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

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.


Introduction
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 ultimately result in blindness [1,2]. Globally, it is estimated that there are over 70 million glaucoma patients [3]. 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. However, the precise mechanisms by which elevated IOP produces neurodegenerative effects in the retina and optic nerve head are not completely understood. A growing body of evidence suggests that endothelin-1 (ET-1), a 21 amino acid vasoactive peptide, is a contributor to the etiology of glaucoma and is one of the factors increased in response to elevated IOP [2,4,5,6,7,8,9]. ET-1 concentrations have been shown to be elevated in the vitreous humor, aqueous humor and plasma of glaucoma patients and also in some glaucoma models in animals including rat, beagle, etc. [5,9,10,11]. Increased ET-1 concentrations were also 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].
ET-1 binds to two classes of receptors namely, endothelin A (ET A ) receptors and endothelin B (ET B ) receptors, which belong to the rhodopsin superfamily of G protein coupled receptors (GPCRs). ET A and ET B receptors are expressed in many types of cells in the central nervous system (CNS) with ET B receptor being the predominant receptor both in neurons and glia in the CNS [13,14]. Both receptors are also highly expressed in various ocular tissues including ciliary body, retina and optic nerve head [15,16,17]. Upregulation of ET B receptor at the mRNA and protein level was reported in retinas and optic nerves from animal models of glaucoma and also in optic nerve head astrocytic processes in human glaucoma [18,19,20]. Our previous study has shown that increased expression of ET B receptor is associated with cell death of RGCs and axon loss in response of elevated IOP, whereas these pathological alterations were greatly attenuated in ET B -deficient rats [20]. Molecular mechanisms responsible for regulation of ET B receptor are gaining increased attention, however there are very few studies addressing ET B receptor gene regulation in ocular cells. Using the Promo3 software, our preliminary analysis indicated six binding sites for Activator protein-1 (AP-1) and forty binding sites for CCAAT/enhancerbinding protein b (C/EBPb) in the promoter of the human ET B receptor gene. Interestingly, increased immunostaining of c-Jun [21] and upregulation of c-Jun and ATF-3 mRNA [22] have been observed in retinas of rats with elevated IOP. In addition, longterm activation of c-Fos and c-Jun in astrocytes was also observed in a monkey model of glaucoma [23]. These observations suggest that AP-1, a transcription factor, may play an important role in gene regulation under glaucomatous conditions. AP-1 is a protein complex comprising of homodimers or heterodimers of basic leucine zipper proteins including Jun, ATF (activating transcription factor), Fos, Maf and JDP, and it is a key transcription factor regulating cell death and survival pathways [24,25]. CCAAT/ enhancer-binding protein (C/EBP) is another member belonging to basic leucine zipper transcription factor family, including C/ EBPa, b, c, d, e and f. Their cellular roles include regulation of cell cycle, growth and differentiation, and C/EBPs act by binding to the CCAAT motif present in the several gene promoter sequences [26,27,28,29]. Studies in the cardiovascular system have shown that both AP-1 and C/EBPb are involved in regulating ET B receptor expression in response to external stimuli in vascular smooth muscle cells [30,31]. However, the functional roles of AP-1 and C/EBPb in regulating the ET B receptor expression in ocular tissues, especially in glaucoma, are still not clear.
The aim of this study was to investigate the role of transcription factors, AP-1 and C/EBPb, in upregulation of ET B receptor expression in human non-pigmented ciliary epithelial cells (HNPE) and determine their status in retinal ganglion cells in retinas of rats with elevated IOP.

