Epigenetic regulation of myofibroblast differentiation by DNA methylation - PubMed (original) (raw)

Epigenetic regulation of myofibroblast differentiation by DNA methylation

Biao Hu et al. Am J Pathol. 2010 Jul.

Abstract

DNA methylation, a key mechanism of repressing gene expression, is of particular relevance in controlling development and cell differentiation. We analyzed the extent and regulation of DNA methylation of the alpha-smooth muscle actin (alpha-SMA) gene to elucidate its potential role in myofibroblast differentiation. These experiments revealed the presence of three CpG islands that were methylated at different levels in fibroblasts, myofibroblasts, and alveolar epithelial type II cells. Coordinately, these cells expressed low, high, or no alpha-SMA, respectively. In addition, inhibition of DNA methyltransferase activity or knock down of DNA methyltransferase using specific small interfering RNA caused significant induction of alpha-SMA in fibroblasts. In contrast, induced overexpression of DNA methyltransferase suppressed alpha-SMA gene expression. Transforming growth factor beta induced myofibroblast differentiation was enhanced or suppressed by knockdown or overexpression of DNA methyltransferase, respectively. Finally, in vitro DNA methylation of the alpha-SMA promoter suppressed its activity. These findings suggest that DNA methylation mediated by DNA methyltransferase is an important mechanism regulating the alpha-SMA gene expression during myofibroblast differentiation.

PubMed Disclaimer

Figures

Figure 1

A: Location of CpG islands in α-SMA gene. DNA sequence of α-SMA gene −2900 to + 3100 from transcriptional start site was submitted to the EMBOSS CpGPlot/CpGReport/Isochore program online. The results revealed three islands between bp 2142 to 2244 (corresponding to −759 to −656 from transcriptional start site, namely promoter region), bp 3095 to 3199 (corresponding to + 195 to + 299 from transcriptional start site, namely first intro region1), and bp 3339 to 3452 (corresponding to + 439 to + 552 from transcriptional start site, namely first intron region 2). B: Methylation status of CpG islands in the α-SMA gene. This was determined by DNA pyrosequencing of the α-SMA gene promoter and the first intron regions 1 and 2 where CpG islands were located. The position of each methylated cytidine from the transcriptional start site was indicated in the x axis. The results were expressed as percentages and shown as the means ± SE from three independent experiments.

Figure 2

Dnmt expression in fibroblasts (RLF) and epithelial cells (AEC). A: Total RNA from the indicated cell type were analyzed by real-time PCR for the indicated Dnmt isoform mRNA. The results were expressed as 2−ΔΔCT with 18s rRNA used as the endogenous control. Data were shown as the mean ± SE from triplicate samples. An asterisk indicated statistically significant difference when compared with the corresponding mean value in AECs. B: DNA methyltransferase assay was conducted using equal amounts of freshly prepared nuclear extracts from either fibroblasts (RLF) or epithelial cells (AEC). The results were expressed as a percentage of the mean activity in fibroblasts. Mean ± SE from triplicate samples are shown.

Figure 3

Effect of DNA methyltransferase inhibitor on α-SMA expression. A: Fibroblasts were treated with the indicated doses of DNA methyltransferase inhibitor, 5-aza-dC for three days. Total RNA was then extracted and analyzed for α-SMA mRNA by real-time PCR. The results were expressed as 2−ΔΔCT using 18s rRNA as the endogenous control. Data were shown as the mean ± SE from triplicate samples. All treated samples showed significantly higher levels of α-SMA expression compared with untreated controls. B: Fibroblasts were treated with 5-aza-dC at the indicated dose for three or five days, and then analyzed for α-SMA protein levels by Western blotting. Equal amounts of total protein were loaded onto the gels for separation. The membrane was striped and reblotted with horseradish peroxidase-conjugated anti-glyceraldehyde-3-phosphate dehydrogenase antibody as a loading control. C: The cells were treated with either control buffer (“None”) or 5-aza-dC for 72 hours. The genomic DNA was then isolated and pyrosequenced using primers corresponding to the α-SMA gene promoter region. The extent of methylation (expressed as percentages) was plotted versus the corresponding position of the CpG island from the transcriptional start site. The results were shown as mean ± SE.

