Gremlin mediates the TGF-β-induced induction of profibrogenic genes in human retinal pigment epithelial cells

Abstract

Proliferative vitreoretinopathy (PVR) is characterised by the contraction and growth of fibrotic membranes on the retina and within the vitreous body. Retinal pigment epithelial (RPE) cells, a major cellular component of the fibrotic membrane, is one of the cell types that have been previously reported to associate with PVR pathogenesis. During PVR, RPE cells undergo increased cell proliferation, migration and the secretion of extracellular matrix molecules, such as fibronectin and type I collagen. A variety of cytokines and growth factors are involved in the formation of the fibrotic membrane. Although gremlin has been reported to serve an important role in the regulation of epithelial-to-mesenchymal transition in PVR, the relationship between gremlin and the expression of profibrogenic factors in human RPE cells remains unclear. In the present study, gremlin promoted RPE cell proliferation and the expression of type I collagen and fibronectin. In addition, knocking down gremlin expression by siRNA significantly suppressed the transforming growth factor (TGF)-β1- and TGF-β2-induced expression of type I collagen and fibronectin in RPE cells. These findings suggest that gremlin may serve an important role in the development of PVR.

Keywords: collagen I; fibronectin; gremlin; proliferative vitreoretinopathy; retinal pigment epithelial cells; transforming growth factor-β.

Figure 1.

Gremlin induces RPE cell proliferation. (A) RPE cell viability was analysed using MTT assay following treatment with 0, 25, 50 or 100 ng/ml gremlin for 48 h. (B) RPE cell viability was analysed by MTT assay following treatment with 100 ng/ml gremlin for 12, 24 and 48 h. (C) RPE cell proliferation was analysed using BrdU assay following treatment with 0, 25, 50 or 100 ng/ml gremlin for 48 h. (D) RPE cell viability was analysed using BrdU assay following treatment with 100 ng/ml gremlin for 12, 24 and 48 h. The data shown represent the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 vs. control. BrdU, 5-bromo-2-deoxyuridine; RPE, retinal pigment epithelial cells.

Figure 2.

Gremlin promotes the expression and secretion of type I collagen and fibronectin in RPE cells. The expression of type I collagen and fibronectin in RPE cells was determined using reverse transcription-quantitative PCR, ELISA and immunofluorescence staining. (A) Relative levels of fibronectin mRNA expression and (B) fibronectin secretion were measured using ELISA following treatment with 25, 50 or 100 ng/ml gremlin for 48 h. (C) Relative levels of type I collagen mRNA expression and (D) type I collagen secretion were measured using ELISA following treatment with 25, 50 and 100 ng/ml gremlin for 48 h. (E) Immunofluorescence staining of fibronectin and (F) type I collagen in RPE cells following treatment with 100 ng/ml gremlin for 48 h. Magnification, ×200. The data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. RPE, retinal pigment epithelial cells.

Figure 3.

TGF-β1 and TGF-β2 increase the expression and secretion of gremlin, fibronectin and type I collagen in human RPE cells. (A) RPE cells were subjected to TGF-β1 and/or gremlin siRNA transfection or (B) TGF-β2 and/or gremlin siRNA transfection for 48 h, following which the relative expression of gremlin mRNA was measured using reverse transcription-quantitative PCR. (C) RPE cells were first treated with 10 ng/ml TGF-β1 for 48 h before fibronectin mRNA expression, (D) fibronectin secretion, (E) type I collagen mRNA expression and (F) type I collagen secretion were measured. (G) RPE cells were first treated with 5 ng/ml TGF-β2 for 48 h before fibronectin mRNA expression, (H) fibronectin secretion, (I) type I collagen mRNA expression and (J) type I collagen secretion were measured. The data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control; ^##P<0.01 vs. respective TGF-β1 or TGF-β2 group; ^ΦP<0.05 vs. Control group. TGF-β, transforming growth factor-β; RPE, retinal pigment epithelial cells; siRNA, small interfering RNA. Con. represents Control.

Figure 4.

Gremlin mediates the TGF-β-induced induction of fibronectin and type I collagen expression and secretion in human RPE cells. The expression and secretion of fibronectin and type I collagen were measured using reverse transcription-quantitative PCR and ELISA, respectively. (A) Following treatment with TGF-β1 and/or gremlin knockdown, fibronectin mRNA expression, (B) fibronectin secretion, (C) type I collagen mRNA expression and (D) type I collagen secretion were measured. (E) Following treatment with TGF-β2 and/or gremlin knockdown, fibronectin mRNA expression, (F) fibronectin secretion, (G) type I collagen mRNA expression and (H) type I collagen secretion were measured. The data shown represent the mean ± SD of three independent experiments. **P<0.01 vs. control; ^#P<0.05, ^##P<0.01 vs. respective TGF-β1 or TGF-β2 group. TGF-β, transforming growth factor-β; RPE, retinal pigment epithelial cells. Con. represents Control.