Promoter-reporter Activities of Different ET B Receptor Promoter Constructs and Increased ET B Receptor Promoter Activity Following Overexpression of c-Jun or C/EBPb in HNPE Cells
The 1258 bp upstream promoter element of the ET B receptor was analyzed using the software Promo 3 (http://alggen.lsi.upc. es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB = TF_8.3/). Six AP-1 binding sites (Fig. 1A) and forty C/EBPb sites (not shown in the diagram) were found on the full length ET B receptor promoter region. Six AP-1 binding sites were located at different regions of ET B receptor promoter, and there were two in 21 to 2300 bp, one in 2301 to 2600 bp and three in 2601 to 21258 bp regions of the promoter (Fig. 1A).
Since no retinal ganglion cell line is currently available, the present study was carried out using the transformed human nonpigmented ciliary epithelial (HNPE) cells. The HNPE cell line has been shown to have ET A /ET B receptor expression, which was confirmed by ET-1 binding assay in our laboratory [32]. In addition, high efficiency of transfection with plasmid constructs was obtained using this cell line. Promoter-reporter assays using the firefly luciferase gene as a reporter were used to examine the transcriptional activity of different human ET B receptor promoter regions. In presence of 300 bp and 600 bp ET B receptor promoter region (Fig. 1B), there are 21.7-and 23.4-fold increase, respectively, in luciferase activity, compared to that of the empty vector.
Furthermore, an 89.2-fold increase in luciferase was detected with 1258 bp promoter region compared to empty vector control, which was a promoter-less vector linked to the luciferase gene. An activity of the positive control vector containing SV40 promoter was about 50-80 fold higher than the full-length ET B receptor promoter vector (not shown in the figures). However, no significant difference was detected in luciferase activity induced by 300 bp-and 600 bp-promoters.
Since the promoter region of ET B receptor (Fig. 1A) was found to have six AP-1 and forty C/EBPb binding sites, co-expression of either c-Jun or C/EBPb with luciferase constructs was carried out and luciferase assays were used to determine the effect of overexpression of these transcription factors binding to the ET B receptor promoter. Without the promoter sequence, overexpressed c-Jun or C/EBPb produced no significant increase in luciferase activity, whereas in the presence of the full length ET B receptor promoter region in the assay, overexpressed c-Jun or C/EBPb boosted the luciferase activity 2.7-and 3.1-fold respectively compared to that of the full length construct alone (Fig. 1C).

Promoter Activities of ET B Receptor Promoter Constructs with Mutations at Different AP-1 Binding Sites in HNPE Cells
In order to study the relative contribution of AP-1 binding sites to the ET B receptor promoter activity, six AP-1 binding sites on the full length ET B receptor promoter construct were mutated or deleted using site-directed mutagenesis. The deletion at 2615 to 2624 bp of ET B receptor promoter region reduced the luciferase activity by 45.3% compared to wild-type promoter (Fig. 2). A modest decrease in activity was also shown in promoter constructs with mutations at 288 to 290 (by 9.1%), 2199 to 2211 (by 4.6%), and 2957 to 2960 (by 11.3%) compared to that of the full length promoter; however, the attenuation effects were not statistically significant, compared with full-length promoter construct. Statistical analysis indicated a significant difference (p,0.01) between constructs with mutation in the regions between 2615 to 2624 bp (by 45.3%), and 2835 to 2838 bp (by 22.3%), when compared to the full-length ET B receptor promoter construct.
The 2615 to 2624 bp Region of the ET B Receptor Promoter is a Crucial AP-1 Binding Site Regulating ET B

Receptor Expression in HNPE Cells
Results of luciferase assays using six ET B receptor mutations at different AP-1 binding sites suggest that the binding site at 2615 to 2624 bp is the most important for ET B receptor transcription. To further address AP-1's binding and interaction, additional luciferase assays using mutated promoter with coexpression of c-Jun were performed. HNPE cells were co-transfected using either the wild-type full-length ET B receptor promoter construct or fulllength ET B receptor promoter containing the deletion at site of 2615 to 2624 bp, with or without c-Jun overexpression and promoter-reporter assays were carried out. Luciferase activity of the 2615 to 2624 bp mutant construct was 54% of the value obtained from wild-type promoter construct (Fig. 3A) without c-Jun protein overexpressed. Following c-Jun overexpression, a similar trend of the attenuating effect (57% of the wild-type promoter construct) was observed. There was no statistically significant difference in decrease in promoter activities between these two groups. Experiments were repeated twice in triplicate, the same trend was obtained. Taken together, overexpression of c-Jun didn't alter the attenuating effect of mutation in this specific binding site to trigger ET B receptor transcription. This suggests that the 2615 to 2624 bp region is a key AP-1 binding site in ET B receptor promoter.
Furthermore, physical interaction of the DNA in the 2615 to 2624 bp region of the ET B receptor promoter and transcription factor AP-1 was confirmed by chromatin immunoprecipitation (CHIP) assays. HNPE cells were transfected with either an empty vector or c-Jun expressing plasmid DNA, cross-linked with formaldehyde and sonicated chromatin fragments of 200-800 bp were obtained from the cells. DNA fragments were immunoprecipitated by incubation with or without c-Jun antibody. A pair of PCR primers was designed to amplify a 150 bp fragment of ET B receptor promoter containing AP-1 binding site 2615 to 2624 bp. The results from real-time PCR showed that there was a 4.1 fold increase of AP-1 binding to this specific region in c-Jun overexpression group compared to vector-transfected control (Fig. 3B). The positive control, promoter region of cyclooxygenase-2 (COX-2), which was upregulated by c-Jun [33], was increased to 9.6 fold in c-Jun overexpression group (Fig. 3B), whereas promoter region of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which served as a negative control showed no appreciable change. Amplification of target fragments was also validated by regular PCR reactions, and PCR products were visualized by a 1.5% agarose gel with SYBR staining (Fig. 3B).

