suggest that siIDH2 knockdown could reverse aberrant ASM phenotypic changes, at least in part through the epigenetic regulation of gene promoter hydroxymethylation (COL3A and TGFB2), in asthmatic ASM cells.
Table 2.
siIDH2 Knockdown Altered 5hmC Enrichment at the Gene Promoter of COL3A, SMAD3, and TGFB2
| Gene* |
siCTL |
siIDH2 |
Versus siCTL | ||
|---|---|---|---|---|---|
| Percentage Change*, Mean | SEM | Percentage Change*, Mean | SEM | P Value† | |
| COL3A | 123.70 | 7.72 | 48.26 | 5.31 | 0.05 |
| SMAD3 | 98.82 | 10.36 | 77.31 | 2.22 | 0.25 |
| TGFB2 | 107.90 | 6.49 | 62.19 | 11.24 | 0.02 |
Definition of abbreviation: 5hmC = 5-hydroxymethylcytosine.
5hmC enrichment at the gene promoter was measured by 5hmC enrichment quantitative PCR. The percentage change of 5hmC enrichment relative to lipofectamine controls was calculated by a 2−ΔΔCt method. All data were expressed as mean values ± SEM. Data were obtained from six asthmatic lung donors.
One-way ANOVA with Tukey’s multiple comparison test was used to determine the statistically significant difference between siCTL- and siIDH2-treated cells.
Our data suggest that increased IDH2 expression in asthmatic ASM cells is associated with increased TET activity and both global and gene-specific DNA hydroxymethylation. We next examined whether the overexpression of IDH2 in human nonasthmatic ASM cells could drive the increased gene-specific DNA hydroxymethylation and its correlated transcription of ASM phenotypic genes. Nonasthmatic ASM cells transfected with the IDH2 plasmid showed a 50-fold increase in the mRNA level of IDH2 (Figure 4A). α-KG production (Figure 4B) showed an increasing trend (P = 0.07), and TET activity (Figure 4C) was significantly increased by 50%. However, there was no difference in the global 5hmC content in cells that overexpressed IDH2 (Figure 4D). In terms of the effect of IDH2 overexpression on the transcription of ASM phenotypic genes, we showed a trend of induction in both mRNA and 5hmC enrichment levels of COL3A, SMAD3, and TGFB2 (these genes were found to be regulated by IDH2-mediated hydroxymethylation; Table 2), but the results were not statistically significant (Tables 3 and 4). Our results indicate that the transient overexpression of IDH2 may drive the induction of TET activity in nonasthmatic ASM cells, although we did not observe any significant increase in the expression levels or promoter hydroxymethylation of these genes. It is believed that long-term overexpression of IDH2 may contribute to a stable epigenetic modification at these promoters, which may contribute to the aberrant ASM cell functions.
Figure 4.
Overexpression of IDH2 showed increased α-KG levels and TET activity in human nonasthmatic ASM cells. (A) mRNA levels of IDH2 were assayed by qPCR. (B) α-KG levels in cells were assayed by a coupled enzyme assay. (C and D) TET activity (C) and global 5hmC levels (D) were examined by ELISA. Each data point represents an individual lung donor. All data are represented as the percentage change relative to LF controls and shown as mean values ± SEM (four lung donors without asthma). *P < 0.05 compared with pCMV6-transfected ASM cells.
Table 3.
Overexpression of IDH2 Altered Airway Smooth Muscle Phenotypic Gene Expression in Human Nonasthmatic Airway Smooth Muscle Cells
| Gene |
pCMV6 |
pCMV6-IDH2 |
Versus pCMV6 | ||
|---|---|---|---|---|---|
| RER*, Mean | SEM | RER†, Mean | SEM | P Value* | |
| Cell hyperplasia |
|||||
| CCND1 | 1.26 | 0.40 | 0.95 | 0.05 | 0.78 |
| PCNA | 1.56 | 0.05 | 1.37 | 0.08 | 0.06 |
| SMA | 1.09 | 0.10 | 1.10 | 0.21 | >0.99 |
| Collagen synthesis |
|||||
| COL1A | 0.87 | 0.03 | 0.73 | 0.08 | 0.18 |
| COL3A | 0.86 | 0.06 | 1.17 | 0.23 | 0.56 |
| Cell contraction |
|||||
| CAMK2D | 0.90 | 0.10 | 1.17 | 0.26 | 0.53 |
| MLCK | 0.91 | 0.15 | 1.21 | 0.24 | 0.5 |
| Growth factors/mediators |
|||||
| SMAD3 | 1.18 | 0.14 | 2.20 | 0.60 | 0.23 |
| TGFB1 | 1.28 | 0.30 | 1.51 | 0.30 | 0.28 |
| TGFB2 | 1.20 | 0.25 | 2.49 | 0.72 | 0.14 |
One-way ANOVA with Tukey’s multiple comparison test was used to determine the statistically significant difference between pCMV6- and pCMV6-IDH2–treated cells.
