Conceived and designed the experiments: FW HL HWW. Performed the experiments: HL QG. Analyzed the data: HL. Contributed reagents/materials/analysis tools: FW XX QG. Wrote the paper: HL HWW.
The authors have declared that no competing interests exist.
The proliferation of retinal pigment epithelium (RPE) cells resulting from an epithelial-mesenchymal transition (EMT) plays a key role in proliferative vitreoretinopathy (PVR), which leads to complex retinal detachment and the loss of vision. Genes of Snail family encode the zinc finger transcription factors that have been reported to be essential in EMT during embryonic development and cancer metastasis. However, the function of Snail in RPE cells undergoing EMT is largely unknown.
Transforming growth factor beta(TGF-β)-1 resulted in EMT in human RPE cells (ARPE-19), which was characterized by the expected decrease in E-cadherin and Zona occludin-1(ZO-1) expression, and the increase in fibronectin and α-smooth muscle actin (α-SMA) expression, as well as the associated increase of Snail expression at both mRNA and protein levels. Furthermore, TGF-β1 treatment caused a significant change in ARPE-19 cells morphology, with transition from a typical epithelial morphology to mesenchymal spindle-shaped. More interestingly, Snail silencing significantly attenuated TGF-β1-induced EMT in ARPE-19 cells by decreasing the mesenchymal markers fibronectin and a-SMA and increasing the epithelial marker E-cadherin and ZO-1. Snail knockdown could effectively suppress ARPE-19 cell migration. Finally, Snail was activated in epiretinal membranes from PVR patients. Taken together, Snail plays very important roles in TGF-β-1-induced EMT in human RPE cells and may contribute to the development of PVR.
Snail transcription factor plays a critical role in TGF-β1-induced EMT in human RPE cells, which provides deep insight into the pathogenesis of human PVR disease. The specific inhibition of Snail may provide a new approach to treat and prevent PVR.
Proliferative vitreoretinopathy (PVR), a scarring process that develops with some retinal detachments (RDs), is the most common cause of surgical failure in the rhegmatogenous RD treatment
EMT is an orchestrated series of events, in which differentiated epithelial cells undergo phenotypic transition to mesenchymal cells, often fibroblasts and myofibroblasts
A wide array of transcription factors, including Snail, Slug (SNAI2), δEF1 (ZEB1), SIP1 and Twist, are involved in regulating EMT
Despite the regulation of Snail transcription factor in EMT during cancer progressive and some fibrotic disorders has been extensively studied, the role of Snail in ocular fibrotic diseases, especially PVR, is rarely defined. In the present study, we first showed that Snail transcription factor plays an essential role in TGF-β1-mediated EMT in human RPE cells. We also report the presence of pathological Snail expression in epiretinal membranes from PVR patients. These findings may help us further understand the molecular mechanisms behind the pathogenesis of PVR, this might provide opportunities to prevent and treat human PVR effectively.
The general morphology of the human RPE cells (ARPE-19) was observed. As showed in
(A) Compared to HGF cells, ARPE-19 cells showed the typical epithelial morphology. Original magnifications 100×. RT-PCR (B) and Western blot (C) analysis of Snail and E-cadherin in ARPE-19, MCF-7 and HGF cells at the mRNA and protein levels respectively. MCF-7 cells were used as a positive control for E-cadherin expression and HGF cells were used as a positive control for Snail expression.
Although various kinds of growth factor and cytokine orchestrate the process of EMT, TGF-β1 is believed to play a major role in this process
ARPE-19 cells were grown on glass coverslips for 24 hr, starved for 24 hr, and then incubated for 24 hr, 48 hr, 72 hr with 10 ng/ml TGF-β1. Compared with control cells (A, E, I, M, Q), stimulated cells displayed an altered mesenchymal morphology by phase-contrast microscopy (B, C, D), with decreasing expression of E-cadherin (F, G, H) and ZO-1 (J, K, L) and an increasing expression of fibronectin (N, O, P) and α-SMA (R, S, T) by immunofluorescence microscopy. The green signal represents the staining of corresponding protein, and the red signal represents the nuclei staining by DAPI. Original magnifications 100× (A–D); 400× (E–T).
