Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells

Antibodies and reagents

Monoclonal rabbit-anti-human PTP1B was purchased from Abcam (Cambridge, UK). Monoclonal rabbit-anti-rat extracellular signal-regulated kinase (Erk)1/2, p-Erk1/2, protein kinase B (Akt; pan), and p-Akt were obtained from Cell Signaling Technology (Danvers, MA). Monoclonal mouse-anti-human α-smooth muscle actin (α-SMA), monoclonal rabbit-anti-human cyclin A and cyclin D1 antibodies, fluorescein isothiocyanate (FITC)-conjugated goat-ant-rabbit, and horseradish peroxidase-conjugated donkey-anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit-anti-human β-actin was obtained from Biomedical Technologies (Stoughton, MA). TCS-401 was obtained from Tocris Bioscience (Tocris, Bristol, UK). PD98059 (an inhibitor of mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/Erk)), LY294002 (an inhibitor of phosphatidylinositol 3-kinase (PI3K)), and propidium iodide (PI) were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA). BSA (BSA) was purchased from Fisher Scientific (Pittsburgh, PA).

Model of retinal detachment

Adult Sprague-Dawley (SD) rats of either gender (180–200 g; Vitalriver Laboratory Animal Equipment Co., Ltd., Beijing, China) were used in this study. Pupils were dilated with a topically applied mixture of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P; Santen Pharmaceutical Co., Ltd., Osaka, Japan). Retinas were detached from the right eyes of SD rats by trans-scleral injection of 1.4% sodium hyaluronate (Healon GV; Pharmacia and Upjohn Co., Kalamazoo, MI) into the subretinal space (SRS) with a 30-gauge needle (BD Biosciences, Franklin Lakes, NJ) [19]. Care was taken not to make a break in the detached retina. Animals were excluded from the study if they developed intraocular hemorrhage. The left eyes of all animals served as normal control eyes. Animals were sacrificed 10 days after surgery. All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Medical Ethics Committee of the Affiliated Hospital, Qingdao University (Permit Number: 00,863). All surgeries were performed under pentobarbital sodium and a topical anesthetic (Oxyben; Santen Pharmaceutical Co., Ltd.). All efforts were made to minimize suffering.

Primary culture of rat RPE cells

Retinal tissues were obtained from adult male SD rats. Rat RPE cells were isolated by modifying the method developed by Edwards [20]. Briefly, enucleated eyes were soaked in phosphate-buffered saline (PBS) solution containing 0.1 mg/mL of streptomycin and 100 U/mL of penicillin. The anterior section of the eye was removed and the vitreous humor was aspirated. The posterior eyecup was incubated in a hyaluronidase (1%) solution for 2 min at room temperature. The remaining neural retina was removed. Exposed RPE cells were trypsinized (0.25% trypsin), incubating the eyecup at 37 °C for 20 min. RPE cells were isolated from Bruch’s membrane using a gentle water jet of buffered saline through a 1 ml pipette. Later, dissociated RPE cells were aspirated and seeded into cell culture flasks. The cells were cultured in Dulbecco’s modified eagle’s medium/nutrient mixture F-12 (DMEM/F12; Invitrogen, Carlsbad, CA) supplemented with 10% FBS. The culture medium was changed every two days. After approximately seven days, the RPE cells were grown to confluence. Primary cultured cells were then trypsinized, resuspended in a culture medium, and seeded into cell culture flasks at a density of 3.0×10⁵ cells per flask. The cells were used between passages 3 and 5.

Immunofluorescence staining

The eyes in the model of retinal detachment were enucleated after cardiac perfusion of 4% paraformaldehyde. The anterior segment of the eye and the vitreous were removed. After neural retinas were removed by forceps, the eyecups were divided into four segments (retinal detachment area and non-detached area were labeled, respectively), and then spread on the slides. The stretched preparations were fixed with 4% paraformaldehyde for 20 min at room temperature. After washing three times with PBS, stretched preparations were pre-incubated for 20 min in 5% BSA to block nonspecific binding, and then incubated with a primary antibody at room temperature for 3 h. The concentration of monoclonal rabbit-anti-human PTP1B was 1:50. To assess the specificity of the staining, the negative control group was processed without the primary antibody. Following incubation of the primary antibody, stretched preparations were washed with PBS, and then incubated for 1 h with a 1:200 dilution of the appropriate FITC-conjugated secondary antibody at 37 °C. After another wash with PBS, slides were coverslipped with glycerol. Positive staining was visualized using a confocal laser scanning microscope (LSM-510; Carl Zeiss, Jena, Germany).

