Aberrant MFN2 transcription facilitates homocysteine‐induced VSMCs proliferation via the increased binding of c‐Myc to DNMT1 in atherosclerosis

Abstract It is well‐established that homocysteine (Hcy) is an independent risk factor for atherosclerosis. Hcy can promote vascular smooth muscle cell (VSMC) proliferation, it plays a key role in neointimal formation and thus contribute to arteriosclerosis. However, the molecular mechanism on VSMCs proliferation underlying atherosclerosis is not well elucidated. Mitofusin‐2 (MFN2) is an important transmembrane GTPase in the mitochondrial outer membrane and it can block cells in the G0/G1 stage of the cell cycle. To investigate the contribution of aberrant MFN2 transcription in Hcy‐induced VSMCs proliferation and the underlying mechanisms. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase in atherosclerotic plaque of APOE−/− mice with hyperhomocystinaemia (HHcy) as well as in VSMCs exposed to Hcy in vitro. The DNA methylation level of MFN2 promoter was obviously increased in VSMCs treated with Hcy, leading to suppressed promoter activity and low expression of MFN2. In addition, we found that the expression of c‐Myc was increased in atherosclerotic plaque and VSMCs treated with Hcy. Further study showed that c‐Myc indirectly regulates MFN2 expression is duo to the binding of c‐Myc to DNMT1 promoter up‐regulates DNMT1 expression leading to DNA hypermethylation of MFN2 promoter, thereby inhibits MFN2 expression in VSMCs treated with Hcy. In conclusion, our study demonstrated that Hcy‐induced hypermethylation of MFN2 promoter inhibits the transcription of MFN2, leading to VSMCs proliferation in plaque formation, and the increased binding of c‐Myc to DNMT1 promoter is a new and relevant molecular mechanism.


