Abstract
Purpose :
A growing body of evidence suggests that endothelins (ETs), a family of 21-amino-acid vasoactive peptides, and their receptors (ETA and ETB receptors) are contributors to the neuronal damage in glaucoma. However, ET’s role in apoptosis of retinal ganglion cells (RGCs) and their signaling mechanisms are not fully understood. The aim of this study is to reveal ET-1-induced signaling pathways in primary rat retinal ganglion cells.
Methods :
Primary RGCs were isolated from postnatal day 4-6 rats by panning with Thy-1 antibody. After maintaining RGCs for 7 days, the culture medium was changed to either RGC culture medium without BDNF, CNTF, and forskolin or DMEM. RGCs were then treated with 100nM ET-1 for different time points: 15 min, 30 min, 1 hour, 6 hours, and 24 hours. After the treatments, immunocytochemistry was used to detect phosphorylated p38/JNK, ATF-1, and ATF-3. Cultured RGCs were also infected with AAV-2 viral particles encoding the ETA or ETB receptor to investigate effects of receptor overexpression on MAP kinases and their substrates. Images were captured at 40x magnification using Cytation 5 Imaging system.
Results :
An increase of p-p38 staining was detected in RGCs treated with ET-1 for 15 and 30 mins. No appreciable changes in the staining of p-JNK were detected in all tested conditions. ET-1 treatment didn’t alter the expression of ATF-1 and ATF-3, which are downstream transcription factors of p38. AAV2-viral particles encoding ETA or ETB receptors resulted in high transduction efficiency and induced protein expression of the ET receptors in RGCs, however, overexpression of ETA or ETB receptors didn’t change the phosphorylation of p38 and ATF-1. An appreciable upregulation of ATF-3 was detected in RGCs following overexpression of either ETA or ETB receptor.
Conclusions :
The results suggests that such rapid phosphorylation of p38 MAP kinase may play an important role in ET-1-induced apoptosis seen in our previous studies. ATF-3 could be tightly associated with ET receptor-mediated signaling pathways. The exploration of endothelins’ roles will help understand the molecular mechanisms underlying glaucomatous changes during ocular hypertension.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.