Regulated ET A and ET B Receptor Expression in Response to Knock-down or Overexpression of c-Jun or C/EBPb in HNPE Cells
In order to explore the role of c-Jun and C/EBPb in the regulation of ET A and ET B receptor, either c-Jun or C/EBPb was overexpressed by transfection of c-Jun or C/EBPb constructs into HNPE cells. Following overexpression, protein level of c-Jun and C/EBPb was detected by immunoblotting and found to be Luciferase assay showed promoter activities using different size constructs of the ET B receptor promoter. 1 mg of promoter construct plasmid DNA was transfected into HNPE cells and luciferase activity was measured using a Luciferase assay kit (Promega). The 300 bp and 600 bp upstream promoter constructs yielded 21.7-and 23.4-fold, respectively, increase in luciferase activity, whereas 1258 bp promoter produced an 89.2-fold increase, compared to the empty vector control without a promoter sequence. C. Promoter activities of full-length ET B receptor promoter in presence of c-Jun or C/EBPb co-expression in HNPE cells. 0.5 mg of the human ET B receptor promoter construct was co-transfected with 0.5 mg of either c-Jun or C/EBPb construct in HNPE cells. The results showed that overexpression of either c-Jun or C/EBPb boosted the luciferase activity 2.7 and 3.1 fold respectively, compared to that of the ET B receptor promoter construct alone (*p,0.05, One Way ANOVA, Student-Newman-Keuls). Bars represented mean 6 SEM, n = 3. doi:10.1371/journal.pone.0079183.g001 appreciably increased in their corresponding overexpression groups. An enhanced protein level of ET B receptor was detected from membrane fraction of HNPE cells in both c-Jun and C/ EBPb overexpression groups (Fig. 4A). On the other hand, overexpression of c-Jun or C/EBPb also triggered a 10-23-fold increase of ET A and ET B receptor mRNA level (Fig. 4B). In a different set of experiments, c-Jun or C/EBPb expression was knocked down by administration of siRNA in HNPE cells. Knockdown of c-Jun or C/EBPb significantly decreased mRNA level of both ET A and ET B receptor by more than 5 fold (Fig. 4C). Effects of siRNA knock-down of c-Jun or C/EBPb were confirmed by Western blot and real-time PCR (Fig. 4C). The c-Jun protein level was decreased in c-Jun siRNA knock-down group (Fig. 4C).
However, knock-down effect of C/EBPb was not readily discernible since the endogenous protein level of C/EBPb was very low and not detectable by Western blot. Interestingly, knockdown of either c-Jun or C/EBPb abolished the mRNA level of both C/EBPb and c-Jun. These observations suggest that AP-1 and C/EBPb are key regulators of ET B receptor expression in HNPE cells.

mRNA Levels of ET A , ET B Receptor and c-Jun were Upregulated in Retinas of Elevated IOP Eyes in Rats
To specifically investigate changes in mRNA expression of ET A , ET B receptor and c-Jun in response to elevated intraocular pressure (IOP) in a rat glaucoma model, IOP was elevated by injection with hypertonic saline into episcleral veins in the left eye in Brown Norway rats. IOP exposure was calculated as the integral product of the extent of IOP elevation and the duration for which rats were maintained following IOP elevation (which is the difference in areas under the curves of IOP-elevated and contralateral eyes to x-axis) and expressed as mm Hg-days. IOP elevation for 2 weeks in Brown Norway rats typically generated IOP exposures between 45 and 100 mmHg-days, depending upon the extent of IOP elevation (Fig. 5A). Ganglion cell layers (GCLs) from retina sections were captured by Laser Capture Microdissection using freshly generated cryosections from rat eyes (Fig. 5B). Real-time PCR was used to detect changes in level of gene expression in mRNA extracted from captured GCL. There was a 3.1-fold, 4.4-fold and 2.2-fold increase in ET A , ET B and c-Jun mRNA levels respectively in IOP elevated eyes compared to contralateral eyes (Fig. 5C).

