Several recent studies suggest that increased expression of the ETB receptor plays a key role in neurodegeneration in glaucoma; however, the gene regulation of ETB receptor is an area that needs further investigation. Knowledge of key regulatory elements mediating ETB receptor upregulation and transcription factors binding to these sites will provide additional tools to block ETB receptor expression, which could be useful to generate neuroprotective approaches. After screening the promoter region of ETB receptor by Promo3 software, six AP-1 binding sites and forty C/EBPβ binding sites were identified. Overexpression of c-Jun or C/EBPβ enhanced the downstream ETB receptor promoter activity by 3 fold compared to the full length promoter control without co-expression of any other transcription factor. This result demonstrated that the AP-1 and C/EBPβ binding sites in the ETB receptor promoter were functional and their cognate transcription factors were able to upregulate gene expression upon binding to these sites. Based on the luciferase activities from truncations, there was no difference in the promoter activity between −300 bp and −600 bp constructs (), which suggested that the AP-1 binding site at the −449 to −456 bp region does not contribute appreciably to regulation of ETB receptor gene expression. Data from the mutation at this site confirmed the conclusion (). Although mutations at three binding sites within −600 to −1258 bp showed differences in their ability to diminish luciferase activities, the AP-1 binding site located at −615 to −624 bp was the most important for ETB receptor promoter activity. This was confirmed by a significant decline in the promoter activity in the construct having a truncation at the −615 to −624 bp site (). Overexpression of c-Jun didn’t alter the attenuating effect of this mutation (), and physical interaction of AP-1 and the −615 to −624 bp site of ETB receptor promoter was further confirmed by CHIP assays (). These observations suggest that AP-1 and C/EBPβ are capable of binding to regulatory sites on the ETB receptor promoter region and trigger ETB receptor expression in HNPE cells.
Since AP-1 and C/EBPβ were found to have a positive regulatory effect on the ETB
receptor promoter, the role of these two transcription factors in regulation of ETA
receptor mRNA and protein levels was investigated. In the current study, knock-down of either c-Jun or C/EBPβ significantly attenuated the mRNA level of both ETA
receptor. On the other hand, overexpression of c-Jun or C/EBPβ boosted the transcription of ETA
receptor and increased protein level of ETB
receptor, suggesting that mRNA expression of both ETA
receptor are regulated mainly by c-Jun or C/EBPβ. Interestingly, upregulation or downregulation of either of these two transcription factors (c-Jun and C/EBPβ) regulated the other in the same manner. It may indicate that there are some interactions between c-Jun and C/EBPβ in HNPE cells. There is evidence to show that the expression of Jun activation-domain binding protein 1 (Jab1), the coactivator of c-Jun, was tightly controlled by binding of C/EBPβ to promoter region of Jab1. Mutations in the C/EBPβ binding site reduced Jab1 promoter activity 
. Furthermore, the inhibition of c-Jun N-terminal kinases (JNKs) abolished the expression of C/EBPβ and its binding activity 
. On similar grounds, the lack of C/EBPβ in C/EBPβ−/−
mice significantly attenuated ERK1/2, JNKs and their phosphorylated forms 
. In addition, the direct interaction between Jun and C/EBPβ, which forms the heterodimer, altered the regulatory role of Jun in expression of downstream genes 
. The detailed mechanisms by which these two transcription factors exert their regulatory roles on each other have not been fully elucidated.
