Abstract
Purpose:
Diabetic Retinopathy (DR), manifests as retinal microvascular defects and neuroretinal dysfunction. The progression of the fibroproliferative element of the disease is accepted to feature growth factor mediated epithelial differentiation in a process analogous to cell fate determination. Manipulating cell differentiation in the context of retinal fibrosis represents a novel approach to targeting fate transitions. We identified a signalling nexus comprising TGFβ, miR-302 and the TGFβ type II receptor (TβRII) that regulates retinal epithelial cell fate. We describe a novel interaction between smad3 and polycomb repressive complexes in miR-302 overexpressing cells that regulate phenotypic responses of retinal epithelial cells to TGFβ.
Methods:
TβRll was validated as miR-302d target by 3' UTR reporter assay, qRT-PCR and Western blot. A polycistronic lentiviral vector was generated for stable expression of miR-302. ARPE19 cells differentiated with TGFβ treatment were transduced with lentivirus and the capacity of the virus to initiate epithelial cell fate specification was assessed by Western blot and ICC using known markers of epithelial and fibroblast fates including e-cadherin, ZO-1, n-cadherin, vimentin and α-smooth muscle actin and their transcriptional regulators (slug, snail). We assessed the expression markers of induced pluipotency (Oct 4, Sox 2 and nanog). Analysis of the Smad 3 interactome was undertaken by mass spectrometry and immunopreciptation studies.
Results:
Silencing of TβRll expression and downstream TGFβ canonical signalling was evidenced by decreased smad phosphorylation. Decreased mesenchymal marker expression and increased in epithelial fate markers suggests a role for miR-302d in regulating epithelial fate. Analysis of the Smad 3 interactome in these cells identified a novel interaction with the polycomb repressive complex EZH2. This interaction was confirmed by immuno-precipitation studies. Treatment of cells with an EZH2 inhibitor recapitulated aspects of epithelial de-differentation.
Conclusions:
These findings suggest polycomb mediated repression is a feature of epithelial differentiation and may be manipulated to restore epithelial phenotype and function in diabetic retinopathy with the possibility of cellular reprogramming. This represents a novel approach to targeting cell fate decisions in disease.
Keywords: 499 diabetic retinopathy •
688 retina •
500 differentiation