Characterization of RPE cells
As shown in Figure 1 (a-c), Positive rates of CK 18 expressions in RPE cells(3rd generation) under fluorescence microscope. Representative fluorescence images showing CK-18 positive (green, Figure 1a) cells counterstained with DAPI (blue, Figure 1b); More than 95% of the cultured RPE cells were positive for CK-18(Figure 1c), which is the typical epithelial surface markers, and showed the typical hexagonal, polygonal or fusiform morphology. The cells were relatively uniform in shape and arranged in a "paved stone" manner.
Blue light exposure decreased the proliferation of RPE cells
Exposure to blue light significantly decreased the viability of RPE cells in an intensity-dependent manner compared to the non-irradiated control (P<0.01; Figure 2a). Consistent with the results of MTT assay, the rate of BrdU incorporation also decreased significantly upon blue light exposure in an intensity-dependent manner (P<0.01; Figure 2b). In both assays, the inhibitory effect of blue light peaked at 2000 Lux, and did not increase further even at 5000 Lux (P>0.05; Figure 2a, 2b). Taken together, blue light significantly inhibits the proliferation of RPE cells in vitro in an intensity-dependent manner.
Blue light exposure downregulated TRPM7 in RPE cells
Blue light irradiation significantly downregulated the TRPM7 mRNA levels in RPE cells in an intensity-dependent manner, with maximum effects seen at 5000 Lux (P<0.01; Figure 2c). Consistent with this, TRPM7 protein levels were also significantly decreased in the irradiated cells compared to the non-irradiated controls, with the lowest levels seen with 5000 Lux (P<0.01; Figure 2d, 2e). Therefore, we hypothesized that the effects of blue light on RPE cells were mediated by downregulating TRPM7.
TRPM7 knockdown downregulated PKC/ERK, Bcl-2 and decreased proliferation of photo-damaged RPE cells
To validate the above hypothesis, we knocked down TRPM7 in RPE cells with the specific siRNA prior to blue light irradiation. As shown in figure 3, the TRPM7, PKC, ERK and Bcl-2 protein levels were decreased to (0.34±0.05), (0.11±0.02), (0.13±0.02), (0.38±0.01)in group PEDF+TRPM7 siRNA compared to control group (2.33±0.08), (0.74±0.07), (0.73±0.08), (0.91±0.02) (P<0.01, Figure 3a-d), while Bax protein levels were increased from (0.55±0.02)in control group to (1.01±0.02)(P<0.01; Figure 3a-d). The TRPM7, PKC, ERK, Bcl-2 protein levels decreased upon photodamage while the Bax protein levels increased, and were restored by 50 ng/ml PEDF (P<0.01, Figure 3a-d).Thus, inhibiting TRPM7 aggravated the effects of blue light.
To determine whether TRPM7 directly affected the proliferation of photo-damaged cells, we analyzed BrdU uptake in the differentially treated cells. As shown in Figure 3e, the proliferation rate decreased significantly in the irradiated cells compared to the control, with the least BrdU uptake seen upon TRPM7 knockdown, which was assuaged when treated with PEDF. Consistent with the results of the BrdU uptake assay, MTT assay showed the lowest viability in the photo-damaged cells with TRPM7-knockdown (P<0.01, Figure 3e), and treatment with PEDF partly rescued the cells (P<0.05, Figure 3e). Taken together, the absence of TRPM7 aggravates the deleterious effects of blue light by blocking the PKC/ERK pathway.
TRPM7 overexpression attenuates the effects of blue light exposure
To further elucidate the protective role of TRPM7 in photo-damaged cells, we overexpressed the protein in RPE cells through a lentiviral system. Interestingly, forced expression of TRPM7 (TRPM7 lentivirus, MOI 100) did not completely restore its levels in blue light-irradiated RPE cells. However, in the PEDF-treated cells, TRPM7 levels were expectedly increased(2.28 ±0.06) in group PEDF + TRPM7 lentivirus compared with control group(1.84±0.05) and blue light group(0.52±0.05)(P<0.01; Figure 4a-b). Consistent with the results far, overexpression of TRPM7 significantly increased the expression levels of PKC (0.59±0.04) and ERK (1.17±0.05) in the photo-damaged RPE cells in group PEDF + TRPM7 lentivirus compared with blue light group(0.06±0.01) and (0.11±0.03)(P<0.01, Figure 4a-b), and PEDF treatment had no significant additional effect compared to the group PEDF +TRPM 7 vehicles (MOI 100) (P>0.05; Figure 4b). Otherwise, overexpression of TRPM7 also increased the expression of Bcl-2 protein levels,(0.87±0.01) in group PEDF + TRPM7 lentivirus and (0.66±0.03) in group blue light, while decreased in Bax, (0.63±0.01) in group PEDF + TRPM7 lentivirus and (1.03±0.03) in group blue light (P<0.01, Figure 4c-d). Finally, the proliferation and viability of photo-damaged RPE cells in group PEDF + TRPM7 lentivirus and group PEDF were also increased compared to group blue light(P<0.01, Figure 4e). Taken together, TRPM7 plays a protective role in blue light-exposed RPE cells, reduces apoptosis and restores their proliferation levels by activating the PKC/ERK pathway.