Endothelin-1 Mediated Endothelin B Receptor Upregulation Contributes to Apoptosis of Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Endothelin-1 (ET-1) administration to the retrobulbar region of the optic nerve has been shown to produce axonal damage similar to that seen in glaucoma. The purpose of this study was to determine if ET-1 treatment could directly contribute to cell death of retinal ganglion cells in culture. Methods: Virally transformed rat retinal ganglion cells (RGC-5 cells) were treated with 100 nM ET-1 for 4 days and apoptotic changes were monitored using TUNEL and MTT (Formazan) assays. Endothelin B (ET_B) receptor expression was studied in RGC-5 cells treated with different doses of ET-1 for 24 hr, by immunoblotting. Electrophoretic mobility shift assays for transcription factor Activator Protein-1 (AP-1) were performed to determine changes in its binding activity in RGC-5 cells treated with 100 nM ET-1. Promoter-reporter assays using two ET_B promoter-luciferase constructs (containing 300 and 600 bp upstream promoter sequences) were carried out to identify the upstream promoter elements confering constitutive and ET-1-inducible expression of the ET_B receptor in RGC-5 cells. Results: ET-1 (100 nM) treatment for 4 days produced a 30% increase in number of cells undergoing cell death. ET-1 treatment for 24 hr produced a dose-dependent increase in ET_B receptor expression in RGC-5 cells. AP-1 binding activity was increased in RGC-5 cells treated for 24 hr with ET-1 (100 nM), which was partially blocked by pretreatment with both ET_A and ET_B receptor antagonists. The 600 bp ET_B upstream promoter construct produced a 8-fold increase in constitutive luciferase activity compared to the 300 bp ET_B upstream promoter construct. Moreover, the 600 bp ET_B upstream promoter construct was also found to confer inducibility of ET_B expression by ET-1 treatment in RGC-5 cells. Conclusions: An increase in ET_B expression preceeds apoptosis of RGC-5 cells treated with ET-1. DNA binding activity of transcription factor AP-1 is increased by ET-1 treatment, which could contribute to the elevated ET_B expression by its actions on upstream promoter elements located between -300 and -600 bp of the ET_B gene.

Keywords: apoptosis/cell death • ganglion cells • molecular biology