Abstract
Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB 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 ETB 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 ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ETB 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, ETA and ETB 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 ETB receptor in a rodent model of ocular hypertension.
Highlights
Glaucoma is an optic neuropathy, characterized by slow degeneration of the optic nerve, cupping of the optic disc, progressive loss of retinal ganglion cells, and visual field deficits that could result in blindness [1,2]
The human non-pigmented ciliary epithelial cells (HNPE) cell line has been shown to have endothelin A (ETA)/endothelin B receptor (ETB) receptor expression, which was confirmed by ET-1 binding assay in our laboratory [32]
Overexpression of c-Jun or C/ EBPb enhanced the downstream ETB receptor promoter activity by 3 fold compared to the full length promoter control without coexpression of any other transcription factor
Summary
Glaucoma is an optic neuropathy, characterized by slow degeneration of the optic nerve, cupping of the optic disc, progressive loss of retinal ganglion cells, and visual field deficits that could result in blindness [1,2]. There are well known risk factors associated with glaucoma, including age, race, sex, hypertension, etc. Among these risk factors, increased intraocular pressure (IOP) is the most significantly correlated with glaucoma, especially in primary open angle glaucoma. ET-1 concentrations have been shown to be elevated in the vitreous humor, aqueous humor and plasma of glaucoma patients and in some glaucoma models in animals including rat, beagle, etc. Increased ET-1 concentrations were found in aqueous humor in the Morrison’s rodent model of ocular hypertension, and ET-1 injected into vitreous induced apoptosis of retinal ganglion cells (RGC) in rats [4,5,12]
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