Gremlin-1: An endogenous BMP antagonist induces epithelial-mesenchymal transition and interferes with redifferentiation in fetal RPE cells with repeated wounds.

PURPOSE

To investigate the role of Gremlin-1, which is an endogenous antagonist of the bone morphogenetic protein (BMP) signaling pathway, in inducing epithelium-mesenchymal transition (EMT) in fetal RPE cells after repeated wounds.

METHODS

Subconfluent repetitive passages in fetal RPE cells were regarded as a model of repeated wounds. A phase contrast microscope was used to observe the morphology and pigment formation in cells. The expression of (Gene ID: 26585; OMIM 603054) and EMT- or RPE-related genes in cells was evaluated with quantitative PCR (qPCR). Recombinant human protein Gremlin-1 (0.1 μg/ml) was added every day to investigate the molecular effects of Gremlin-1 on fetal RPE cells. The cell migration rate was investigated using a cell wound scratch assay, and western blotting was used to analyze the representative proteins (P-cadherin, ZO-1, vimentin, Smad4, and phosphorylated-Smads). In addition, transfection of siRNA was used to explore the rescue effects on EMT cells through the downregulation of . Finally, LDN193189, which is a type of pan-inhibitor of BMP receptors, was used to verify whether complete blocking of the BMP pathway interferes with the redifferentiation in low-passage fetal cells, even if the cells were treated with transforming growth factor beta 1 (TGF-β) inhibitors.

RESULTS

In fetal RPE cells, the expression of were gradually upregulated with repetitive passages, and at the same time, the function-specific genes in fetal RPE cells (, , , , and ) were downregulated while the EMT-specific genes were upregulated. In addition, had a similar expression pattern as , which is a key transcription factor to trigger EMT. Recombinant human Gremlin-1 promoted EMT with the upregulation of and elevated the cell migration rate in a cell scratch assay, as well as decreased the expression of two key transcription factors of RPE embryonic development ( and ) and the RPE marker, . Furthermore, the EMT marker, vimentin, and the TGF-β pathway downstream transcription factor phosphorylated-Smad2 (p-Smad2) increased, but the epithelial marker, ZO-1, was reduced. Additionally, Smad4, which plays a role as a Snail1 cooperator by binding Smad3, was also increased. In contrast, silencing increased the expression of and , which means there was better redifferentiation in subconfluent fetal RPE cells, but it had little influence on p-Smad2 compared to the negative control group. Finally, by adding LDN193189, the BMP signaling pathway was blocked, and this block led to poor redifferentiation in low-passage cells, although the cells were treated with TGF-β inhibitors. In addition, as positive feedback to block the BMP pathway, was subsequently upregulated.

CONCLUSIONS

In fetal RPE cells, Gremlin-1 induces EMT and inhibits redifferentiation by promoting the TGF-β pathway and inhibiting the BMP pathway. silencing alleviates EMT and increases the redifferentiation of cells by relieving the blockade of the BMP pathway. However, silencing has no effects on the TGF-β pathway. Thus, Gremlin-1 may serve as a novel target to treat proliferative vitreoretinopathy (PVR) and inhibit subretinal fibrosis, which is a risk factor for influencing the therapeutic effects of anti-vascular endothelial growth factor (anti-VEGF) on neovascular age-related macular degeneration (nAMD).