Immunostaining of c-Jun and C/EBPb was Significantly Increased in Retinal Ganglion Cells in Rat Eyes with IOP Elevation for Two Weeks
Since overexpression of c-Jun or C/EBPb was found to upregulate ET B receptor expression (Figures 1 and 4) immunostaining for c-Jun and C/EBPb was carried out in retina sections from rats with elevated IOP to determine if these factors are upregulated in vivo in the retina following ocular hypertension. Briefly, IOP was elevated in the left eye of six retired breeder Brown Norway rats, while the right eye served as the corresponding contralateral control eye. Following IOP elevation, rats were maintained for 2 weeks, and IOP values obtained and plotted as a function of time. A representative plot of IOP elevation in a Brown Norway rat is shown in Figure 6B which yielded an IOP exposure of 66.2 mm-Hg days. After maintaining rats with elevated IOP for 2 weeks, they were sacrificed. Five micron retina sections were obtained and immunohistochemical staining for c-Jun and C/ EBPb was carried out. Immunohistochemical analyses revealed that there was a significant increase of c-Jun and C/EBPb immunostaining in IOP-elevated eyes (Fig. 6A). Based upon a quantitative analysis of fluorescent intensity measurements, a 2.1and 9.1-fold increase in immunostaining for c-Jun and C/EBPb respectively was obtained in retinas from IOP elevated eyes, compared to contralateral eyes (Fig. 6C). The increased staining was observed primarily in GCL, suggesting that increased levels of c-Jun and C/EBPb may contribute to upregulation of ET B receptor expression in retinal ganglion cells following elevation of IOP.

