Conclusions
Expression of key proteins having regulatory roles in apoptosis, calcium homeostasis, cell signaling, and matrix remodeling were altered by treatment with endothelins. The elucidation of molecular mechanisms underlying endothelins' actions in RGCs will help understand endothelin-mediated neurodegenerative changes during ocular hypertension.
Methods
Primary RGCs isolated from rat pups were treated with 100 nM of ET-1, ET-2, or ET-3 for 24 hours. Total RNA was extracted followed by cDNA synthesis. Changes in gene expression in RGCs were detected using Affymetrix Rat Genome 230 2.0 microarray and categorized by DAVID analysis. Real-time PCR was used to validate gene expression, and immunocytochemistry and immunoblotting to confirm the protein expression of regulated genes.
Purpose
A growing body of evidence suggests that the vasoactive peptides endothelins (ETs) and their receptors (primarily the ETB receptor) are contributors to neurodegeneration in glaucoma. However, actions of ETs in retinal ganglion cells (RGCs) are not fully understood. The purpose of this study was to determine the effects of ETs on gene expression in primary RGCs.
Results
There was more than 2-fold upregulation of 328, 378, or 372 genes, and downregulation of 48, 33, or 28 genes with ET-1, ET-2, or ET-3 treatment, respectively, compared to untreated controls. The Bcl-2 family, S100 family, matrix metalloproteinases, c-Jun, and ET receptors were the major genes or proteins that were regulated by endothelin treatment. Immunocytochemical staining revealed a significant increase in ETA receptor, ETB receptor, growth associated protein 43 (GAP-43), phosphorylated c-Jun, c-Jun, and Bax with ET-1 treatment. Protein levels of GAP-43 and c-Jun were confirmed by immunoblotting. Conclusions: Expression of key proteins having regulatory roles in apoptosis, calcium homeostasis, cell signaling, and matrix remodeling were altered by treatment with endothelins. The elucidation of molecular mechanisms underlying endothelins' actions in RGCs will help understand endothelin-mediated neurodegenerative changes during ocular hypertension.