| INTRODUC TI ON
Atherosclerosis is a chronic progressive disease which is characterized by the formation of atheromatous plaque in the intimal layer that mainly derived from the deregulation of cell behaviour, such as the activation of macrophages and abnormal vascular smooth muscle cells (VSMCs) proliferation and so on. 1,2 It has been indicated that homocysteine (Hcy) is an independent risk factor for atherosclerosis and it can induce endothelial dysfunction, foam formation and promote VSMC proliferation. 3,4 As an important component of the medial layer of blood vessels, VSMCs migration and proliferation with subsequent formation of intimal thickening is important for the development of atherosclerotic lesions. However, little is known about the underlying mechanisms regarding the aberrant VSMCs proliferation during atherosclerotic plaque formation.
Mitochondrial GTPase mitofusin-2 (MFN2), also known as a hyperplasia suppressor gene, is widely distributed in mammalian. 5 Recently, it was reported that MFN2 plays an essential role in mitochondrial fusion, which regulates mitochondrial morphology and function in multiple cell types. 6 Meanwhile, MFN2 has been demonstrated to be low expressed in various types of human malignant tumours, such as gastric cancer, hepatocellular cancer, colorectal cancer and breast cancer. 7,8 Apart from its role in cancer, clinical evidence revealed that dysfunction of MFN2 is also involved in the pathophysiology of several cardiovascular diseases including hypertension, restenosis after angioplasty, cardiac hypertrophy and cardiac oxidative stress injury. 9,10 Intriguingly, MFN2 was found to exert an anti-proliferative effect by inducing more cells at the G0/G1 phase in cultured MCF-7 cells and a rat carotid artery balloon-injury model. 11 These data led us to suggest that destabilization of MFN2 may play a role in the excessive proliferation of VSMCs during the formation of atherosclerotic plaque.
Hcy is a product of the methionine cycle, it is involved in one-carbon methyl group-transmethylation pathway and acts as a methyl donor when it is converted to S-adenosyl-methionine (SAM). 12 Accumulating evidences demonstrated that aberrant DNA methylation induced by Hcy is associated with various diseases including atherosclerosis, osteoporosis, uraemia and alcoholism. 13,14 DNA methylation could directly modulate gene transcription via recruiting chromatin remodelling proteins and modulating the binding affinities of specific transcription factors. 15 In eukaryotes, it is well accepted that transcriptional activity of the specific genes was influenced by epigenetic marks and interplay between transcription factors and the cis-elements of specific promoters in time and space, which is closely related to gene expression. 16 MFN2 expression has been reported to be regulated by a series of transcription factors that control mitochondrial biogenesis and functions, including ERRalpha and MEF2. As a highly conserved transcription factor, c-Myc could drive cell stress, proliferation and apoptosis through integrating multiple cellular signals and mediating a transcriptional response. 17 Previous studies demonstrated that c-Myc is involved in the transcriptional regulation of the specific genes in different ways, and one of its regulatory functions involved gene transcription driven by the binding to E-box sequences located on the gene promoter regions. 18 In addition to its function as an activator, c-Myc also can repress transcription of genes through interaction with epigenetic modification such as DNA methylation.
c-Myc may orchestrate DNMTs to regulate common target genes linked to multiple networks in the development and progression of diseases. In lung cancer cells, DNMT3b could be recruited to the promoter region of RAS association domain family1A (RASSF1A) by c-Myc to silence its expression through DNA hypermethylation. 19 A comprehensive understanding of this dynamic interplay will set the stage, not only for the design of novel treatment strategies, but also for the discovery of pan-cellular transcription factor regulatory strategies to predict disease risk, therapy response and patient prognosis 20 and the dynamics of the mode of binding to DNA has changed this postulate and paved the way for new therapies targeted against VSMCs proliferation.
In this study, we aimed to elucidate the role of MFN2 in Hcy-induced VSMCs proliferation during the formation of atherosclerotic plaque and the relevant molecular mechanisms. Our findings revealed that c-Myc binding to DNMT1 promoter positively regulates DNMT1 expression, and DNMT1-mediated DNA hypermethylation of MFN2 promoter thereby inhibits MFN2 expression in VSMCs proliferation induced by Hcy. These findings shed new insight into the mechanism of Hcy-induced VSMCs proliferation in atherosclerosis and may be a therapeutic tool in the treatment of Hcy-induced cardiovascular diseases.

| Chemicals and reagents
Cell-Light EdU Apollo 567 In Vitro Imaging Kit was from RiboBio

| Animals
Six-week-old male APOE −/− mice with C57BL/6J genetic background were provided by Animal Center of Peking University (Beijing, China). They were housed in a temperature-controlled (24°C) facility with a 12 hours light/dark cycle. After 1 week of acclimatization, APOE −/− mice were randomly divided into two groups (n = 6 each) and fed with regular diet (APOE −/− +NC) or fed with regular diet plus 1.7% methionine (APOE −/− +HMD). After 15-week experimental diets, mice were killed with pentobarbital (50 mg/kg body weight), and aortic tissues were frozen in liquid nitrogen and stored at 80°C until further analysis. All animal experiments were approved by the Ethics of Animal Experiments of the Health Science Center of Ningxia Medical University.

| Haematoxylin and eosin (HE) and oil red O staining
The aortas in APOE -/mice were flushed with saline and embedded in OCT after sacrifice. Frozen sections were cut in 4 μm thickness, followed by staining with haematoxylin and eosin (HE) and Oil Red O staining. Details about the staining can be found in our previous study, 3 and lipid-stained lesions were measured by digitizing morphometry and reported in mm 2 per lesion.

| Detection of serum Hcy
Blood samples collected from the mice were centrifugated at 3000 g for 10 min at 4°C after standing at room temperature for 30 min, then serum concentrations of Hcy were measured by automatic biochemistry analyzer (SIEMENS, Germany).