Zinc-finger transcription factor Snail plays an essential role in regulating EMT during tumor progression and organ fibrosis
Total RNA was extracted from ARPE-19 cells at the time points as indicated after TGF-β1 (10 ng/ml) treatment. (A) The increasing expression of Snail mRNA was time-dependent. The increasing expression of mesenchymal markers fibronectin (B) and α-SMA (C) and the decreasing expression of epithelial markers E-cadherin (D) and ZO-1 (E). Values were normalized to GAPDH mRNA levels and represent fold change as compared to untreated cells. Data shown as mean ± SD of two independent experiments each performed in triplicate. (F) Western blot analysis of Snail, fibronectin, α-SMA, E-cadherin and ZO-1 with proteins extracted from ARPE-19 cells treated with 10 ng/ml TGF-β1 at differential time points. (G) Imunofluorescence analysis and subcellular localization of Snail (green) in ARPE-19 cells treated with 10 ng/ml of TGF-β1 for 48 hr. Nuclei were stained with DAPI (red). Original magnifications 400×.
Control of the nuclear localization of transcription factors is an important process in response to external stimuli because transcription factors can not function until they translocate to the nucleus
Since TGF-β1 stimulation can induce EMT and up-regulate Snail expression in ARPE-19 cells, we speculated that snail might be involved in EMT of ARPE-19 cells. To understand the specific biological functions that Snail exerts during TGF-β1 induced EMT, we knocked down Snail in ARPE-19 cells transfected with a Snail-targeting siRNA-expressing plasmid. The plasmid pSuper-Snail-shRNA contains a small hairpin RNA (shRNA) transcription unit which can be processed into Snail-targeting siRNA in cells
(A) Immunofluorescence staining of Snail in Snail-shRNA or control-shRNA transfected cells which were starved for 24 hr in serum-free media and treated with 10 ng/ml of TGF-β1 for 48 hr. The green signal represents the staining of Snail protein, the red signal represents the nuclei staining by DAPI. (B) Photomicrographs of Snail-shRNA or control-shRNA transfected ARPE-19 cells are showing different cell morphologies. Cells transfected with Snail-shRNA or control-shRNA plasmid were starved for 24 hr in serum-free media and treated with 10 ng/ml of TGF-β1 for 48 hr. The expression of Snail, fibronectin, a-SMA, E-cadherin and ZO-1 were analyzed by Western blot (C) and real-time PCR (D). Values represent fold change as compared to control cells. Data shown as mean ± SD of two independent experiments each performed in triplicate. Original magnifications 400× (A); 100× (B).
EMT can increase the cell motility. To determine the functional changes in ARPE-19 cells behavior that occurred following suppression of Snail, we used Transwell chamber assays to gauge the migratory ability of Snail-knockdown cells in the presence or absence of TGF-β1. Snail expression was blocked by shRNA, and then equal numbers of control-shRNA or Snail-shRNA transfected ARPE-19 cells were added to transwell inserts and allowed to migrate in the presence or absence of TGF-β1 (10 ng/ml). TGF-β1 promoted an obviously increase in migration of control cells, whereas this process was significantly reduced by Snail knockdown in cells transfected with Snail-shRNA (
(A) Cells transfected with control-shRNA or Snail-shRNA were allowed to migrate transwell chambers for 18 hr in the presence or absence of TGF-β1. After 18 hr, the migrated cells were fixed, stained, and photographed. (B) The number of migrated cells. Data shown represent the average of three independent experiments. *P<0.05, compared with control-shRNA, TGF-β1(−) samples and control-shRNA, TGF-β1(+) samples.
To assess whether Snail involved in the pathological process of PVR, we investigated Snail protein distribution and expression in the PVR epiretinal membranes. Immunofluorescent staining detected the presence of Snail in epiretinal membranes from PVR patients (
(A) Immunofluorescent staining of Snail (green) in epiretinal membranes. Nuclei were stained with DAPI (red). Arrowheads indicated colocalization of Snail expression and nuclei. Original magnifications 50× (a); 200× (b–e). (B) 50 µg and 80 µg total protein extracted from PVR epiretinal membranes were detected by Western blot. GAPDH was used as internal control.
