BMP-4 and BMP-7 Inhibit EMT in a Model of Anterior Subcapsular Cataract in Part by Regulating the Notch Signaling Pathway

Abstract

Purpose: The proliferation, migration, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are believed to be the pathological mechanisms underlying anterior subcapsular cataract (ASC). Bone morphogenetic proteins (BMPs) inhibit transforming growth factor-beta (TGF-β)-induced fibrosis in the lens. Herein, we aimed to further clarify the roles of BMP-4/BMP-7 in the progression and the underlying mechanisms of fibrotic cataract.

Methods: BMP-4/BMP-7, TGF-β2, jagged-1 peptide, or DAPT were applied in a mouse injury-induced ASC model and in the human LEC cell line SRA01/04. The volume of opacity was examined by a slit lamp and determined by lens anterior capsule whole-mount immunofluorescence. Global gene expression changes were assessed by RNA sequencing, and the levels of individual mRNAs were validated by real-time PCR. Protein expression was determined by the Simple Western sample dilution buffer. Cell proliferation was examined by CCK8 and EdU assays, and cell migration was measured by Transwell and wound healing assays.

Results: Anterior chamber injection of BMP-4/BMP-7 significantly suppressed subcapsular opacification formation. RNA sequencing of the mouse ASC model identified the Notch pathway as a potential mechanism involved in BMP-mediated inhibition of ASC. Consistently, BMP-4/BMP-7 selectively suppressed Notch1 and Notch3 and their downstream genes, including Hes and Hey. BMP-4/BMP-7 or DAPT suppressed cell proliferation by inducing G1 cell cycle arrest. BMP-4/BMP-7 also inhibited TGF-β2-induced cell migration and EMT by modulating the Notch pathway.

Conclusions: BMP-4/BMP-7 attenuated ASC by inhibiting proliferation, migration, and EMT of LECs via modulation of the Notch pathway, thereby providing a new avenue for ASC treatment.

Figure 1.

BMP-4 and BMP-7 suppressed the formation of injury-induced ASC in mice. (A) Representative images of anterior segment with slit-lamp of mice 7 days after surgery. White arrows show subcapsular opacity. (B) Immunofluorescence of lens anterior capsule whole mount with α-SMA (red) for the EMT marker and DAPI (blue) for nuclei. The horizontal pictures show the section with the largest area and the orthogonal view displays the thickness of the opacity. Scale bar = 100 µm. (C, D) Quantification and statistical analysis of the volume of subcapsular plaques and α-SMA-positive cells. Twelve capsules were collected in each group in three independent experiments. (E) Output electrophoretogram by Simple Western with the HDR exposure of the relative protein expression of E-cadherin, N-cadherin, and fibronectin of mouse lenses. (F) Quantification and statistical analysis of relative protein expression. Data from three independent experiments are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

RNA sequencing analysis identified the Notch pathway as a possible mechanism underlying BMP-4- and BMP-7-mediated EMT inhibition in vivo. (A) GO enrichment analysis between the BMP-4-treated and control groups shown by a dot plot. (B) GO enrichment analysis between the BMP-7-treated and control groups shown by a dot plot. GeneRatio shows the ratio of the genes found enriched in the specific pathway dataset to the total genes in the dataset. Color grading from blue to red represents the P value. The number of genes enriched in the specific pathway set is shown by the size of the dot (count). (C) Heatmap of DEGs related to the Notch signaling pathway, cell migration, cell proliferation, and cell differentiation. Color gradation from orange to blue indicates relative gene expression. Dots of different colors show DEGs related to Notch signaling pathway (green), cell migration (yellow), cell proliferation (blue), and cell differentiation (red). Each group had four biological replications. (D) Results of RT‒PCR show the relative expression of Notch1, Notch2, Notch3, Hes1, Hes5, Hes6, Hes7, Hey1, Hey2, and Heyl. β-actin was used as a reference gene. (E, F) Images and statistical analysis of Simple Western showed the relative protein expression of Notch1 and Notch3 normalized to β-actin. Data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

BMP-4 and BMP-7 inhibited the Notch pathway in vitro. (A, B) Images and statistical analysis of the results of Simple Western showed the relative protein expression of Notch1 and Notch3. (C) Real-time PCR results of the relative mRNA expression levels of Hes1, Hes2, Hes3, Hes4, Hes5, Hes6, Hes7, Hey1, Hey2, and Heyl. β-actin was used as a reference gene. (D, E, F, G) Images in traditional Western blot style and statistical analysis of Simple Western displayed the relative protein expression of Notch3 normalized to β-actin after cotreatment with JAG-1 or DAPT. Data from three independent experiments are presented as the mean of the relative fold change ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no statistical significance.

Figure 4.

BMP-4 and BMP-7 attenuated the proliferation of LECs by inducing cell cycle arrest. (A) OD reading at 450 nm after treatment with BMP-4 or BMP-7 for 72 hours. (B) Relative expression of PCNA normalized to β-actin shown by Simple Western assay. (C) Quantification and statistical analysis of protein expression. (D) Representative immunofluorescence pictures of the EdU assay. DAPI (blue) represents the nuclei. Scale bar = 50 µm. (E) Statistical analysis of the percentage of EdU-positive cells per visual field. (F, G) Simple Western results and quantification of relative protein expression of CDK2, CDK4, p21, and p27 normalized to that of β-actin. (H, I) Simple Western results and quantification and statistical analysis of the protein levels of PCNA, CDK4, p27, and p21 normalized to that of β-actin. Data represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

BMP-4 and BMP-7 inhibited the migration and EMT of LECs induced by TGF-β₂. (A) Representative pictures of scratch width at 0 hours and 24 hours. Straight black lines represent wound edges at 0 hours. (B) Quantification and statistical analysis of relative cell migration into the scratch area. (C) Representative pictures showing cells that migrated through the pore and clung to the bottom membrane after 24 hours. (D) Quantification of the transmigrated cells per field. Pictures were taken by an inverted microscope. Scale bar = 200 µm. (E) Simple Western results showed the relative protein expression of fibronectin. (F) Quantification of relative protein expression. (G) Immunofluorescence of LECs stained with DAPI (blue) representing the nuclei and fibronectin (red). Scale bar = 20 µm. (H) Mean relative cell fluorescence of fibronectin. Three independent experiments were carried out in triplicate. (I, J) Simple Western results and quantification as well as statistical analysis of the relative expression of fibronectin in LECs treated with TGF-β₂, BMPs and JAG-1, or DAPT. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Schematic illustration of the interaction among TGF-βs, BMPs, and the Notch signaling pathway in the regulation of the proliferation, migration, and EMT of LECs. TGF-β ligands, such as TGF-β₂, bind to the receptors, which initiate the following signaling pathways, including the Notch signaling pathway. The ligand of a cell binds to the receptor of its adjacent cell, which brings about proteolysis of the receptors, leading to the release of an active NICD from a membrane tether and the activation of target genes, such as Hes and Hey. Upon binding to BMP-4 or BMP-7, BMP receptors initiate the attenuation of the Notch signaling pathway to inhibit Hes and Hey and consequently reverse the proliferation, migration, and EMT of LECs.