Gpr91, a pro-angiogenic receptor for succinate, is upregulated in hemojuvelin-null mouse retina via increased BMP6 signaling

Abstract

Purpose: The succinate receptor GPR91 is expressed in retinal ganglion cells and retinal pigment epithelium (RPE) and its signaling promotes retinal angiogenesis through increased expression of vascular endothelial growth factor (VEGF). Retinal Gpr91 expression is upregulated in Hfe-/- mouse model of hemochromatosis. Here we studied retinal Gpr91 expression in Hjv-/- mouse model of hemochromatosis. Hemojuvelin (HJV), encoded by HFE2 gene, is a co-receptor for bone morphogenetic proteins (BMPs) and facilitates the expression of the iron-regulatory hormone hepcidin via BMP signaling. Deletion of Hjv in mice disrupts BMP signaling. Therefore, we also investigated the involvement of BMP signaling in the alterations of Gpr91 expression in Hjv-/- mouse retina and RPE.

Methods: Gpr91 expression was examined by RT-PCR, qPCR, immunofluorescence, and Western blot analysis in wild type and Hjv-/- mouse retinas and in primary RPE (pRPE) cells isolated from wild type and Hjv-/- mice. The influence of excessive iron (exposure to ferric ammonium citrate) and BMP signaling (exposure to BMP6) on GPR91 expression was investigated in ARPE19 cells and in wild type and Hjv-/- pRPE cells. Co-treatment with succinate was used to determine the changes in Gpr91 signaling and the resultant VEGF expression. BMP6 signaling was studied by monitoring the expression of Smad1/5/8 and phospho-Smad4 (Western blot and immunofluorescence) and the BMP target gene Id1 (qPCR).

Results: Gpr91 expression was higher in Hjv-/- mouse retina and Hjv-/- pRPE than in wild type mouse retina and pRPE. These changes were accompanied with increased BMP signaling as evident from increased expression of Smad1/5/8 and phospho-Smad4, and also from increased expression of Id1. Exposure to excessive iron or BMP6 also increased GPR91 expression and concomitant VEGF expression in ARPE19 cells as well as in pRPE cells. These effects were observed both in wild type and Hjv-/- pRPE.

Conclusions: Gpr91 expression is increased in Hjv-/- mouse retinas and RPE through BMP signaling. This represents the first report on the regulation of retinal Gpr91 expression by BMP6 and on increased BMP signaling in Hjv-/- retinas. Studies by other investigators have shown that BMP signaling is decreased in Hjv-/- mouse liver, but our studies demonstrate the opposite effect on BMP signaling in the retina as a result of Hjv deletion.

Keywords: 688 retina • 701 retinal pigment epithelium • 674 receptors