Epithelial to mesenchymal transition is an essential morphological conversion occurring during embryonic development, and this process is re-engaged in adults during wound healing, tumor progression and organ fibrosis
TGF-β1 has been showed to play central roles in initiating EMT in models of metastatic tumour development and in fibrogenesis in progressive kidney disease
The E-cadherin suppressor, Snail, has been considered as the key regulator of EMT in normal development and tumor progression
After identifying the relationship between TGF-β1 stimulation and the expression and nuclear translocation of Snail in ARPE-19 cell, we then determined whether Snail was sufficient to induce EMT through knockdown experiments. Down-regulation of Snail by a siRNA-expressing vector decreased the expression of mesenchymal markers fibronectin and α-SMA, indicating that Snail is involved in maintaining the mesenchymal properties of transformed ARPE-19 cells. Simultaneously, our results showed that Snail knockdown dramatically increased the protein levels of E-cadherin and ZO-1, suggesting that Snail is required for suppressing these important cell-cell adhesion molecules. In addition, down-regulation of Snail significantly reduced the ability of ARPE-19 cells migration in response to TGF-β1. These results indicated that there was a functional linkage between Snail expression and TGF-β1-mediateed EMT in RPE cells. However, knockdown of Snail did not reverse the full EMT that induced by TGF-β1 treatment in ARPE-19 cells. Previous studies have demonstrated the function of another zinc-finger transcription factor Slug, a close relative of Snail, in regulating EMT during early development and cancer progression
In conclusion, we showed that induction of EMT in human RPE cells led to up-regulation of Snail expression, and inhibition of Snail limited the development of EMT. Specifically, the expression of Snail was detected in epiretinal membranes from human PVR, and that, this implicates the role of Snail in PVR pathogenesis. These findings demonstrate that Snail transcription factor plays a critical role in TGF-β1-induced EMT in human RPE cells and Snail may be partially responsible for the formation of epiretinal membranes found in patients with PVR. The specific inhibition of Snail may provide a new approach for the prevention and treatment of human PVR.
The current research involving human participants has been approved by the ethics committee of Shanghai First People's Hospital with written consents. The study followed the tenets of Declaration of Helsinki for the use of human subjects, and informed consents were obtained from all patients.
Human retinal pigment epithelial cell line ARPE-19 was routinely maintained in a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) and Ham's F12 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). Human breast cancer cell line MCF-7 and human gingle fibroblast (HGF) cells were kindly provided by Biochemistry and Molecular biology Institute, Shanghai Jiao Tong University Affiliated First People's Hospital and Shanghai Jiao Tong University Affiliated Ninth People's Hospital, respectively. MCF-7 cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) with 10% FBS and insulin. HGF cells were grown in high DMEM with 5% FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. The medium was changed twice a week. For some experiments, equal numbers of ARPE-19 cells were plated and cultivated in serum-free medium for 24 hours before stimulation with 10 ng/ml of TGF-β1 at various time points.
Human recombinant TGF-β1 was purchased from HumanZyme (Chicago, US). Antibodies used in Western blot analysis and immunofluorescene were as follows: Antibodies against human E-cadherin and fibronectin were obtained from R&D systems (Minneapolis, MN). Snail antibody was purchased from Abcam Ltd (Cambridge, UK). α-SMA antibody was purchased from Sigma-Aldrich (MO, USA). ZO-1 antibody was purchased from Invitrogen (Carlsbad, CA). Puromycin, FITC-conjugated sheep anti-rabbit and FITC-conjugated sheep anti-mouse antibodies and 4′-6′-Diamidino-2-phenylindole (DAPI) were from Sigma (St Louis, MO). Sheep anti-mouse or anti-rabbit horseradish peroxidase (HRP)-labeled secondary antibodies and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were from Chen Kang, Inc. (China).
Total RNA was extracted from the different cell lines (TRIzol, Invitrogen-Gibco, Carlsbad, USA), according to the manufacturer's protocol. RT-PCR was performed as follows: 1 µg of total RNA was reversely transcripted using random primer (Tiangen, Shanghai, China) and reverse transcriptase (ReveTra Ace; Promega, Madison, USA), according to the manufacturer's instructions. The resulting cDNAs were employed as templates for specific PCR reactions using Taq DNA Polymerase (TAKARA, Osaka, Japan). RT-PCR of GAPDH was used as an internal control. The sequences for PCR primers were as follows: human Snail sense
For Real-Time PCR analysis, quantification was performed using the 7500 Fast Real-time PCR System (Applied Biosystems, Foster, USA). The specificity of the amplification reactions was confirmed by melting curve analysis. The primer sequences used were as follows: human Snail sense
Different time periods after treatment with 10 ng/ml TGF-β1, cells were lysed in lysis buffer (25 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 5 mM sodium orthovanadate, 50 mM NaF, 0.5 mg/ml AEBSF, and 10 µg/ml pepstatin). The samples were clarified by centrifugation at 12,000 rpm for 15 min at 4°C and boiled for 10 min with sample buffer containing 100 mM NaF. Samples were quantified and separated by 8%, 10% or 12% SDS–PAGE gel and then transferred onto a polyvinyl difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Membranes were blocked in 5% nonfat milk (diluted in Tris-buffered saline and 0.1% Tween-20) for 2 hr at room temperature. The blots were incubated with primary antibodies overnight at 4°C with rabbit anti-snail (1∶1000), anti-E-cadherin (1∶500), anti-ZO-1 (1∶1000), and mouse anti-fibronectin (1∶1000), anti-α-SMA (1∶500). This procedure was followed by incubation with sheep anti-mouse or anti-rabbit HRP-labeled secondary antibody (1∶2000) for 2 hr at room temperature. The Immunoreactive bands were visualized with chemiluminescence detection reagent (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA).
