MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Abstract Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.


| INTRODUC TI ON
One of serious complications of ocular trauma and failed repair of primary rhegmatogenous retinal detachment is proliferative vitreoretinopathy (PVR). [1][2][3] PVR is an exaggerated wound healing process and a major cause of vision loss due to trauma. The essential pathogenesis of PVR is poorly understood, and there is no effective cure or prevention of PVR, so the loss of vision due to PVR has a serious socioeconomic effect.
Although inflammatory cells and glial cells participate in the formation of PVR, it is known that retinal pigment epithelium (RPE) cells are the key player in the pathogenesis of PVR. [1][2][3] In PVR, RPE cells are undergone epithelial-mesenchymal transition (EMT) and differentiate into a myofibroblast phenotype cell that migrates and proliferates to form retinal fibrotic membranes, [4][5][6][7][8]  It is known that TGF-β is the major inducer of EMT and retinal fibrosis. 7,10,11 TGF-β binds its receptor and activates Smad2/3 signalling and regulates several transcription factors that mediate the expression of α-SMA and ECM such as collagen and fibronectin (FN) deposition and fibrosis. [12][13][14][15] In addition, increased TGF-β activity is associated with the down-regulation of the genes of trans-differentiation inhibitor such as peroxisome proliferator-activated receptor-γ (PPAR-γ), 16,17 which is a key negative regulator of TGF-β signalling.
Inhibition of TGF-β signalling via knocking down Smad2/3 has been demonstrated to suppress the expression of α-SMA, collagen and experimental PVR in mice. 12,13 Recent studies suggest that wound healing process including fibrosis is also regulated by epigenetic factors including the factors of DNA methylation, histone modification [18][19][20][21] and non-coding RNA; specifically, the methyl-CpG-binding protein 2 (MeCP2) has been demonstrated to play a pivotal role in fibrosis of systemic diseases. 19,[22][23][24][25] MeCP2 binds to methylated DNA and has been considered as a classic epigenetic factor that regulates gene expression.
In a rat liver fibrosis model, MeCP2 expression is predominately associated with myofibroblastic cells and was found to selectively expressed in fibrotic tissues 19 ; however, in non-trans-differentiated cells MeCP2 expression is low, 19,22 and furthermore, MeCP2 acts as powerful epigenetic regulator for the genes related to myofibroblast trans-differentiation, knocking down MeCP2 by siRNA increases the expression of PPAR-γ. 19,22 Cell migration is also inhibited by silencing MeCP2. 21 The expression of MeCP2 is regulated by a number of factors. 26 There are many sites of serine phosphorylation in MeCP2 molecular; notably, most known MeCP2 that mediated gene activation is related to its phosphorylation at Serin421, [27][28][29][30][31] and it is known that phosphorylation of MeCP2 leads to its dissociation with sin3 and HDAC and gene activation. 32 Therefore, MeCP2 not only functions as a transcription repressor, but also acts as gene expression activator. 27,[31][32][33] Although the expression of MeCP2 in PVR membranes has been reported, 21 the relevance of its phosphorylation especially P-MeCP2-421 PVR remained unclear. In the current study, we sought to investigate the role of P-MeCP2-421 in the pathogenesis of PVR.

| PATIENTS AND ME THODS
All procedures conformed to the Declaration of Helsinki for research involving human subjects. The Institutional Review Board of Henan Eye Institute approved the use of human specimens. Informed consent was obtained from all individuals. Retinal sections from patients with PVR 10 eyes and retinal sections from the eyes without PVR (6 eyes) were included in the study. The medical records of all patients were reviewed retrospectively. The PVR membranes were obtained from the patients with PVR who had undergone pars plana vitrectomy and membrane peeling by retinal specialist in our eye hospital.
Patients who had received intraocular injections of steroids or any other drugs before surgery were excluded.

| Immunohistochemistry
The surgically excised PVR membranes from the patients were fixed in 4% paraformaldehyde, and 3-µm paraffin sections were obtained.
The methods of the details of the Immunohistochemistry were described before. 27 Briefly, antigen retrieval was performed and the tissue section was blocked with 5% normal goat serum for 30 minutes.
Anti-phosphorylated MeCP2-S80 (Thermo Fisher Scientific) and anti-phosphorylated MeCP2-S421 (ABGENT) were applied to the sections for 1 hour in room temperature and then followed by biotinylated secondary anti-rabbit antibody (1:400; Vector Laboratories) and streptavidin peroxidase for 30 minutes, respectively. Between each step, the slides were washed three times with PBS (PH.7.2). An aminoethyl carbazole kit (Zymed) was used to detect the immunoreactivity. Isotype-matched primary antibody was used as a negative control. Haematoxylin was applied to the slides for contrast staining.
Retinal sections from patients with eyelid tumour orbital extension who underwent exenteration were used as control.

| Immunofluorescence double labelling
For double labelling, anti-phospho-MeCP2-421 antibodies were applied to the sections and incubated at 4°C overnight. Next day, a rhodamineconjugated secondary antibody (red colour) was used for additional 30-minutes incubation. After washing with PBS, the sections were incubated with the antibodies specific for a-SMA (monoclonal mouse anti-a-SMA, 1:100 dilution, clone 1A4; Sigma-Aldrich Corp.), cytokeratin (Abcam), TGF-β (Santa Cruz Biotechnology) and PPAR-γ (Biosis) for 1 hour. The sections were washed again with PBS and then incubated with secondary antibody conjugated to fluorescein isothiocyanate for 30 minutes. After PBS washing, the slides were mounted with Hoechst mounting medium (Solarbio). The double labelling was observed by confocal laser-scanning microscopy (Nikon C1 Si). Isotype-matched primary antibody was used for negative controls.