For immunofluorescent analysis of cultured cells, cells were seeded onto chamber slides at a density of 1×10⁵ cells per chamber. After washing three times with PBS, cells were fixed in 95% ethanol at room temperature for 10 min, followed by a further wash with PBS. The fixed cells were then permeabilized with 1% Triton X-100 in PBS for 10 min. Later cells were blocked, incubated with the primary antibody overnight at 4 °C, and washed in PBS. The concentration of anti-α-SMA was 1:100. The FITC-conjugated secondary antibody was applied for 1 h with a 1:200 dilution at 37 °C. Cells were later washed with PBS and mounted in a medium containing PI for visualizing the nuclei. Positive staining was visualized using the confocal laser scanning microscope.

MTT assay

Cell proliferation was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetracolium bromide (MTT; Roche Molecular Biochemicals, Mannheim, Germany) assay. The 5×10³ cells grown in a 96-well plate for 24 h were partially starved in DMEM/F12 supplemented with 1% FBS for 12 h, and then stimulated with various concentrations of TCS-401 for an additional 24 h. MTT was added to the culture medium, and the cells were incubated for an additional 4 h. The formazan crystals formed were then dissolved by adding dimethyl sulfoxide (100 μL per well). Absorbance at 490 nm was measured using a microplate reader (Model 550; Bio-Rad, Tokyo, Japan).

Protein extraction and immunoblotting

Protein preparation and western blot analysis were performed as described previously [15]. Cultured cells grown in culture flasks were trypsinized; suspended in a lysis buffer containing 1% Triton X-100, 250 mM sodium chloride, 2 mM EDTA, 50 mM tris (hydroxymethyl) aminomethane (Tris)-HCl, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride (all from Sigma-Aldrich); and homogenized. Equal amounts of extracted protein were loaded on 4%–12% polyacrylamide gels (Invitrogen) for sodium dodecyl sulfate PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA). Nonspecific binding was blocked by overnight incubation at 4 °C with 5% nonfat dry milk in PBS. The membranes were then incubated at room temperature for 2 h with anti-cyclin A, anti-cyclin D1, anti-α-SMA, anti-Erk, anti-p-Erk, anti-Akt, or anti-p-Akt antibodies at a dilution of 1:500. Anti-β-actin was used as an internal control for the immunoblot. The membranes were washed three times with PBS with Tween-20 and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at a final dilution of 1:1000. After final washes with 0.1% Triton X-100 in PBS, signals were detected by enhanced chemiluminescence following the manufacturer’s instructions (Pierce, Rockford, IL) and exposed to autoradiographic film.

Migration assay

Cell migration was determined using a transwell assay: 1×10⁴ cells were placed in the upper chamber (Costar, Cambridge, MA) with a volume of 200 μl serum-free medium with various concentrations of TCS-401. Next, DMEM/F12 with 10% FBS was placed in the bottom chamber, with a volume of 600 μl per well. After 12 h incubation, the cells were fixed in 95% ethanol for 10 min, stained with hematoxylin for 5 min, and washed in Dulbecco’s calcium and magnesium free PBS (Gibco®; Invitrogen). The remaining cells on the upper surface of the filter were removed by wiping with a cotton swab. Then the filters were cut off, dehydrated using graded ethanol, hyalinized by dimethylbenzene, and fixed by neutral resins. Cell migration was quantified by the number of cells that migrated across the filter toward the lower surface in five random fields per filter under microscope. All migration assays were performed in triplicate.

Statistical analysis

Statistical analysis was performed using SPSS version 15.0 (SPSS, Chicago, IL). Data are expressed as mean±standard deviation. Statistical analysis was performed using one-way ANOVA and the student’s t test. p<0.05 was considered statistically significant.