The morphology of the cells was observed under an inverted phase-contrast microscope (Olympus). The photographs were taken at 100× magnifications by a digital camera.
The cells were seeded and cultured in 24-well chamber slides (Invitrogen, Carlsbad, USA) in serum-free medium for 24 hr and then were exposed to 10 ng/ml of recombinant TGF-β1 for the indicated time. Cells were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 min and washed in PBS. Slides were incubated with the indicated primary antibodies at optimal dilution for 2 hr at 37°C. After three washes, the coverslips were incubated with the appropriate fluorescent secondary antibodies diluted 1∶500 for 1 hr at room temperature. Nuclei were stained with DAPI (1∶3000). Cells were observed at timely intervals for 72 hours and pictures were obtained using a fluorescence microscopy (Carl Zeiss Inc., Oberkochen, Germany). Primary antibodies included: rabbit anti-snail (1∶100), anti-E-cadherin (1∶100), anti- ZO-1 (1∶200), and mouse monoclonal anti-fibronectin (1∶100), anti-αSMA (1∶200).
Plasmids containing Snail short hairpin RNA (shRNA) transcription unit (pSuper-Snail-shRNA) and control shRNA (pSuper-control) were kindly gifted to us by Dr. S. J. Weiss (Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.). Transfection of cells, at 90% confluency, was performed by Lipofectamine 2000 (Invitrogen, Carlsbad, USA) as recommended by the manufacture. Briefly, palsmids (1 µg per 24-well chamber slides) were mixed with Lipofectamine 2000 (2 µl per 24-well chamber slides) in 50 µl Opti-MEM I Reduced Serum Medium before addition to the ARPE-19 cells. Subsequently, the mixture of DNA and Lipofectamine reagent was added to the well with 1×106 cells and incubated for 48 hr. Then medium was replaced with selective medium containing 1 µg/ml of Puromycin for cells transfected with pSuper-Snail-shRNA and pSuper-control, respectively. Stable transfectants were selected by picking the surviving colonies 3–4 weeks later. Single colonies were amplified and continually grew in medium containing antibiotics. Transfected cells were then collected, lysised, or treated with TGF-β1 for the following experiments.
Cell migration assays were performed using Transwell chambers (8-µm pores, Costar, Conning, USA). Serum-starved transfected cells were treated by TGF-β1 (10 ng/ml) for 48 hr or untreated, and then trypsinized and counted. A total of 1×106 cells were plated into the insert in 100 µl DMDM/F12 containing 0.5% FBS and allowed to migrate from upper compartment to lower compartment toward a 10% FBS gradient for 18 hr. After migration, non-migratory cells on the upper membrane surface were scrubbed off and the migratory cells attached to the bottom surface of the membrane were stained with H&E for 20 min at room temperature. The migrated cells were then enumerated. Statistical analysis was performed by two-tailed unpaired Student's t-test using the statistical software program SPSS17.0 (Chicago, IL). Data are represented as mean ± SD, with P<0.05 considered to be statistically significant.
Epiretinal membranes were obtained from 6 eyes undergoing vitreoretinal surgery for the treatment of rhegmatogenous retinal detachment complicated by PVR. Membranes were put into PBS (pH 7.4) during the operation and then snap frozen in liquid nitrogen or fixed in 4% paraformaldehyde. Frozen membranes were cut into small pieces and homogenized in 0.1 ml lysis buffer. The methods of protein extraction and detection were consistent with the above description.
Immunofluorescent staining was performed by a reported method
We gratefully thank Biochemistry and Molecular Biology Insititute of First People's Hospital for MCF-7 cells, Wenwen Yu for human gingle fibroblast cells and Ye Feng for excellent technical assistance with transfection.