title: Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.
abstract: OBJECTIVE
  Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States 2Department of Periodontics, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Pachiappan
authors.initials: P
authors.lastname: Arjunan
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Jaya P
authors.initials: JP
authors.lastname: Gnanaprakasam
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Sudha
authors.initials: S
authors.lastname: Ananth
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Romej
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Veeranan-Karmegam
authors.initials: VK
authors.lastname: Rajalakshmi
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Puttur D
authors.initials: PD
authors.lastname: Prasad
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Martin
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Mariappan
authors.initials: M
authors.lastname: Gurusamy
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Thangaraju
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States 3Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, United States.
authors.forename: Yangzom D
authors.initials: YD
authors.lastname: Bhutia
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, Georgia, United States 3Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, United States.
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
mesh_terms: D000818:Animals; D015153:Blotting, Western; D055418:Bone Morphogenetic Protein 6 / Q000378:metabolism*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004195:Disease Models, Animal; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006432:Hemochromatosis / Q000151:congenital* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D012160:Retina / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: C525817:Bmp6 protein, mouse; D055418:Bone Morphogenetic Protein 6; C531499:GPR91 protein, mouse; D043562:Receptors, G-Protein-Coupled; D012313:RNA