The mRNA level of ASM phenotypic genes was examined by real-time PCR and normalized to the housekeeping gene RPL19. RER values were calculated by a 2−ΔΔCt method, and RER was assigned a value of 1.00 for lipofectamine controls. All data were expressed as mean values ± SEM. Data were obtained from four lung donors without asthma.
Table 4.
Overexpression of IDH2 Altered 5hmC Enrichment at the Gene Promoter of COL3A, SMAD3, and TGFB2
| Gene |
pCMV6 |
pCMV6-IDH2 |
Versus pCMV6-AC | ||
|---|---|---|---|---|---|
| Percentage Change*, Mean | SEM | Percentage Change*, Mean | SEM | P Value† | |
| COL3A | 111.20 | 8.71 | 157.80 | 25.42 | 0.3 |
| SMAD3 | 119.30 | 14.12 | 156.80 | 44.03 | 0.57 |
| TGFB2 | 104.70 | 6.70 | 132.10 | 14.78 | 0.25 |
5hmC enrichment at the gene promoter was measured by 5hmC enrichment quantitative PCR. The percentage change of 5hmC enrichment relative to lipofectamine controls was calculated by a 2−ΔΔCt method. All data were expressed as mean values ± SEM. Data were obtained from four lung donors without asthma.
One-way ANOVA with Tukey’s multiple comparison test was used to determine the statistically significant difference between pCMV6- and pCMV6-IDH2–treated cells.
Discussion
In this study, we showed increased global DNA hydroxymethylation and TET activity in asthmatic ASM cells as compared with nonasthmatic cells. In addition, we provided new evidence that the upregulation of TET could be modulated by IDH2, a protein involved in the citric acid cycle. Using an experimental asthma model chronically exposed to house allergens, we demonstrated that increased airway inflammation and airway remodeling were associated with increased Tet1 expression and global 5hmC levels in whole lung tissues and isolated tracheal ASM cells (16). Somineni and colleagues reported that CpG-site–specific hypomethylation at the TET1 promoter in nasal epithelial samples was associated with childhood asthma status (n = 12, P = 0.014) in an African-American sibling cohort (26). The loss of DNA methylation patterns at the TET1 promoter was consistent across different tissues, including peripheral blood mononuclear cells and saliva. In addition, this finding was validated on saliva DNA from an independent cohort that included children with asthma (n = 158) and children without allergy or asthma (n = 28). An in vitro model of treating human bronchial epithelial cells with DNA demethylating agent (5aza) showed an association between TET1 demethylation and increased TET1 mRNA and global 5hmC levels. Our data suggested that ASM cells from subjects with asthma had increased levels of TET1 mRNA and 5hmC compared with those obtained from subjects without asthma. Together, these findings suggest that increased TET1 expression levels and global DNA hydroxymethylation are associated with asthmatic status. However, the regulation of TET protein, as well as its mediated hydroxymethylation, was not investigated in these studies (16, 26).
TET proteins facilitate the oxidation of 5mC to 5hmC (12–14). Cofactors such as α-KG have been shown to play an important role in regulating TET activity and DNA hydroxymethylation (12, 18, 19). In this study, asthmatic ASM cells showed an increase in α-KG levels, which was associated with the increase in TET activity and global 5hmC level. In addition, we provided evidence that the increase in α-KG levels in asthmatic ASM cells could be due to the increased expression of IDH2, which is a mitochondrion-specific IDH and functions in the conversion of isocitrate in the citric acid cycle (27). We did not find any difference in the mRNA levels of the cytosolic IDH1 and another mitochondrial IDH3A (catalytic subunit) between nonasthmatic and asthmatic ASM cells, suggesting that the upregulation of IDH2 may contribute to the increase in α-KG levels seen in asthmatic ASM cells. Other studies have suggested that IDH1/2 mutation causes changes in the production of the oncogenic metabolite 2HG (R-2-hydroxyglutarate), which inhibits IDH1/2 functions (18, 28–30). In addition, these mutations may contribute to alterations in epigenetics such as DNA methylation (31, 32) and DNA hydroxymethylation (19, 33). The ASM cells used in this study did not show any mutation in IDH1 (codon 132) or IDH2 (codon 172), as determined by pyrosequencing (Table E2). This indicates that the increases in the global 5hmC level and TET activity seen in asthmatic ASM cells are not due to genetic variations of IDH1 and IDH2. Furthermore, asthmatic ASM cells treated with siIDH2 did not show any difference in 2HG levels as compared with those treated with siCTL (Figure E5). This suggests that knockdown of IDH2 altered the production of α-KG, but not 2HG, which in turn affected TET activity and 5hmC production because of the low availability of α-KG substrate.