| Cell culture
Human VSMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin. Cells at 80% confluence were subsequently treated with Hcy at the concentrations of 0 (control), 50, 100, 200 and 500 μmol/L for 72 hours, medium were changed every 12 hours due to the short half-life of Hcy. Recombinant adenoviruses expressing DNMT1 or c-Myc gene were purchased from HANBIO (Shanghai, China), the plasmid expressing MFN2, siRNAs specifically targeting MFN2, DNMT1, c-Myc and control siRNA were synthesized by Gene Pharma (Shanghai, China), and they were transfected into cells according to the manufacturer's protocol.

| EdU proliferation assay
VSMCs proliferation was evaluated using Cell-Light EdU Apollo 567 in vitro Imaging Kit according to the manufacture's instruction. Cells in confocal dish were treated as above and incubated with EdU solution for 2 hours. Then they were fixed with 4% paraformaldehyde for 20 minutes, and treated with 0.5% Triton-X-100 for another 20 minutes at room temperature. After washing with PBS, cells were incubated with 1× Apollo® reaction cocktail for 30 minutes. Subsequently, nuclei were counterstained with Hoechst 33342 stain solution for 30 minutes at room temperature. Images were captured by OLMPUS FV3000 confocal laser scanning microscope (Tokyo, Japan), and the proliferation rate of cells was assessed with the proportion of EdUpositive nucleus (red) to blue fluorescent nucleus by counting six microscopic fields randomly in each well in three separate experiments.

| Immunofluorescent staining
The cold acetone of aortas root in APOE −/− mice were fixed with for 30 minutes, permeabilized with 0.2% Triton X-100 for 8 minutes, blocked with PTS (1% goat serum in PT) at 4°C and then incubated with primary antibodies (PCNA, p27, Ki-67, α-SMA, c-Myc and DNMT1) respectively overnight at 4°C. Subsequently, the specimens were incubated with corresponding TRITC-or FITC-conjugated secondary antibody at 37°C for 1 hour, nuclei were stained with DAPI for 5 minutes at room temperature. Digital images were acquired with OLMPUS FV3000 confocal laser scanning microscope (Tokyo, Japan).

| Cell cycle analysis
VSMCs treated with different concentrations of Hcy were trypsinized and washed with cold PBS for three times, and then fixed in 75% ethyl alcohol at 4°C overnight. After washing with PBS, cells were incubated with 1 mg/mL RNase A at 37°C for 30 minutes, then stained with PI for 1 hour in the dark. Cell cycle was analysed in a

| qRT-PCR
Total RNA was isolated from cells with RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol, and the cDNA was synthesized by the Revert Aid first strand cDNA synthesis kit (Fermentas, USA). Primer sequences of PCNA, p27, MFN2, DNMT1, c-Myc and GAPDH were listed in Table 1. qRT-PCR was performed using a miScript SYBR Green PCR Kit (DBI® Bioscience, Germany).
The expression levels of mRNA were normalized using GAPDH as a reference gene. All experiments were done in triplicate.

| Western blot
Whole cell lysates were prepared as described previously. 15 A total of 30 μg protein were separated by 8% SDS-PAGE, and then electro-transferred onto PVDF membrane (Millipore, USA). Membranes were blocked with 5% non-fat milk in PBST and incubated with indicated antibody at 4°C overnight. After washing with PBST for three times, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Protein bands were visualized using ECL solution after PBST washing, and the relative expression of each target protein was measured using β-actin as the reference.

| MassArray methylation analysis
Genomic DNA was purified from cultured cells using a genomic DNA isolation kit (Thermo Scientific, USA) followed by bisulfite

| ChIP qPCR assay
ChIP assay was performed using a ChIP assay kit (Millipore, USA) according to the manufacturer's instruction. Briefly, the specimens were cross-linked with 1% formaldehyde at 37°C for 8 minutes, and then

| Dual-Luciferase reporter assay
Dual-Luciferase reporter assays were performed using Luciferase

| Co-immunoprecipitation (Co-IP) assay
Cells were lysed in a lysis buffer containing protease inhibitor on ice.
After centrifugation, the supernatant was incubated with an anti-DNMT1, anti-c-Myc or normal rabbit IgG respectively at 4°C overnight followed by incubation with Dynabeads Protein G. Immune complex was separated by SDS-PAGE and proceeded for Western blot analysis.

