UC Santa Barbara

As retinal pigmented epithelium (RPE) cells are passaged, they undergo an irreversible epithelial-to-mesenchymal transition (EMT). We have shown previously that growth differentiation factor 6 (GDF6), a member of the transforming growth factor-beta (TGFβ) family, is highly upregulated in RPE cells that have lost the capacity to obtain an epithelial phenotype. We hypothesize that GDF6 plays an integral role in the irreversible transition of an RPE cell from an epithelial state to a mesenchymal state.

To test this hypothesis, we overexpressed GDF6 in differentiation competent RPE and assessed the effects on phenotype and gene expression. To evaluate what receptors and signaling pathways might mediate GDF6’s effects, cells were also treated with an Alk5 inhibitor (Repsox), an Alk2/3/6 inhibitor (LDN-193189), or both in combination. Passage 0 RPE transduced with GDF6 produce significantly less pigmentation than cells infected with an empty vector control. This reduction in pigmentation is accompanied with a change in cell morphology; the control cells maintain a cuboidal morphology and the GDF6 expressing cells have a spindle-like morphology. Quantitative PCR analysis reveals that RPE cells transduced with GDF6 significantly upregulate known EMT markers like ACTA2, CTGF, and COL1A1 and downregulate classical RPE markers such as LRAT and PMEL compared to control cells. Both RepSox and LDN-193189 have the ability to reverse the GDF6 phenotype. While the phenotype is recovered, RNA-seq analysis reveals GDF6-mediated regulation of genes that are unaffected by inhibitors, such as TGFβ1, MSX2, and CDH1.

GDF6 is involved in the EMT process in RPE. We believe GDF6 upregulates TGFβ1, which in turn promotes EMT. The ability to revert back to an epithelial cell is inhibited by GDF6. Cells exposed to GDF6 will prematurely undergo EMT, simultaneously downregulating traditional RPE markers while upregulating EMT and wound response markers. Inhibition of the BMP receptors rescues the GDF6 phenotype, therefore inhibition of GDF6 may be integral in prolonging the integrity and functional lifespan of the RPE. As such, inhibition of GDF6 may help restore RPE to their epithelial state in diseases like proliferative vitreoretinopathy and age-related macular degeneration.