Retinal ganglion cells (RGC) are output neurons located in the innermost layer of the retina, which receive inputs from bipolar cells and fire action potentials which are transmitted to the brain. The percentage of RGCs is less than 1% of total neurons in the retina of human eyes. In order to study gene expression in RGCs in vivo
, specialized techniques such as Laser Capture Microdissection (LCM) provide us the ability to isolate the retinal ganglion cell layer for a more selective analysis of changes in gene expression in a cell population of interest. In this study, the tissue obtained from LCM still contained other cell types and layers including amacrine cells and nerve fiber layer (); however, RGCs were the major components in the LCM-captured tissue. In the past two years, Morrison’s group 
employed the q-PCR and microarray to detect gene expression from LCM captured RGC layers from rat eyes with IOP elevation for five weeks. The authors found an increase in expression of ATF3 (which interacts with members of the c-Jun family) in retinas of rats with elevated IOP. Since AP-1 proteins belong to immediate early gene families, which are activated rapidly in response to a variety of stimuli, we focused on an early time point of IOP elevation (two weeks) and assessed changes in c-Jun and C/EBPβ. The mRNA levels of c-Jun in RGC layer obtained from the 2 week IOP elevated eye were increased to 2.2 fold of control levels, which is consistent with the published results 
, where a 1.4-fold increase of c-Jun was measured in the whole retina and a 5.1-fold increase in RGC layer by real-time PCR from eyes with 5-weeks of IOP elevation. A prominent increase in staining intensity of c-Jun that was observed in IOP-elevated eye could indicate higher protein expression of c-Jun in response to IOP elevation. A similar staining pattern of C/EBPβ was also detected in RGC layer (). However, the mRNA of C/EBPβ was not detectable with several pairs of different primers. Although the promoter assays in the current study showed that c-Jun and C/EBPβ are upstream regulators to bind the promoter of ETB
receptor and activate the transcription of ETB
receptor, the direct functional roles of these factors in vivo
in glaucoma experimental eyes are still unclear. Recently, lack of JNK2/3 signaling due to deficiency of JNK2/3 or Jun in mice has been shown the protective effects from optic nerve crush-induced RGC death 
. However, the mechanisms that Jun-mediated pathways activate apoptosis of RGC have not been elucidated. It is possible that ETA
receptors are downstream targets of JNK activation and gene expression and some of the protective effects of JNK inactivation may be through attenuation of ETB
receptor expression. On the other hand, MAPK, JNK, PKC and PI3K pathways are involved in signaling transduction activated via endothelin receptors, and these pathways subsequently trigger downstream signaling and activate transcriptional factors, such as c-Myc, Elk-1, c-Fos, c-Jun, AP-1, etc. 
. For instance, ET-1-mediated activation of c-Jun and JNK via endothelin receptors was proved in a variety of cell types and tissues including astrocyte, smooth muscle cells, endothelial cells 
. Therefore, besides the pathways which regulate the expression of ETA
receptor through AP-1, activated endothelin receptors also modulate the expression of transcription factors in response to the treatment of endothelins and other external stimuli.
There was a more than 4.4-fold increase detected in ETB
receptor mRNA level from IOP-elevated eyes. Yang et al., (2007) reported an increase in mRNA level of ETB
receptor in whole retinas in a glaucoma rat model using a laser-induced photocoagulation of the trabecular meshwork to elevate IOP 
. The current study further identified the localization of the increase of ETB
receptor gene expression to RGC layer. In addition, a 3.1-fold increase of ETA
receptor mRNA was also detected in RGC layer in elevated IOP eyes by real-time PCR. Therefore, it is possible that in addition to ETB
receptor may also be involved in RGC death. Application of bosentan, an antagonist to both ETA
receptor, significantly attenuated glaucomatous alterations in DBA/2J mouse model without changes in blood pressure, IOP elevation and onset of glaucoma 
. However, the exact role that the ETA
receptor plays in the pathogenesis of glaucoma remains to be understood.
In this study, the roles of transcription factors, AP-1 and C/EBPβ, in regulation of ETB receptor was investigated in the HNPE cell line and in an in vivo rat model of glaucoma. The 1258 bp upstream promoter region was found to be important for constitutive expression of the human ETB receptor gene. The −615 to −624 bp region is the key binding site of AP-1 in ETB receptor promoter and was found to be crucial for inducible ETB receptor expression. Increased expression of c-Jun and C/EBPβ was associated with upregulation of ETB receptor expression in rat retinas in response to elevated IOP. A comprehensive understanding of the role of AP-1 and C/EBPβ in ETB receptor regulation in glaucoma would help develop molecular tools to control inappropriate ETB receptor expression for neuroprotective approaches in glaucoma.