Increased retinal expression of the pro-angiogenic receptor GPR91 via BMP6 in a mouse model of juvenile hemochromatosis

PURPOSE. Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone orphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv^-/-mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv^-/- retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. METHODS. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv^-/- mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv^-/- pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. RESULTS. Expression of GPR91 was higher in Hjv^-/- retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv^-/- retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv^-/- retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. CONCLUSIONS. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.