Involvement of AP-1 and C/EBPβ in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma

Shaoqing He, Alena Z. Minton, Hai Ying Ma, Dorota Luiza Stankowska, Xiang Le Sun, Raghu Krishnamoorthy

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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 ET B 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.

Original languageEnglish
Article numbere79183
JournalPLoS ONE
Volume8
Issue number11
DOIs
StatePublished - 12 Nov 2013

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Endothelin B Receptors
glaucoma
endothelins
Transcription Factor AP-1
Glaucoma
Rodentia
rodents
Up-Regulation
receptors
eyes
Intraocular Pressure
promoter regions
Epithelial Cells
epithelial cells
Assays
Endothelins
Retinal Ganglion Cells
Genetic Promoter Regions
Messenger RNA
Microdissection

Cite this

@article{a0e89ec3283c4bbca11e7749f1a53388,
title = "Involvement of AP-1 and C/EBPβ in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma",
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 ET B 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.",
author = "Shaoqing He and Minton, {Alena Z.} and Ma, {Hai Ying} and Stankowska, {Dorota Luiza} and Sun, {Xiang Le} and Raghu Krishnamoorthy",
year = "2013",
month = "11",
day = "12",
doi = "10.1371/journal.pone.0079183",
language = "English",
volume = "8",
journal = "PLoS ONE",
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number = "11",

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Involvement of AP-1 and C/EBPβ in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma. / He, Shaoqing; Minton, Alena Z.; Ma, Hai Ying; Stankowska, Dorota Luiza; Sun, Xiang Le; Krishnamoorthy, Raghu.

In: PLoS ONE, Vol. 8, No. 11, e79183, 12.11.2013.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Involvement of AP-1 and C/EBPβ in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma

AU - He, Shaoqing

AU - Minton, Alena Z.

AU - Ma, Hai Ying

AU - Stankowska, Dorota Luiza

AU - Sun, Xiang Le

AU - Krishnamoorthy, Raghu

PY - 2013/11/12

Y1 - 2013/11/12

N2 - 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 ET B 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.

AB - 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 ET B 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.

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