| Statistical analysis
Results are expressed as the mean ± SD from at least three independent experiments. The data were analysed using one-way ANOVA and additional analysis using the Student Newman-Keuls test for multiple comparisons within treatment groups or t-test for two groups. P < 0.05 was considered to be statistically significant.

| Hcy promotes VSMCs proliferation in atherosclerotic plaque formation
Plaques formation is a major event in atherosclerosis related activa- respectively. 22 We next conducted double immunofluorescent staining with antibodies against (PCNA, Ki-67 and p27) and α-smooth muscle actin (α-SMA), a marker for smooth muscle cells. As shown in Figure 1D, Hcy induced a substantial increase in the number of PCNA and Ki-67 puncta, which also co-localized well with α-SMApositive cells. Conversely, an opposite effect was observed on p27, suggesting that VSMCs proliferation induced by Hcy might dependent on G0/G1 arrest. Moreover, Western blot was used to analyse the VSMC proliferation marker in the mice aorta and we found that the protein expression of PCNA and Ki-67 were obviously enhanced in aorta of APOE −/− HMD mice compared with APOE −/− NC mice, while the protein level of p27 apparently reduced ( Figure 1E). To further support our observation, VSMCs were incubated with different concentrations of Hcy and subjected to EdU incorporation assays, the results showed that incorporation of VSMCs was accelerated by Hcy and the significant effects was observed at the concentration of 100 μmol/L ( Figure 1F). In addition, the proliferation of VSMCs was assessed by flow cytometry analysis. The results showed that Hcy could result in the transition from G0/G1 to the S phase in VSMCs, particular at the concentration of 100 μmol/L ( Figure 1G). In agreement with the results above, the expression of PCNA was also significantly elevated in response to Hcy, and the expression of p27 was suppressed ( Figure 1H). Collectively, these results suggested that Hcy enhances VSMCs proliferation via promoting G1/S transition in the atherosclerotic plaque formation.

| MFN2 attenuated VSMCs proliferation in the atherosclerotic plaque formation
MFN2 is involved in a set of biological processes, including apoptosis and proliferation, and it was reported that overexpression of MFN2 results in a cell-cycle arrest at G0/G1 phase. 11 To determine whether MFN2 participates in VSMCs proliferation during atherosclerotic plaque formation, double immunofluorescent staining using antibodies against MFN2 and α-SMA was conducted in APOE −/− mice.
As shown in Figure

| Hcy inhibited MFN2 transcriptional activity via DNMT1 leading to VSMCs proliferation
Transcriptional activity is key to determine gene expression levels in eukaryotic organisms, which is susceptible to various factors such as epigenetic modification including DNA methylation. 23 To get insight into the underlying mechanism of MFN2 down-regulation in VSMCs proliferation induced by Hcy, we analysed the sequence of the MFN2 promoter using the UCSC Human Genome Browser and NCBI gene bank (http://www.ncbi.nlm.nih. gov/pubmed/), and one CpG island between −885 and −783 at the proximal promoter of MFN2 relative to the TSS was found, meaning that MFN2 promoter has the potential to be methylated, which may alter its transcriptional activity ( Figure 3A). Subsequently, several fragments of MFN2 the SssI, showed the greatest repression of MFN2 promoter activity ( Figure 3D). These results implied that MFN2 proximal promoter activity could be abrogated by DNA methylation.
DNA methylation in mammals is catalyzed by DNA methyltransferases (DNMTs) including maintenance (DNMT1) and de novo methyltransferases (DNMT3a, DNMT3b). 24 Therefore, VSMCs were exposed to the specific inhibitor of these three DNMTs, DC-05, TFD and nanao-mycinA respectively when the cells were treated with Hcy, to make

