The complex interplay between ERK1/2, TGFβ/Smad, and Jagged/Notch signaling pathways in the regulation of epithelial-mesenchymal transition in retinal pigment epithelium cells

Abstract

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is a major pathologic change in the development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. ERK1/2 pathway has been reported to play a key role in the carcinogenesis, cancer metastasis, and multiple fibrotic diseases. We hypothesized that ERK1/2 signaling could cross-interact with transforming growth factor β2 (TGFβ2)/Smad and Notch signaling pathways in the regulation of EMT in RPE cells. Here, we demonstrated that ERK1/2 signaling was activated in TGFβ2-induced EMT in human RPE cells, while blockade of the canonical TGFβ2/Smad2/3 signaling with SB431542 could not inhibit TGFβ2-induced the activation of ERK1/2. Meanwhile, blockade of ERK1/2 signaling with a specific MEK/ERK1/2 inhibitor U0126 strongly prevented TGFβ2-induced the downregulation of P-cadherin, and the upregulation of α-SMA, collagen type IV, N-cadherin and fibronectin in RPE cells. In addition, we also identified that blockade of ERK1/2 signaling could inhibit not only the canonical TGFβ/Smad signaling, but also the Jagged/Notch pathway. Finally, we found that blockade of Notch pathway with a specific inhibitor DAPT could inhibit TGFβ2-induced the activation of ERK1/2 pathway conversely. Therefore, our study provides evidence that ERK1/2 signaling can cross-interact with the canonical TGFβ/Smad and the Jagged/Notch signaling pathways in RPE cells EMT. ERK1/2 inhibitor may have therapeutic value in the prevention and treatment of PVR and other fibrotic diseases.

Figure 1. Blockade of ERK1/2 pathway by U0126 prevents TGFβ2-induced EMT in RPE cells.

RPE cells were cultured in the absence or presence of TGFβ2 (5 ng/ml) with U0126 (10.0 µM) or DMSO for 24 h. (A) Cell morphology was examined using phase contrast microscope under ×100 magnification. Scale bar = 80 µm. (B) Immunofluorescence analysis of α-SMA (red), Col IV (green), and FN (red) using confocal microscopy. Representative images are shown (scale bar = 40 µm).

Figure 2. U0126 prevents TGFβ2-induced EMT through upregulating epithelial cell marker and downregulating EMT markers expression.

RPE cells were cultured in the absence or presence of TGFβ2 (5 ng/ml) with U0126 (2.5, 5.0, 10.0, 20.0 µM) or DMSO for 24 h. (A) The mRNA expression levels of α-SMA, Col IV, N-cadherin and FN were determined by real-time quantitative PCR. Gene expression levels were normalized to the GAPDH control. *, P<0.05 v. TGFβ2 treated with DMSO group. (B) The protein expression levels of α-SMA, Col IV, N-cadherin, P-cadherin and FN were detected by western blot. (C) Quantification of protein levels from three independent experiments. *, P<0.05 v. TGFβ2 treated with DMSO group.

Figure 3. TGFβ2-induced ERK1/2 activation is independent of TGFβ/Smad pathway and U0126 inhibits TGFβ2-induced phosphorylation of Smad2.

(A) RPE cells were cultured in the absence or presence of TGFβ2 for 15min, 30 min and 60 min, the expression of p-ERK1/2 and ERK1/2 were determined by western blot. (B) RPE cells were cultured in the absence or presence of TGFβ2 with U0126 (10.0 µM) or SB431542 (10.0 µM) for 30 min, the expression of p-ERK1/2 and ERK1/2 were determined by western blot. (C) Quantification of protein levels from three independent experiments. *, P<0.05 v. TGFβ2 treated with DMSO group. (D) The phosphorylation and the total levels of Smad2 and Smad3 were detected by western blot after 60 min treatment. (E) Quantification of protein levels from three independent experiments. *, P<0.05 v. TGFβ2 treated with DMSO group.

Figure 4. U0126 prevents TGFβ2-induced EMT via suppressing the Notch pathway.

(A) RPE cells were treated with TGFβ2 in the presence of U0126 (2.5, 5.0, 10.0, 20.0 µM) or DMSO for 24 h, the mRNA expression levels of Jagged-1and Notch-3 were detected by real-time PCR. Gene levels were normalized to control GAPDH. *, P<0.05 v. TGFβ2 treated with DMSO group. (B) The protein expression levels of Jagged-1and Notch-3 were detected by western blot. (C) Quantification of protein levels from three independent experiments. *, P<0.05 v. TGFβ2 treated with DMSO group.

Figure 5. U0126 suppresses Notch target genes expression and blockade of Notch pathway inhibits ERK1/2 pathway activation.

(A) RPE cells were treated with TGFβ2 in the presence of U0126 (2.5, 5.0, 10.0, 20.0 µM) or DMSO for 24 h, the mRNA expression levels of Notch target genes Hes-1and Hey-1 were detected by real-time PCR. Gene levels were normalized to control GAPDH. *, P<0.05 v. TGFβ2 treated with DMSO group. (B) RPE cells were cultured in the absence or presence of TGFβ2 with DAPT (1.0, 2.5, 5.0, 10.0 µM) or DMSO for 30 min, the expression of p-ERK1/2 and ERK1/2 were determined by western blot. (C) Quantification of protein levels from three independent experiments. *, P<0.05 v. TGFβ2 treated with DMSO group.