Discussion
Several recent studies suggest that increased expression of the ET B receptor plays a key role in neurodegeneration in glaucoma; however, the gene regulation of ET B receptor is an area that needs further investigation. Knowledge of key regulatory elements mediating ET B receptor upregulation and transcription factors binding to these sites will provide additional tools to block ET B receptor expression, which could be useful to generate neuroprotective approaches. After screening the promoter region of ET B receptor by Promo3 software, six AP-1 binding sites and forty C/ EBPb binding sites were identified. Overexpression of c-Jun or C/ EBPb enhanced the downstream ET B receptor promoter activity by 3 fold compared to the full length promoter control without coexpression of any other transcription factor. This result demonstrated that the AP-1 and C/EBPb binding sites in the ET B 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 2300 bp and In the absence of c-Jun overexpression, luciferase activity obtained from the mutant construct was 54% of the value from wild-type promoter construct. Following c-Jun overexpression, a similar trend of decrease (57% of the wild-type promoter) in promoter activity was observed in the mutant construct. Bars represented mean 6 SEM, n = 3. B. CHIP assays were performed in HNPE cells, which were transfected with vector control or c-Jun overexpression plasmid DNA. DNA fragments were immunoprecipitated by incubation with or without c-Jun antibody. The data obtained by real-time PCR showed that there was a 4.1 fold increase of AP-1 binding to this specific region of ET B receptor promoter in c-Jun overexpression group compared to vector-transfected control (*p,0.05, student's t-test). The positive control, promoter region of cyclooxygenase-2 (COX-2) was increased 9.6 fold in c-Jun overexpression group (**p,0.005, student's t-test), whereas promoter region of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which served as a negative control showed no appreciable change. Bars represented relative fold increased in data obtained from real-time PCR, n = 4-6. A representative agarose gel shows the amplified DNA from regular PCR at the end of 31 cycles. doi:10.1371/journal.pone.0079183.g003 2600 bp constructs (Fig. 1), which suggested that the AP-1 binding site at the 2449 to 2456 bp region does not contribute appreciably to regulation of ET B receptor gene expression. Data from the mutation at this site confirmed the conclusion (Fig. 2). Although mutations at three binding sites within 2600 to 21258 bp showed differences in their ability to diminish luciferase activities, the AP-1 binding site located at 2615 to 2624 bp was the most important for ET B receptor promoter activity. This was confirmed by a significant decline in the promoter activity in the construct having a truncation at the 2615 to 2624 bp site (Fig. 2). Overexpression of c-Jun didn't alter the attenuating effect of this mutation (Fig. 3A), and physical interaction of AP-1 and the 2615 to 2624 bp site of ET B receptor promoter was further confirmed by CHIP assays (Fig. 3B). These observations suggest that AP-1 and C/EBPb are capable of binding to regulatory sites on the ET B Morrison's model of glaucoma in rat was developed by injection of hypertonic saline into episcleral veins in the left eye in Brown Norway rats. IOP was monitored twice a week using a Tonolab tonometer. IOP exposure was calculated as the integral product of extent of IOP elevation and the duration for which rats were maintained following IOP elevation and expressed as mm Hg-days. A plot of IOP from treated eye (open circle) and contralateral eye (closed circle) for 3 weeks following surgery indicates the extent of IOP exposure to be 49.7 and 61.3 mmHg days for two rats respectively. The values of IOP obtained are plotted as mean 6 SD, n = 10 (10 values was obtained at each time point). B. Eyes were enucleated from rats following two-week IOP-elevation in left eyes, while the corresponding right eyes served as contralateral controls. The ganglion cell layers were captured from retina cryosections by Laser Capture Microdissection. C. Equal amounts of total RNA extracted from ganglion cell layers were transcribed to cDNA (Pico RNA Extraction, Applied Biosystem). Real-time PCR was employed to detect gene expression using cDNA generated from both IOP elevated and control eyes. Two separate tissue captures and total RNA isolations were carried out for each eye. The analysis showed a 3.1-, 4.4-and 2.2-fold increase in mRNA level of ET A , ET B receptor and c-Jun in RGC layer from IOP-elevated eyes, compared to control eyes. Results are shown as mean 6 SEM, n = 4. *indicates statistical significance (p,0.01, student's t-test). doi:10.1371/journal.pone.0079183.g005 receptor promoter region and trigger ET B receptor expression in HNPE cells.
Since AP-1 and C/EBPb were found to have a positive regulatory effect on the ET B receptor promoter, the role of these two transcription factors in regulation of ET A and ET B receptor mRNA and protein levels was investigated. In the current study, knock-down of either c-Jun or C/EBPb significantly attenuated the mRNA level of both ET A and ET B receptor. On the other hand, overexpression of c-Jun or C/EBPb boosted the transcription of ET A and ET B receptor and increased protein level of ET B receptor, suggesting that mRNA expression of both ET A and ET B receptor are regulated mainly by c-Jun or C/EBPb. Interestingly, upregulation or downregulation of either of these two transcription factors (c-Jun and C/EBPb) regulated the other in the same manner. It may indicate that there are some interactions between c-Jun and C/EBPb 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/ EBPb to promoter region of Jab1. Mutations in the C/EBPb binding site reduced Jab1 promoter activity [34]. Furthermore, the inhibition of c-Jun N-terminal kinases (JNKs) abolished the expression of C/EBPb and its binding activity [35]. On similar grounds, the lack of C/EBPb in C/EBPb 2/2 mice significantly attenuated ERK1/2, JNKs and their phosphorylated forms [36]. In addition, the direct interaction between Jun and C/EBPb, which forms the heterodimer, altered the regulatory role of Jun in expression of downstream genes [37,38]. 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 Figure 6. Immunostaining of c-Jun and C/EBPb was significantly increased in retinal ganglion cells in eyes with IOP elevation for two weeks. The Morrison's glaucoma model was developed in Brown Norway rats and rats with IOP elevation were maintained for 2 weeks. Paraformaldehyde-fixed retina sections from these rats were stained using specific antibodies to c-Jun and C/EBPb (Santa Cruz Biotech). Eight to ten images were captured using Z-scan in a Zeiss 510meta confocal microscope from each view and stacked. A. Stained RGCs are indicated by white arrows. Red: c-Jun; Green: C/EBPb; Blue: DAPI. Intense staining of c-Jun and C/EBPb (indicated by arrows) was detected mainly in RGC layer from retinas of rats and increased staining was observed in retinal sections from IOP elevated rat eyes. B. A representative plot from a Brown Norway rat which was subjected to IOP elevation and maintained for 2 weeks. IOP was measured twice a week and values were plotted as mean 6 SD (n = 10) for each measurement. The IOP elevation generated 66.2 mm Hg-days of IOP exposure in this rat. C. Fluorescent intensity was measured at 10 different regions in the ganglion cell layers using NIH ImageJ. The fluorescent intensity values are shown as mean 6 SEM, n = 10. doi:10.1371/journal.pone.0079183.g006 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 (Fig. 5B); however, RGCs were the major components in the LCM-captured tissue. In the past two years, Morrison's group [39,40] 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/EBPb. 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 [39,40], 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/EBPb was also detected in RGC layer (Fig. 6A). However, the mRNA of C/EBPb was not detectable with several pairs of different primers. Although the promoter assays in the current study showed that c-Jun and C/EBPb are upstream regulators to bind the promoter of ET B receptor and activate the transcription of ET B 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 [41]. However, the mechanisms that Jun-mediated pathways activate apoptosis of RGC have not been elucidated. It is possible that ET A and ET B receptors are downstream targets of JNK activation and gene expression and some of the protective effects of JNK inactivation may be through attenuation of ET B 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. [42,43,44,45,46]. 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 [4,33,47,48,49,50]. Therefore, besides the pathways which regulate the expression of ET A and ET B 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 ET B receptor mRNA level from IOP-elevated eyes.  reported an increase in mRNA level of ET B receptor in whole retinas in a glaucoma rat model using a laser-induced photocoagulation of the trabecular meshwork to elevate IOP [22]. The current study further identified the localization of the increase of ET B receptor gene expression to RGC layer. In addition, a 3.1fold increase of ET A receptor mRNA was also detected in RGC layer in elevated IOP eyes by real-time PCR. Therefore, it is possible that in addition to ET B receptor, ET A receptor may also be involved in RGC death. Application of bosentan, an antagonist to both ET A and ET B receptor, significantly attenuated glaucomatous alterations in DBA/2J mouse model without changes in blood pressure, IOP elevation and onset of glaucoma [51]. However, the exact role that the ET A receptor plays in the pathogenesis of glaucoma remains to be understood.
In this study, the roles of transcription factors, AP-1 and C/ EBPb, in regulation of ET B 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 ET B receptor gene. The 2615 to 2624 bp region is the key binding site of AP-1 in ET B receptor promoter and was found to be crucial for inducible ET B receptor expression. Increased expression of c-Jun and C/EBPb was associated with upregulation of ET B receptor expression in rat retinas in response to elevated IOP. A comprehensive understanding of the role of AP-1 and C/EBPb in ET B receptor regulation in glaucoma would help develop molecular tools to control inappropriate ET B receptor expression for neuroprotective approaches in glaucoma.

