Expression and iron-dependent regulation of succinate receptor GPR91 in retinal pigment epithelium - PubMed (original) (raw)
Expression and iron-dependent regulation of succinate receptor GPR91 in retinal pigment epithelium
Jaya P Gnana-Prakasam et al. Invest Ophthalmol Vis Sci. 2011.
Abstract
Purpose: 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.
Figures
Figure 1.
Expression and localization of GPR91 in mouse retina. (A) RT-PCR analysis of GPR91 mRNA in neural retina and RPE/eyecup. HPRT1 was used as an internal control. (B) Immunofluorescence localization of GPR91 protein in mouse retina. Left: staining in the retina using GPR91 primary antibody; middle: nuclear staining; right: merged image. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (C) Confocal analysis of the polarized expression of GPR91 in RPE. MCT1 was used as a marker for the RPE apical membrane. GPR91 was detected with a secondary antibody conjugated to Alexa Fluor 488 (green), and MCT1 was detected with a secondary antibody conjugated to Alexa Fluor 568 (red). Hoechst was used as a nuclear stain. Merging of the fluorescent signals in the RPE cell layer indicates the coexpression of the two proteins in RPE apical membrane.
Figure 2.
Upregulation of GPR91 in retina in an _Hfe_−/− hemochromatosis mouse model. (A) RT-PCR analysis of GPR91 mRNA in WT and _Hfe_−/− (KO) primary RPE cells. HPRT1 was used as an internal control. (B) Western blot analysis of GPR91 protein in whole retinas from WT and _Hfe_−/− (KO) mice. β-Actin was used as an internal control. (C) Immunofluorescence analysis of GPR91 protein in 8-month-old and 18-month-old WT and _Hfe_−/− (KO) mouse retinas.
Figure 3.
Upregulation of GPR91 in CMV-infected mouse retina and RPE cells. (A) Mice were infected in vivo by intraocular injection of MCMV. Control mice were injected with PBS. RT-PCR was performed for GPR91 with RNA samples from whole retinas of PBS-injected and MCMV-injected mice. HPRT1 was used as an internal control. (B) Primary mouse RPE cells were infected in vitro with MCMV, and the levels of GPR91 protein were analyzed by Western blot in control and MCMV-infected cells. β-Actin was used as an internal control. (C) Mice were infected in vivo by intraocular injection of MCMV. Control mice were injected with PBS. Retinal sections were prepared on day 7 after infection and used for immunofluorescence detection of GPR91 protein.
Figure 4.
Upregulation of GPR91 in mouse and human RPE cells by CMV-US2. (A) RT-PCR analysis of Hfe, GPR91, CMV-US2, and HPRT1 with RNA from control RPE cells (ARPE-19 cells and primary mouse RPE cells) and from RPE cells treated with lentivirus carrying the CMV-US2 gene. HPRT1 was used as an internal control. (B) Western blot using protein lysates from control RPE cells and from RPE cells treated with lentivirus carrying the CMV-US2 gene. The blots were developed using primary antibody specific to Hfe or GPR91. β-Actin was used as an internal control.
Figure 5.
Upregulation of GPR91 in mouse and human RPE cells by treatment with FAC. Human RPE cell lines ARPE-19 and HRPE and mouse primary RPE cells were treated with or without FAC (100 μg/mL) for 72 hours. RNA prepared from these cells was used for RT-PCR analysis of GPR91 mRNA with HPRT1 as an internal control.
Figure 6.
Expression of proangiogenic factors VEGF, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) in retina in WT and _Hfe_−/− (KO) mice. RNA was isolated from whole retinas of 8-month-old, 18-month-old, and 24-month-old WT mice and _Hfe_−/− mice. mRNA levels for GPR91, Ang-1, and Ang-2 were determined by RT-PCR, with HPRT1 as an internal control (A). VEGF mRNA levels were determined by real-time PCR (B). Three RNA samples from three mice for each genotype and age were used. Data for the real-time PCR are presented as the mean ± SE for the three independent RNA samples.
Figure 7.
Induction of VEGF mRNA expression by FAC and succinate in ARPE-19 cells with or without GPR91 knockdown. (A) ARPE-19 cells were subjected to lentivirus-mediated knockdown of GPR91 expression with four different gene-specific shRNAs. The magnitude of knockdown was monitored by RT-PCR, with 18S RNA as an internal control. Data (normalized to the internal control) are presented as mean ± SE from three RNA preparations. (B) Control ARPE-19 cells (vector only) and shRNA-expressing ARPE-19 cells were treated with FAC (100 μg/mL) for 72 hours; the final portion, 16 hours, of this 72-hour period occurred with or without succinate (2 mM). RNA from the cells with and without the treatment was used for RT-PCR to analyze the levels of VEGF mRNA. 18S RNA was used as an internal control. Data (normalized to the internal control) are presented as the average of two independent experiments.
References
- Wrighting DM, Andrews NC. Iron homeostasis and erythropoiesis. Curr Top Dev Biol. 2008;82:141–167 -PubMed
- Beutler E. Hemochromatosis: genetics and pathophysiology. Annu Rev Med. 2006;57:331–347 -PubMed
- Fleming RE, Britton RS. Iron imports, VI: HFE and regulation of intestinal iron absorption. Am J Physiol Gastrointest Liver Physiol. 2006;290:G590–G594 -PubMed
- Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996;13:399–408 -PubMed
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