Involvement of AP-1 and C/EBPβ in Upregulation of Endothelin B (ET(B)) Receptor Expression in a Rodent Model of Glaucoma

Previous studies showed that the endothelin B receptor (ET(B)) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ET(B) receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ET(B) promoter region was found to be essential for constitutive expression of ET(B) receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The −300 to −1 bp and −1258 to −600 bp upstream promoter regions of the ET(B) receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at −615 to −624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPβ enhanced the ET(B) receptor promoter activity, which was reflected in increased mRNA and protein levels of ET(B) receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ET(B) and ET(A) receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ET(B) receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPβ in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ET(A) and ET(B) receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ET(B) receptor in a rodent model of ocular hypertension.