Our data demonstrated that siIDH2 treatment significantly reduced 5hmC levels by 50% in asthmatic ASM cells (Figure 2D) but did not affect TET1 expression (Figure E2). This suggests that IDH2 alters 5hmC production by regulating the activity (via availability of α-KG), but not the transcription of TET1. Although the mRNA level of IDH2 was reduced by siIDH2 to 25% of that of siCTL-treated cells, we did not observe a massive reduction of 5hmC in the siIDH2-treated cells. We may speculate that this was due to the increased TET1 expression in asthmatic ASM cells. Knockdown of TET1 in asthmatic ASM cells decreased 5hmC by 25–30% (Figure E4). This suggests that, other than IDH2 induction, increased TET1 mRNA level in asthmatic ASM cells also contributes to increased 5hmC production. Cotreatment of siTET1 and siIDH2 caused a greater decrease (70–80%) in 5hmC levels in asthmatic ASM cells (Figure E4). This indicates that 5hmC production in asthmatic ASM cells could be modulated by both IDH2-mediated TET activity (via availability of α-KG) and TET1 expression. These results provide new insights into combining treatments to target the aberrant TET-mediated gene hydroxymethylation changes and cell functions of asthmatic ASM cells.
We showed that the modulation of IDH2 expression could affect the aberrant epigenetic changes seen in asthmatic ASM cells. The loss of IDH2 caused a significant decrease in 5hmC binding at the promoters of COL3A and TGFB2, which may result in a decrease in their gene expression levels. COL3A encodes type III collagen, which was shown to be upregulated in the house dust mite–induced mouse asthma model (34). TGFB2 is involved in TGFB2 signaling, which contributes to airway remodeling (35–38). This finding is consistent with our experimental asthma model, in which we found that the promoter demethylation of Tgfb2 in mouse ASM cells was associated with increased AHR (16). Our current findings suggest that inhibiting IDH2 expression in ASM cells may abrogate collagen synthesis and TGFB2 signaling, at least through the DNA hydroxymethylation of genes. The epigenetic regulation of these genes may help reverse the aberrant cell phenotypes (such as cell proliferation) seen in individuals with asthma; however, this remains to be addressed by future functional studies of these gene promoters in asthmatic ASM cells.
We did not observe any changes in the mRNA levels of AHR-related genes involved in cell hyperplasia (CCND1, PCNA, and SMA), collagen synthesis (COL1A), cell contraction (CAMK2D and MLCK), and growth factor signaling (SMAD3 and TGFB1) upon inhibition of IDH2. This suggests that the regulation of these AHR phenotypic genes may be modulated by other post-transcriptional and/or post-translational mechanisms instead of IDH2-mediated hydroxymethylation. On the other hand, in nonasthmatic ASM cells, overexpression of IDH2 upregulated TET activity but did not cause any changes in the global 5hmC level and gene-specific DNA hydroxymethylation of TGFB2, SMAD3, and COL3A. We might argue that the transient transfection of IDH2 may not upregulate TET activity to a level that induces significant epigenetic changes at these promoters. Perhaps, in the future, we can determine whether the stable transfection of IDH2 could modulate the promoter hydroxymethylation of these genes seen in asthmatic ASM cells.
Changes in DNA methylation at an individual CpG can be crucial in affecting the DNA binding specificities of transcription factors (TFs) leading to gene transcription. Bisulfite sequencing cannot distinguish between 5mC and 5hmC; therefore, we performed oxBS-seq (39) to examine the hydroxymethylation status of the gene promoter. In this study, we demonstrated that siIDH2 knockdown decreased DNA hydroxymethylation at CpG sites 1 (P < 0.01) and 9 (P = 0.05) of the TGFB2 promoter as compared with the siCTL-treated samples. By performing an; in silico search of the TRANSFAC database (40), we found that CpG site 9 encompasses the putative binding site for TFs such as c-MYC and USF1 (upstream stimulatory factor 1). We did not find any putative binding site of TFs located at CpG site 1. c-MYC contributes to cell proliferation, promotion of cell transformation, and glutamine metabolism (41). USF1 interacts directly with histone-modifying enzymes and plays a role in regulating chromatin barrier insulators (42). A decrease in the DNA hydroxymethylation level occurring at the recognition sites of TFs may suppress gene transcription by inhibiting the recruitment of either TFs or histone modification enzymes to the promoter. Although future studies are needed to examine the transcriptional regulation of the TGFB2 promoter, our data provide insights into the development of specific inhibitors of TGFB2 that may act by targeting the hydroxymethylation pattern at specific CpGs, which may contribute to a reduction in gene expression levels.