HNPE Cells Culture, Transfection of Plasmid DNA and siRNA Knock-down
Human non-pigmented ciliary epithelial (HNPE) cells, a kind gift from Dr. Miguel Coca-Prados (Yale University) [52], were propagated using DMEM containing 10% fetal bovine serum in the presence of Penicillin (100 mg/ml) and Streptomycin (100 units/ml). To study the effects of c-Jun or C/EBPb overexpression, HNPE cells cultured in 100-mm dish were transfected with 10 mg plasmid DNA constructs encoding either c-Jun or C/EBPb open reading frame using Roche FuGene 6 (Roche Applied Science, Indianapolis, IN). Cells collected 24 hours after transfection were used for RNA isolation/real-time PCR analysis and protein detection. To study the effects of c-Jun or C/EBPb knockdown, cells cultured in 35 mm dish were transfected with 100 pmole siRNA of c-Jun or C/EBPb (Santa Cruz Biotechnologies Inc. Santa Cruz, CA) using LipoFectamine 2000 (Invitrogen, Grand Island, NY). Cells collected 24 hours after transfection were used for RNA extraction. Total RNA was extracted using Trizol method (Invitrogen, Grand Island, NY), and RNA quality and quantity were monitored using a nano drop spectrometer. One microgram of total RNA was transcribed to cDNA which served as template for real-time PCR analysis to detect gene expression of ET A receptor, ET B receptor, c-Jun and C/EBPb. Human cyclophilin A served as an internal control. Primers were used as shown below.
hc-Jun (NM_002228. Relative fold-increase in gene expression was calculated by first normalizing to the corresponding cyclophilin A levels and then a ratio to gene expression in the empty vector control was calculated.

