Downregulation of TNFRSF10A promotes RPE cell death via PKC deactivation

Abstract

Purpose : We previously reported that downregulation of TNFRSF10A, a susceptibility gene for age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC), can cause retinal pigment epithelium (RPE) cell death in vitro and Tnfrsf10knockout mice. In this study, we investigated the molecular mechanism of RPE cell death associated with TNFRSF10A downregulation.

Methods : All in vitro experiments were performed using primary fetal human RPE (hRPE) cells. After transfection with siRNA targeting TNFRSF10A, we treated the cells with or without different cell death inhibitors or phorbol 12-myristate 13-acetate (PMA),a PKC activator, and cell viability change were evaluated by Cell Counting Kit-8 and staining with FITC-conjugated annexin V/PI followed by flow cytometric analysis. Realtime RT-PCR was performed to examine the transcriptional levels of cell cycle regulatory proteins in the cells.

Results : Knockdown of TNFRSF10A by siRNA significantly reduced RPE cell viability and increased the percentages of annexin V+/PI- early apoptotic cells (P<0.0001). PMA inhibit the cell death induced by TNFRSF10A knockdown, while apoptosis inhibitors, a necroptosis inhibitor, a feroptosis inhibitor and a pyroptosis inhibitor did not. TNFRSF10A knockdown changed mRNA levels of cell cycle regulatory proteins, downregulation of cyclinA1 (P<0.05) and upregulation of p27 (P<0.005), which were reversed by PMA treatment.

Conclusions : TNFRSF10A downregulation may contribute to the pathogenesis of AMD and CSC by promoting RPE cell death via PKC pathway.

This is a 2021 ARVO Annual Meeting abstract.