Detection of PTP1B in rat RPE cells in vivo

At present, many PTP isoforms have been identified in various cells, but not all tissues express all isoforms. There was no report on the expression of any PTPs in RPE cells until now. In this study, ICC was used to determine the relative expression of PTP1B in RPE stretched preparations (normal and retinal detachment areas). As shown in Figure 1A, retinal (one-half to one-third) detachments were created in the right eyes of the rats. In vivo, intense staining of PTP1B was observed in the cytoplasm of RPE cells (Figure 1B). However, the expression of PTP1B in RPE cells located at the retinal detachment area (Figure 1C) was significantly lower than that in RPE cells located at the non-detachment area (Figure 1B).

Inhibition of PTP1B increases proliferation of RPE cells in vitro

Experiments were performed to evaluate whether TCS-401 had any effect on the proliferation of RPE cells with MTT. Cells were incubated with TCS-401 at concentrations of 0.5, 1, and 2 μM for 24 h. Among the various concentrations of TCS-401 tested, TCS-401 at a concentration of 0.5, 1, or 2 μM was observed to significantly increase the proliferation of RPE cells compared to the control group (Figure 2A).

The effects of TCS-401 on protein expression levels of cyclin A and cyclin D1 in RPE cells were examined. Contact-inhibited RPE cells were stimulated with fresh growth medium with various concentrations of TCS-401 (0.5, 1, and 2 μM) and incubated for 24 h. The results demonstrate that the basal levels of cyclin A and cyclin D1 were minimal. TCS-401 significantly increased the expression of cyclin A and cyclin D1 at the concentrations of 1 and 2 μM in a concentration-dependent manner (Figure 2B-D).

Inhibition of PTP1B regulates differentiation of RPE cells

The alteration in the cell phenotype was demonstrated by the expression of α-SMA, which is characteristic of the mesenchymal phenotype. Immunofluorescence and western blot evaluated α-SMA in rat RPE cells treated with TCS-401 at concentrations of 0.5, 1, and 2 μM for 24 h. The results demonstrate that the normal RPE cells exhibited little immunoreactivity and protein expression for α-SMA. However, the immunopositive reaction (Figure 3A) and protein expression (Figure 3B) were strengthened along with the increased concentration of TCS-401. Furthermore, Figure 3A-C and Figure 3A-D show that, like the effect of SOV studied in our previous research [15], the treatment of confluent cultures with TCS-401 also induced a concentration-dependent release of cell-cell contacts, resulting in the appearance of gaps between cells and a gradual loss of monolayer integrity.

Inhibition of phosphatase improves migration activity of RPE cells

Results of an in vitro migration assay are shown in Figure 4. Cells were measured in a transwell chamber in which RPE cells migrated through a porous membrane. The mean number of migrated cells in the TCS-401-treated RPE cells was significantly higher than the mean number of migrated control cells (Figure 4). The mean number increased along with the increased concentration of TCS-401 (0.5, 1, and 2 μM).

Inhibition of PTP1B activates MEK/Erk and PI3K/Akt signaling pathways in RPE cells

To investigate whether a PTP1B blockade promotes Erk and Akt phosphorylation in RPE cells, various concentrations (0.5, 1, and 2 μM) of TCS-401 were added to these cells. It was found that TCS-401 at concentrations of 0.5, 1, and 2 μM significantly increased phosphorylation of Erk and Akt compared to the control group (Figure 5A-B). The activation of Erk and Akt by TCS-401 was blocked by pretreatment with PD98059 and LY294002, respectively (Figure 5C-F).

MEK/Erk and PI3K/Akt signaling pathways mediate the effect of inhibition of PTP1B on proliferation and migration of RPE cells

Having found that TCS-401 treatment activated the MEK/Erk and PI3K/Akt signaling pathways and induced proliferation, differentiation, and migration in RPE cells, it was examined whether the activation of the MEK/Erk and PI3K/Akt signaling pathways played a vital role in TCS-401-induced activation of RPE cells. MTT and migration assay were used to evaluate the role of MEK/Erk and PI3K/Akt signaling pathways on the proliferation and migration of RPE cells. As shown in Figure 6A, pretreatment of RPE cells with PD98059 or LY294002 significantly inhibited TCS-401-induced proliferation at a concentration of 2 μM. In addition, fewer migrated cells were observed in the pretreatment groups (treated with PD98059 or LY294002) than non-pretreatment group when the cells were treated with TCS-401 at a concentration of 2 μM (Figure 6B-C).