| Elevated binding of c-Myc to DNMT1 promoter suppressed MFN2 transcription in VSMCs
To further explore the underlying mechanism of c-Myc in the regula- to explore the mechanism for c-Myc-mediated MFN2 repression. The results showed that the distribution of DNMT1 was almost completely cytoplasm, while c-Myc was distributed throughout both the nucleus and the cytoplasm as reported previously, 25 and the co-localization of these proteins in the cells remained unchanged under Hcy treatment ( Figure 5C). We also employed protein co-immunoprecipitation F I G U R E 2 Down-regulation of MFN2 is required for the VSMCs proliferation induced by Hcy. A, Representative immunofluorescence images of MFN2 (green) co-localized with α-SMA (red) in APOE −/− mice. Nuclei were stained with DAPI (blue  The results showed that DNMT1 promoter activity could be attenuated when mutated in either of the two binding sites, and mutation of both binding sites will lead to the further reduction of DNMT1 promoter activity ( Figure 5H). Collectively, these data suggested that the increased binding of c-Myc to DNMT1 promoter region is responsible for the negative regulation of c-Myc on MFN2 transcription in VSMCs proliferation induced by Hcy ( Figure 6).

| D ISCUSS I ON
Over the past decade, a wealth of studies showed that abnormal VSMCs proliferation is a key event in the development of atherosclerotic plaque. 27 Given its importance, a detailed understanding of the molecular mechanism factors on aberrant VSMCs proliferation during atherosclerotic plaque formation is imperative.
Here we presented evidence about the critical role and molecular mechanism of MFN2 in regulating VSMCs proliferation during the formation of atherosclerotic plaque. Our data demonstrated that the binding of c-Myc to DNMT1 promoter facilitates MFN2 hypermethylation and suppresses MFN2 transcription activity. This finding provides a new insight into Hcy-associated VSMCs proliferation in the process of atherosclerotic plaque formation.
Atherosclerosis is a complex disease which begins with eccentric thickening of the intima, which is predominantly composed of VSMCs, mesenchymal intimal cells and inflammatory cells. 2 In recent years, VSMCs proliferation has been considered as an important pathological factor in atherosclerosis. 2 Hcy is an independent risk factor of atherosclerosis. 28 Evidences revealed a marked stimulatory effect of Hcy on VSMC proliferation. 13 It was well accepted that the transition of cell cycle from G0/G1 phase to S phase is one of the important characteristics of cell proliferation, which is accompanied by a great increase in DNA synthesis and abnormal expression of cell cycle proteins. 29,30 p27 can block the cell cycle in G0/G1 phase by negatively regulation of cyclin/CDK complexes, and PCNA is a well-known molecular marker for cell proliferation because of the role of replication in S phase, which could coordinate with p27 in the regulation of cell cycle. 31,32 In this study, we found Raf-ERK1/2 signalling pathway. 38 These previous observations and this study suggest that Hcy can promote proliferation of VSMCs during the formation of atherosclerotic plaque, which is at least partially dependent on the down-regulation of MFN2 expression.
Given the fact that the proximal promoter and 5′-untranslated region of MFN2 is enriched with CpG sites, we found that the methylation levels are significantly elevated in response to Hcy, which is accompanied by the inhibition of MFN2 transcription. DNA methylation is catalyzed by DNMTs including DNMT1, DNMT3a and DNMT3b. 24,39 Here, we found that MFN2 transcription was modulated by DNMT1, as exhibited by the notably up-regulated MFN2 expression when VSMCs were exposed to the specific DNMT1 inhibitor DC_05. This observation is consistent with the previous report that promoter hypermethylation is associated with transcriptional suppression, 40 43 Recently, c-Myc has been considered as a key factor in the transcriptional response to induce the transition of hepatocytes from G0/G1 to the S phase. 44 In accordance with the previous findings, 45 our study showed that c-Myc expression was up-regulated in VSMCs treated with Hcy and atherosclerotic plaque in APOE −/− mice which were fed with high-methionine diet.
Additionally, the regulation of c-Myc on MFN2 expression was in-

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.