Promoter Constructs, Mutagenesis and Luciferase Assay
The upstream promoter fragments located at 1258 bp, 600 bp and 300 bp from the transcriptional start site of the human ET B receptor promoter were generated by PCR amplification from human genomic DNA and inserted into pGL3 promoter vector carrying the firefly luciferase reporter gene (Promega, Madison, WI). The construct with 1258 bp promoter region served as a full length promoter, the pGL3 with SV40 promoter as the positive control and empty pGL3 without a promoter sequence as a negative control. Six AP-1 binding sites on 1258 bp full length ET B receptor promoter region were identified using the software Promo3 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/ promoinit.cgi?dirDB = TF_8.3). Site-directed mutagenesis was employed to mutate each predicted binding site by deletion or change of nucleotides using QuikChange II Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA). All the ET B receptor promoter constructs were confirmed by DNA sequencing. The plasmid DNA constructs were transfected into HNPE cells for 24 hours following which cells were disrupted using a lysis buffer (Promega, Madison, WI). The resultant cell lysate (20 ml) was mixed with luciferase reagent (50 ml). Mixture was vortexed briefly and luminescent value was measured immediately in Luminometer (Turner Biosystems, Promega, Madison, WI). Assays were carried out in triplicate and mean values were normalized with corresponding protein amount. The relative fold increase in reporter activity was obtained by calculating the ratio of the activity of the promoter construct to that of the empty vector control.

Chromatin Immunoprecipitation (ChIP) Assays
ChIP assays were performed to determine if transcription factor AP-1 interacts with the 2615 to 2624 bp site within the ET B receptor promoter region to provide further evidence to strengthen the data obtained from the luciferase assays. HNPE cells transfected with either the empty vector or the c-Jun overexpressing plasmid DNA for 24 hours were cross-linked by 1% formaldehyde for 10 min at room temperature and subsequently quenched with glycine. Cells were lysed and nuclei were isolated from the transfected cells. The resultant chromatin was resuspended in Lysis Buffer 3 (10 mM Tris-HCl, pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, and a cocktail of protease inhibitors) and sonicated to obtain DNA fragments at 200-800 bp. The samples were treated with 1% Triton X-100 (final concentration) and subjected to centrifugation at 20,000 g at 4uC for 10 min. The supernatant of samples were incubated overnight at 4uC on rotator with either 4 mg of c-Jun antibody (Santa Cruz Biotechnologies Inc. Santa Cruz, CA) or without antibody which served as the sham control. Following incubation, 25 ml of magnetic A/G beads (Thermo Scientific, Rockford, IL) was added to each reaction and suspension was incubated for 3 hours at 4uC with rotation. The beads were washed four times with RIPA buffer (10 mM Tris-HCl, 0.25 M LiCl, 0.5% NP-40 and 0.5% sodium deoxycholate, pH 7.5) and two times with TE buffer supplemented with 50 mM NaCl. The cross-linking was disrupted by treatment with 10% Chelex-100 by boiling. The samples were treated with RNase A and Proteinase K. The resultant DNA was used as template to analyze AP-1 binding regions within promoter of genes. The following PCR primers were used to assess AP-1 binding to their respective promoters: ET B receptor promoter (flanking region containing 2615 to 2624 bp site): Forward: 59-GGGTAAAGGAAGGAGCGCG Reverse: 59-CTACTCCCTGGCTGGCTGAG COX-2 promoter (positive control: containing AP-1 binding site) [33]: Forward: 59-CCCCACCGGGCTTACG Reverse: 59-GTCGCTAACCGAGAGAACCT GAPDH promoter (negative control: containing no AP-1 binding site) [53]: Forward: 59-ATGGTTGCCACTGGGGATCT Reverse: 59-TGCCAAAGCCTAGGGGAAGA.
The abundance of promoter sequences that were bound by c-Jun was analyzed by real-time PCR and PCR using 2 ml of DNA as template and SsoAdvanced SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Experiments were repeated at least 4 times in duplicate or triplicate. Fold increase in DNA bound by c-Jun in each experimental group was obtained by subtracting C T values obtained from sham bead control and normalized to GAPDH negative control. The results were confirmed by regular PCR and PCR products were subjected by 1.5% agarose gel with SYBR visualization.

