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

Junming Wang,Shaoqing He,Hai-Ying Ma,Thomas Yorio,Raghu R Krishnamoorthy,Tudor C Badea

doi:10.1371/journal.pone.0185390

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.

Highlights

Glaucoma is a chronic eye disease affecting 70 million people [1] globally and is expected to reach 118 million by 2040 [2]
Since upregulation and co-localization of c-Jun and CCAAT/enhancer binding protein β (C/EBPβ) were observed in the retinal ganglion cell layers of rats with elevated intraocular pressure (IOP) [10], we tested c-Jun and C/EBPβ protein levels by immunocytochemistry in primary rat retinal ganglion cells (RGCs) treated with ET-1 or ET-3 for 24 hours
Since c-Jun and C/EBPβ were found to upregulate both ETA and endothelin B (ETB) receptor expression in our previous study [10], we proposed that C/EBPβ could be one of factors regulating the expression of c-Jun

Summary

Introduction

Glaucoma is a chronic eye disease affecting 70 million people [1] globally and is expected to reach 118 million by 2040 [2]. An elevation of ETB receptor expression was found in the Morrison’s ocular hypertension rat model [8], and the increased ETB expression was associated with upregulation of transcription factors, AP-1 and C/EBPβ [10]. Both transcription factors have been shown to have regulatory roles in the cell cycle, growth, differentiation, proliferation and apoptosis [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The phosphorylation of ERK1/ 2 is a key step in triggering downstream signaling and potential activation of transcriptional factors, such as c-Myc, Elk-1, c-Fos, AP-1, etc. [28,29,30,31,32,33,34]

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Conclusion