title: Expression and iron-dependent regulation of succinate receptor GPR91 in retinal pigment epithelium.
abstract: OBJECTIVE
  GPR91, a succinate receptor, is expressed in retinal ganglion cells and induces vascular endothelial growth factor (VEGF) expression. RPE also expresses VEGF, but whether this cell expresses GPR91 is not known. Excessive iron is also proangiogenic, and hemochromatosis is associated with iron overload. Therefore, we examined the expression and iron-dependent regulation of GPR91 in the RPE.
  
  
  METHODS
  GPR91 expression was examined by RT-PCR and immunohistochemistry. Hemochromatosis mice, cytomegalovirus (CMV) infection of retina, expression of CMV-US2 in RPE, and exposure of RPE to ferric ammonium citrate (FAC) were used to examine the iron-dependent regulation of GPR91 expression. VEGF expression was quantified by qPCR. Knockdown of GPR91 in ARPE-19 cells was achieved with shRNA.
  
  
  RESULTS
  GPR91 was expressed in RPE but only in the apical membrane. Retinal expression of GPR91 was higher in hemochromatosis (Hfe(-/-)) mice than in wild-type (WT) mice. Primary RPE cells from Hfe(-/-) mice had increased GPR91 expression compared with WT RPE cells. Iron accumulation in cells induced by CMV infection, expression of CMV-US2, or treatment with FAC increased GPR91 expression. VEGF expression in the Hfe(-/-) mouse retina was increased at ages younger than 18 months, but the expression was downregulated at older ages. The involvement of GPR91 in succinate-induced expression of VEGF in RPE cells was confirmed with GPR91-specific shRNA.
  
  
  CONCLUSIONS
  GPR91 is expressed in the RPE with specific localization to the apical membrane, indicating that succinate in the subretinal space serves as the GPR91 agonist. Excessive iron in the retina and RPE enhances GPR91 expression; however, VEGF expression does not always parallel GPR91 expression.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, USA.
authors.forename: Jaya P
authors.initials: JP
authors.lastname: Gnana-Prakasam
authors.forename: Sudha
authors.initials: S
authors.lastname: Ananth
authors.forename: Puttur D
authors.initials: PD
authors.lastname: Prasad
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Sally S
authors.initials: SS
authors.lastname: Atherton
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Martin
authors.forename: Sylvia B
authors.initials: SB
authors.lastname: Smith
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005260:Female; D005290:Ferric Compounds / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology; D006432:Hemochromatosis / Q000378:metabolism; D006566:Herpesviridae Infections / Q000378:metabolism / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D007501:Iron / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018146:Muromegalovirus / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D012173:Retinitis / Q000378:metabolism / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019802:Succinic Acid / Q000494:pharmacology; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D005290:Ferric Compounds; C531499:GPR91 protein, mouse; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D019802:Succinic Acid; D007501:Iron; C013531:ferric ammonium citrate