| siRNA transfection
The RPE cells were cultured in 50% confluence condition. MeCP2 siRNA transfection was performed as previously publication . Briefly, the RPE cells were transfected with 10 nmol/L MeCP2 siRNA (2 hours) (stB-0002766c) or scrambled siRNA (siN05815122147) (Guangzhou RiboBio Co., LTD) using HiPerFect Transfection Reagent (QIAGEN) as instructed by the manufacture. 48 hours after the transfection with or without TGF-β (1-5 ng/mL) treatment for additional 48 hours, the cells were harvested and protein was extracted from the cells and the expressions of Phosph-Smad2/3, collagen I, FN and PPAR-γ were analysed by Western blot.

| Western Blot
The proteins from RPE cells were lysed using RIPA buffer and centrifuged at 12 000 ×g for 20 minutes, and protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad). Proteins were resolved on Tris-HCl 4-12% polyacrylamide gels at 110 V. The proteins were transferred to PVDF blotting membrane (Millipore, Bedford, MA). The membranes were blocked in 5% non-fat milk and probed with antibody specific for Phosph-Smad2/3 (Abcam), α-SMA After stripping, the membranes were re-probed with anti-GAPDH antibody (Millipore) for protein loading control (Li, et al) 27 .

| Chip assay
ChIP assay was performed using the Imprint Chromatin Immunoprecipitation Kit (Sigma-Aldrich) according to the manufacturer's instructions. Chromatin from the RPE cells was first fixed with 1% formaldehyde for 10 minutes, and cells were fragmented

| P-MeCP2-421 and 80 expressions in PVR membranes
All the PVR membranes (10 patients, the average age was 38.9 years old) included in the study were positive for the expression of P-MeCP2-421 and 80 by immunohistochemistry analysis (Figure 1E, F); however, the intensity of the immunoreactivities of P-MeCP2-421 was much stronger than P-MeCP2-80 ( Figure 1E). In contrast, the immunoreactivities of P-MeCP2-421 and 80 in normal retinal section were similar ( Figure 1B and C).

| Co-localization of P-MeCP2-421 with α-SMA, cytokeratin, TGF-β and PPAR-γ in PVR membranes
Because we found that the expression of P-MeCP2-421 was dominated expressed in the PVR membranes, therefore, all double labelling was performed for the co-localization of P-MeCP2-421 with the important molecules often to be seen in PVR membranes

| Binding of MeCP2 to TGF-β
To confirm interaction of MeCP2 with TGF-β gene in the RPE cells, specific antibody for MeCP2 antibody was used in a ChIP assay in the samples from the RPE cells. MeCP2 and TGF-β complexes were immunoprecipitated. The eluted DNA from the immunoprecipitation was used for PCR magnification using TGF-β promoter-specific primer. As shown in Figure 6, a 465-bp DNA fragment was amplified by PCR using the sample from MeCP2 antibody precipitated DNA, suggesting MeCP2 was bound to TGF-β promoter in the RPE cells.

| The effects of TGF-β on the expression of PPAR-γ expression
There were abundant immunoreactivity of PPAR-γ in the RPE cells without TGF-β addition; however, the treatment of the RPE cells with TGF-β (10 ng/mL) inhibited the expression of PPAR-γ as shown in Figure 7A by Western blot, and PPAR-γ protein was reduced even at the concentration of 5ng/ml of TGF-β, up to 10 ng/mL of TGF-β treatment on PPAR-γ was almost undetectable (Figure 7. A). On the other hand, treatment of the RPE cells with MeCP2 inhibitor 5-AZA increased PPAR-γ expression ( Figure 7B). Figure 7C showed the relevance of reduction of PPAR-γ to MeCP2 by immunofluorescent

| D ISCUSS I ON
MeCP2 is critically important for normal cell function maintaining, and abnormal expression of MeCP2 is related to many pathologic conditions including EMT and fibrosis. 19,21,22,28,30 The expression of non-phosphorylated MeCP2 in numerous fibrotic tissue such as liver, 19,22 lung, 24 heart 23 and PVR membranes 21 has been re- can binds to α-SMA gene and enhance α-SMA expression. 24 Taken together, MeCP2 might be an enhancer of α-SMA expression and participates in the formation of fibrosis and PVR.
As both P-MeCP2-421 and TGF-β are rich in PVR membranes, therefore we asked if there is an interaction between MeCP2 and TGF-β in the RPE cell, a ChIP assay was performed using anti-MeCP2 antibody and then DNA from the protein-DNA complex was amplified by human TGF-β2 primer. As seen in Figure 6, a DNA fragment One of important phenomenon in the formation of fibrotic tissue is the reduction of PPAR-γ, 18,19,22,39,40 and decreased expression of PPAR-γ is seen in numerous systemic fibrotic diseases, implying that there is the loss of PPAR-γ in the process of fibrotic tissue formation. 18,19,22,39,40 The rational for us to study PPAR-γ was because in the current study, we found that the expression of PPAR-γ in PVR In summary, our study demonstrates a novel role of P-MeCP2-421 in retinal fibrosis specifically in the pathogenesis of PVR and identifies critical intracellular targets (such as α-SMA, Sm ad2/3, collagen I, FN, PPAR-r) in the formation of fibrosis that is regulated by MeCP2, suggesting MeCP2 could be potentially targeted for therapy in the treatment of PVR and other fibrotic diseases.

ACK N OWLED G EM ENT
We thank Min Yuan and Ruijie Yin for technical assistance.

CO N FLI C T O F I NTE R E S T S
All authors declare that no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data were included in the manuscript.