Morrison's Rat Model of Glaucoma
Wild-type Brown Norway rats (male, retired breeder, 200-300 g; Charles River, Wilmington, MA) were used in experiments. All procedures were carried out in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research under the guidelines of the UNTHSC Institutional Animal Care and Use Committee (IACUC). The animal experimental procedures were reviewed and approved by IACUC with Protocol number: 2011/12-51-A05. The rats were anesthetized using an anesthesia cocktail during surgery and sacrificed with overdose of sodium pentobarbital to minimize suffering. Eight Brown Norway rats were anesthetized using a cocktail of ketamine, xylazine and acepromazine, and surgery was performed in the left eye to elevate intraocular pressure using the procedure developed by Dr. John Morrison as described below [54]. In brief, episcleral veins in rat eye were injected with approximately 50 ml of hypertonic (1.8 M) saline which resulted in scarring of the trabecular meshwork, and produced resistance in the outflow pathway thereby producing IOP elevation. IOP was surgically elevated in the left eyes of rats while the corresponding right eyes without any treatment served as contralateral controls in our experiments. IOP elevation was monitored using a Tonolab tonometer (Icare Finland Oy, Espoo, Finland) twice a week. The rats were sacrificed 2 weeks following IOP elevation with overdose of sodium pentobarbital. Eyes from six rats were fixed with 4% paraformaldehyde and retina sections were obtained for immunofluorescent staining. In a separate experiment, eyes from two Brown Norway rats were used for cryosection for laser capture microdissection to prepare samples for mRNA extraction and realtime PCR.

Immunoflourescent Staining
The Morrison's method to elevate IOP was carried out in Brown Norway rats and rats were maintained for two weeks following IOP elevation after which they were sacrificed. Rat eyes were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 4 hours. After paraffin embedding, five-micron sagittal sections of eyes were obtained and immunofluorescent staining was carried out. Briefly, slides were de-paraffinized in xylene, rehydrated using ethanol washes and PBS wash. After permeabilization using sodium citrate (0.1%) and 0.1% Triton X-100, the sections were washed with PBS and non-specific binding was blocked by incubation with 5% bovine serum albumin and 5% normal donkey serum in PBS for 1 hour. The sections were incubated with combinations of primary antibodies of target proteins (c-Jun and C/EBPb, Santa Cruz Biotechnologies Inc. Santa Cruz, CA) overnight at 4uC, followed by PBS washes and corresponding secondary antibody (donkey anti-mouse Alexa 488 or donkey anti-rabbit 647-conjugate) incubations. Control sections incubated with only secondary antibodies served as background control. The slides were overlaid by coverslips in antifade medium (FluorSave; Calbiochem, La Jolla, CA). Nearly 8-10 images were captured for each view using Zeiss 510meta confocal microscope with Z-scan, obtained images from each Z-scan were stacked. Fluorescent intensities was measured at 10 different regions in the RGC layer using NIH ImageJ and the mean fluorescence intensities were compared between elevated IOP eyes and contralateral eyes.

Laser Capture Microdissection and RNA/cDNA Sample Preparation
Brown Norway rats were used for IOP elevation in one eye, while the companion eye served as the corresponding contralateral control. After maintaining the rats with elevated IOP for 2 weeks, the rats were sacrificed by an overdose of pentobarbital and rat eyes were enucleated. The eyes were rinsed with PBS and frozen immediately in Optimal Cutting Temperature (OCT) at 280uC. Cryosections (20 mm) were prepared and stained using Hematoxylin and Eosin (H&E) staining according the instruction of HistoGene Staining Solution (Arcturus, Cat# KIT0415, Sunnyvale, CA). The ganglion cell layers were captured from retina cryosection by Laser Capture Microdissection system (Arcturus, Molecular Devices, Sunnyvale, CA). Total RNA from RGC layer was extracted using the PicoPure RNA Isolation Kit (Arcturus, Cat# KIT0204) and cDNA was prepared from equal amount of total RNA using iScript Reverse Transcription Kit (BioRad, Cat# 170-8890). Two separate tissue captures and total RNA isolations were performed for each eye. Real-time PCR (Applied Biosystems, Cat# 4312704) was performed using cDNA as template. Amplification of cyclophilin A served as an internal control and ET A receptor, ET B receptor and c-Jun were amplified using following primers.
rat-ET A receptor (NM_012550. Quantitation of fold-increase in gene expression was done by first normalizing to the internal control, cyclophilin A, and the ratio of gene expression between the elevated IOP eye and the contralateral eye were plotted as histograms.