A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells

Abstract

c-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein β (C/EBPβ) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPβ in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPβ as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPβ augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPβ was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPβ and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPβ appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.

Fig 1. Co-localization of c-Jun and C/EBPβ in RGCs.

Primary rat RGCs were cultured on glass coverslips for 7 days followed ET-1 or ET-3 treatment (100nM) for 24 hours. c-Jun and C/EBPβ (CE) protein levels were detected using immunocytochemistry in primary rat RGCs. Images were taken by using Zeiss Meta 510 con-focal microscope, and fluorescent intensity of staining was measured using NIH ImageJ. Flourescent staining of both proteins were overlaid in the merged images. The immunostaining of both factors was increased with treatment of ET-1 or ET-3. Bars represent mean and standard deviation (#: p< 0.01, one-way ANOVA, n = 6 from 2 individual experiments).

Fig 2. ET-1 induced upregulation of c-Jun and JNK.

HNPE cells were treated with 100nM ET-1 at several time points, and cell lysates were subjected to detect c-Jun and JNK using western blot. Calnexin served as a loading control for total protein. ET-1 produced an increase of c-Jun and JNK expression from 2–24 hours post treatment. A set of representative data is shown from three repeats. Densitometry of western blot bands was analyzed using Bio-Rad Image Lab software. Bars represent mean and standard deviation (*: p< 0.05 and #: p< 0.01, one-way ANOVA, n = 3).

Fig 3. ET-1 treatment enhanced AP-1 binding.

The nuclear fraction was isolated from HNPE cells following treatment with 100nM ET-1 at different time points. An EMSA assay was used to identify AP-1 DNA binding. (A) ET-1-induced c-Jun/AP-1 binding was tested using an EMSA assay. c-Jun overexpression served as a positive control for AP-1 binding, and the specificity of binding was confirmed by a binding reaction using non-biotin-labelled AP-1 oligonucleotides (NoBio) to compete the radio-labelled oligo. (C/E: C/EBPβ) A set of representative results was shown here from three repeats. (B) Supershift assay for EMSA was carried out by pre-incubation with either the phosphor-c-Jun antibody or the c-Jun antibody during the EMSA reaction system, and the specific binding of AP-1 complex was identified by the supershift (slow migration) of a complex of AP-1, labelled oligoes and antibody. Supershift is indicated with arrows in the figure (NE: nuclear protein extraction).

Fig 4. Overexpression of ET_A and ET_B receptor upregulated c-Jun expression.

(A) SYBr-based-real-time PCR was used to detect c-Jun mRNA levels in HNPE cells treated with ET-1 with/without transient overexpression of the ET_A or ET_B receptor. Cyclophilin A served as an internal control, and the relative expression of genes was normalized to control (One way ANOVA, *: p<0.05, n = 3). (B) & (C) The protein levels of c-Jun were detected by western blot to confirm the results of real-time PCR. Calnexin served as a loading control for total protein. Densitometry of western blot bands was analyzed using Bio-Rad Image Lab software. Bars represent mean and standard deviation (*: p< 0.05, one-way ANOVA, n = 3). (D) c-Jun promoter region was cloned into pGL3 luciferase vector. Cell lysates from transiently co-overexpression of either ET_A or ET_B and pGL3-c-Jun vector in HNPE cells for 24 hours were used to detect luciferase activity. Luciferase reporter assay was carried out using Dual-Light System (Applied Biosystems, Bedford, MA) according to the manufacturer’s instruction. The luminescence was normalized by the protein amount of samples and triplicates were used in the assay. Luciferase activity was increased more than 5.1 fold in the group with ET_A receptor overexpression (* p<0.05, One way ANOVA, n = 3; Bars represent mean and standard deviation) and 2.3 fold in the group with ET_B overexpression compared to control which was transfected with luciferase construct without promoter.

Fig 5. C/EBPβ binds to c-Jun promoter region and triggers c-Jun expression.

(A) ET-1 treatment enhanced C/EBPβ binding. C/EBPβ binding was determined using EMSA assay. Overexpression of C/EBPβ served as a positive control and the specificity of binding was confirmed by a binding reaction using non-biotin-labelled C/EBPβ oligonucleotides (NoBio) to compete the radio-labelled oligos. (B) C/EBPβ binding to the c-Jun promoter region was determined by the luciferase-reporter assay using overexpression of C/EBPβ in HNPE cells. The relative luciferase activity was normalized by protein amount of the samples. (* p<0.05, One way ANOVA, n = 3; Bars represent mean and standard deviation).

Fig 6. The interaction between c-Jun and C/EBPβ was confirmed using co-IP.

(A) co-IP was carried out to confirm the physical interaction of c-Jun and C/EBPβ. The cell lysate was immunoprecipitated with mouse anti-C/EBPβ and subjected to western blot to detect c-Jun using a rabbit c-Jun antibody. Rabbit IgG or mouse IgG was incubated with samples from c-Jun or C/EBPβ overexpression to serve as a negative control. The representative result is shown from three repeats with a similar pattern. (B) One twentieth of the cell lysate amount that was used in co-IP was subjected to western blot to confirm the input proteins of c-Jun and/or C/EBPβ.

Fig 7. ET-1 induced the apoptosis of HNPE cells.

HNPE cells treated with 100nM ET-1 for 24 hours induced greater apoptosis compared to control. (A) ET-1-induced apoptosis or necrosis in HNPE cells was determined using Annexin V/PI staining (Biotool, Inc. Houston, TX). Images were captured using the Cytation 5 (BioTek, Inc.). Green staining represents apoptosis from Annexin binding and red staining is indicative of necrosis from PI binding. A partial region of merged images was shown at higher magnification. (B) The apoptotic, necrotic cells, and total cells were counted and the results were shown as cell number/mm2 and cell number/100 total cell number. The significance was analyzed using One-way ANOVA. Grey bar represents apoptotic cell counting and black bar represents necrotic cell counting. (*, # represents p<0.05, n = 4. *: versus control; #: receptor overexpression + ET-1 treatment versus receptor overexpression).

Fig 8. A diagram depicting a feed-forward regulatory loop between ET receptor and c-Jun expression.

ET-1 binds to ET_A and ET_B receptors and activates them, leading to the upregulation of c-Jun and C/EBPβ expression and an increase in DNA binding ability of both transcription factors. Moreover, c-Jun and C/EBPβ were found to promote the expression of ET_A and ET_B receptors. The direct interaction between c-Jun and C/EBPβ was also firstly reported in this study. C/EBPβ plays a regulatory role in expression of c-Jun. In addition, overexpression of ET_A and ET_B receptors induces the upregulation of c-Jun as well. Taken together, our results suggest that ET-1 triggers the upregulation of c-Jun through both receptors, and conversely c-Jun also has a feed-forward role in elevating endothelin receptor expression.