Here, we show that IDH2 modulates TET activity and DNA hydroxymethylation, at least through an alteration in α-KG production in ASM cells. In addition to TET enzymes, there are more than 60 α-KG–dependent dioxygenases that participate in cell physiology and differentiation (17). Histone demethylases, prolyl hydroxylases, and collagen prolyl-4-hydroxylases are known as α-KG–dependent dioxygenases (43–46). The inhibition of α-KG production by either mutation of IDH1/2 or competitors of α-KG was shown to modulate histone demethylases and consequent changes in the methylation of DNA and histones (19, 43–46). We found that inhibition of IDH2 showed a trend toward reductions in the relative mRNA levels of SMAD3 (P = 0.06), but not in 5hmC enrichment at its promoter. This suggests that IDH2 may regulate the transcription of SMAD3 through epigenetic mechanisms other than DNA hydroxymethylation; however, further study is needed to prove this hypothesis. On the other hand, the production of α-KG is known to alter collagen synthesis via the hydroxylation of collagen through prolyl-3 and prolyl-4 hydroxylases in a nonepigenetic manner (45). We found that siIDH2 knockdown decreased the mRNA level of COL3A. It is plausible that the inhibition of IDH2 may decrease the hydroxylation of collagens in addition to the TET-mediated effect, resulting in a decreased collagen synthesis in asthmatic ASM cells.
In this study, we demonstrated that the effect of IDH2 in human asthmatic ASM cells on alterations in the DNA hydroxymethylation level is exerted via the TET1 enzyme, but not TET2, as we did not observe any changes in the mRNA level of TET2 as compared with nonasthmatic ASM cells. In the future, an experimental asthma model should be used to validate the effect of IDH2 on TET1-mediated methylation changes and the subsequent changes in asthma pathogenesis. Whether the effect of IDH2 on the lung epigenome is cell, time, or exposure specific remains to be determined in further studies. Here, we demonstrated that IDH2 modulates the activity of TET by regulating the availability of α-KG. However, we did not exclude other possible components or signaling pathways that may be altered by IDH2 or citrate metabolism and subsequently affect TET activity and 5hmC production. More studies are required to demonstrate the mechanisms underlying the regulation of IDH2. IDH2 enzymatic activity can be inhibited by acetylation at lysine residues. SIRT3, a mitochondrial NAD+-dependent class III histone deacetylase, facilitates IDH2 activity, at least through the deacetylation of K413 (47). Further studies are needed to investigate a possible link among mitochondrial redox, citrate metabolism, and epigenetic changes. Optimization of energy metabolism with mitochondria-targeted therapeutics has been suggested to ameliorate chronic lung disease symptoms (48).
In this study, we found that overexpression of IDH2 in asthmatic ASM cells was associated with an increase in 5hmC levels. We acknowledge that our sample size was relatively small. It is possible that these changes would not be consistently observed in a larger study of ASM cells, owing to differences in age, sex, environmental exposures, allergen sensitivities, severity of disease, or medication use. Future studies with larger sample sizes and a well-defined population of humans with asthma may help to improve our understanding of the role of IDH2 in the epigenetic regulation of ASM cell functions. Taken together, many lines of evidence indicate that energy metabolism can modulate epigenetic changes of the genome by regulating DNA methylation and histone modifications, which may ultimately affect the development of various diseases, including cancer (reviewed in Reference 49). Future investigation of the relationship between IDH2 and TET-mediated hydroxymethylation in asthma pathogenesis by means of omic studies is highly recommended. This will allow us to target the epigenetic changes and metabolic pathways in specific lung cell types at the right time. Moreover, the results should provide new insights into the development of precise epigenetic therapeutic approaches for lung disease. IDH enzymes are already targets in cancer therapy (50). We showed that the inhibition of IDH2 epigenetically modified the transcription of AHR-phenotypic genes in asthmatic ASM cells. Targeting IDH2 or α-KG production (50) may serve as a new approach to reverse the aberrant phenotypes seen in individuals with asthma.
Supplementary Material
Acknowledgments
Acknowledgment
The authors thank the donors, families, and staff of the Gift of Hope Organ and Tissue Donor Network for their generous donation of lung tissues used in this study.
Footnotes
Supported by National Institute of Environmental Health Sciences grant ES-024784 (W.-y.T.) and U.S. National Heart, Lung, and Blood Institute grant HL-114471 (S.S.A.).
Author Contributions: Participated in research design and performed data analysis: B.H.Y.Y. and W.-y.T. Conducted experiments: B.H.Y.Y. Contributed reagents and cell lines: J.H., S.S.A., J.S., and W.-y.T. Wrote or contributed to the manuscript: B.H.Y.Y., J.H., S.S.A., J.S., W.M., and W.-y.T.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2019-0323OC on March 9, 2020
Author disclosures are available with the text of this article at www.atsjournals.org.
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