Figure 4

Effect of Dnmt siRNAs on fibroblast α-SMA expression. A: Fibroblasts were transfected with the indicated siRNA construct or constructs or all three constructs (“ALL”) for 48 hours and then analyzed for α-SMA mRNA by real-time PCR. The results were expressed as 2−ΔΔCT using 18s rRNA as the endogenous control. Data were shown as the mean ± SE from triplicate samples. Mean values of cells treated with Dnmt siRNA were significantly higher than that of cells treated with the control vector. B: The indicated siRNA construct or all three constructs (“ALL”) were transfected into fibroblasts and then stained with phosphatidylethanolamine-conjugated anti-α-SMA antibody followed by flow cytometric analysis. The dashed line indicated the IgG control, while the solid line represented the cells transfected with control siRNA and stained with phosphatidylethanolamine-conjugated anti-α-SMA antibody. The shaded region represented cells transfected with the indicated Dnmt siRNA construct(s) stained with phosphatidylethanolamine-conjugated anti-α-SMA antibody. C: Fibroblasts were transfected with the indicated individual Dnmt siRNA or all three siRNAs together (“ALL”) and treated with either buffer only (“Buffer”) or TGFβ (4 ng/ml) for 72 hours. On harvest, equal amounts of total protein extracts were loaded onto gels for Western blotting. The membrane was striped and reblotted with HRP conjugated anti-glyceraldehyde-3-phosphate dehydrogenase antibody as a loading control.

Figure 5

Effect of Dnmt overexpression on α-SMA expression. A: Fibroblasts were transfected with indicated the indicated Dnmt isoform expression plasmid or all three plasmids (“ALL”) for 48 hours. They were then analyzed for α-SMA mRNA levels by quantitative real-time PCR. The results were expressed as 2−ΔΔCT using 18s rRNA as the endogenous control. Data were shown as the mean ± SE from triplicate samples. All samples treated with Dnmt plasmid(s) were significantly different from the sample treated with the control vector. B: The cells were transfected with the indicated individual Dnmt isoform expression plasmid or all three plasmids together (“ALL”) and treated with either buffer only (“Buffer”) or TGFβ for 72 hours and then analyzed for α-SMA content by Western blotting. The membrane was stripped and reblotted with HRP conjugated anti-glyceraldehyde-3-phosphate dehydrogenase antibody as a loading control. C: Rat lung fibroblasts were treated with 4 ng/ml TGFβ or buffer only (“Buffer”) for 6 hours and then total RNA was extracted and analyzed for mRNA of the indicated dnmt gene by real-time PCR. The results were expressed as mean ± SE with N = 3. The inhibition by TGFβ treatment was significant only for dnmt1 and dnmt3a mRNA.

Figure 6

Effect of DNA methylation on α-SMA promoter activity. α-SMA promoter constructs without the first intronic region (α-SMApro-Luc) and the α-SMA promoter constructs with the first intronic region (α-SMApro-intron-Luc) were methylated in vitro and transfected into rat lung fibroblast (diagrams of constructs in A). B: The transfected cells were treated with either TGFβ or buffer only for 48 hours and tested for the promoter activity in terms of luciferase activity in relative light units. Experiments with each construct were repeated two to four times and the results were expressed as mean ± SE.

References

1. Gabbiani G, Ryan GB, Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia. 1971;27:549–550. - PubMed
1. Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML, Gabbiani G. The myofibroblast: one function, multiple origins. Am J Pathol. 2007;170:1807–1816. - PMC - PubMed
1. Phan SH. The myofibroblast in pulmonary fibrosis. Chest. 2002;122:286S–289S. - PubMed
1. Orimo A, Weinberg RA. Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle. 2006;5:1597–1601. - PubMed
1. Hinz B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol. 2007;127:526–537. - PubMed

Epigenetic regulation of myofibroblast differentiation by DNA methylation - PubMed (original) (raw)