The MAPK signaling pathway mediates the GPR91-dependent release of VEGF from RGC-5 cells

Hu,Jianyan; Li,Tingting; Du,Shanshan; Chen,Yongdong; Wang,Shuai; Xiong,Fen; Wu,Qiang

doi:10.3892/ijmm.2015.2195

The MAPK signaling pathway mediates the GPR91-dependent release of VEGF from RGC-5 cells

Authors:
- Jianyan Hu
- Tingting Li
- Shanshan Du
- Yongdong Chen
- Shuai Wang
- Fen Xiong
- Qiang Wu
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Affiliations: Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, P.R. China
Published online on: April 23, 2015 https://doi.org/10.3892/ijmm.2015.2195
Pages: 130-138
Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Vascular endothelial growth factor (VEGF) is one of the major regulatory molecules in diabetic retinopathy (DR). In our previous study, we demonstrated that succinate levels were elevated in the retinas of diabetic rats and that the knockdown of the succinate receptor, G-protein-coupled receptor 91 (GPR91), inhibited the release of VEGF and attenuated retinal vascular disorder in the early stages of DR. In the present study, we examined the signaling pathways involved in the GPR91-dependent release of VEGF in the retinal ganglion cell line, RGC-5. The cells were infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91). Immunofluorescence staining revealed that GPR91 was predominantly localized in the cell bodies of the RGC-5 cells. RT-qPCR, western blot analysis and ELISA indicated that succinate exposure upregulated VEGF expression, activated the extracellular signal-regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways and led to the release of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The knockdown of GPR91 inhibited ERK1/2 and JNK activity, but did not inhibit the activation of the p38 MAPK pathway. The increase in COX-2 expression and the release of PGE2 were inhibited by transduction with LV.shGPR91 and ERK1/2, JNK and COX-2 inhibitors. The expression and release of VEGF showed similar results. Cell Counting Kit-8 (CCK-8) assays revealed that the shRNA-mediated knockdown of GPR91 decreased the proliferation of RF/6A cells cultured in succinate-conditioned medium. Our data suggest that GPR91 modulates the succinate-induced release of VEGF through the MAPK/COX-2/PGE2 signaling pathway.

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1	Nolan CJ, Damm P and Prentki M: Type 2 diabetes across generations: from pathophysiology to prevention and management. Lancet. 378:169–181. 2011. View Article : Google Scholar : PubMed/NCBI
2	Witmer AN, Blaauwgeers HG, Weich HA, Alitalo K, Vrensen GF and Schlingemann RO: Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey. Invest Ophthalmol Vis Sci. 43:849–857. 2002.PubMed/NCBI
3	Zheng Z, Chen H, Wang H, et al: Improvement of retinal vascular injury in diabetic rats by Statins is associated with the inhibition of mitochondrial reactive oxygen species pathway mediated by peroxisome proliferator-activated receptor coactivator 1. Diabetes. 59:2315–2325. 2010. View Article : Google Scholar : PubMed/NCBI
4	Neufeld G, Cohen T, Gengrinovitch S and Poltorak Z: Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13:9–22. 1999.PubMed/NCBI
5	Duh E and Aiello LP: Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes. 48:1899–1906. 1999. View Article : Google Scholar : PubMed/NCBI
6	Dahrouj M, Alsarraf O, McMillin JC, Liu Y, Crosson CE and Ablonczy Z: Vascular endothelial growth factor modulates the function of the retinal pigment epithelium in vivo. Invest Ophthalmol Vis Sci. 55:2269–2275. 2014. View Article : Google Scholar : PubMed/NCBI
7	Krizova D, Vokrojova M, Liehneova K and Studeny P: Treatment of corneal neovascularization using anti-VEGF Bevacizumab. J Ophthalmol. 2014:1781322014.PubMed/NCBI
8	Sapieha P, Sirinyan M, Hamel D, et al: The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis. Nat Med. 14:1067–1076. 2008. View Article : Google Scholar : PubMed/NCBI
9	He W, Miao FJ, Lin DC, et al: Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature. 429:188–193. 2004. View Article : Google Scholar : PubMed/NCBI
10	Li T, Hu J, Du S, Chen Y, Wang S and Wu Q: ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes. Mol Vis. 20:1109–1121. 2014.PubMed/NCBI
11	Correa PR, Kruglov EA, Thompson M, Leite MF, Dranoff JA and Nathanson MH: Succinate is a paracrine signal for liver damage. J Hepatol. 47:262–269. 2007. View Article : Google Scholar : PubMed/NCBI
12	Aguiar CJ, Andrade VL, Gomes ER, et al: Succinate modulates Ca²⁺ transient and cardiomyocyte viability through PKA-dependent pathway. Cell Calcium. 47:37–46. 2010. View Article : Google Scholar
13	Rubic T, Lametschwandtner G, Jost S, et al: Triggering the succinate receptor GPR91 on dendritic cells enhances immunity. Nat Immunol. 9:1261–1269. 2008. View Article : Google Scholar : PubMed/NCBI
14	Robben JH, Fenton RA, Vargas SL, Schweer H, Peti-Peterdi J, Deen PM and Milligan G: Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells. Kidney Int. 76:1258–1267. 2009. View Article : Google Scholar : PubMed/NCBI
15	Matsumoto M, Suzuma K, Maki T, Kinoshita H, Tsuiki E, Fujikawa A and Kitaoka T: Succinate increases in the vitreous fluid of patients with active proliferative diabetic retinopathy. Am J Ophthalmol. 153:896–902.e1. 2012. View Article : Google Scholar : PubMed/NCBI
16	Hu J, Wu Q, Li T, Chen Y and Wang S: Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91. Exp Eye Res. 109:31–39. 2013. View Article : Google Scholar : PubMed/NCBI
17	Toma I, Kang JJ, Sipos A, et al: Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney. J Clin Invest. 118:2526–2534. 2008.PubMed/NCBI
18	Vargas SL, Toma I, Kang JJ, Meer EJ and Peti-Peterdi J: Activation of the succinate receptor GPR91 in macula densa cells causes renin release. J Am Soc Nephrol. 20:1002–1011. 2009. View Article : Google Scholar : PubMed/NCBI
19	Schulte G and Fredholm BB: Human adenosine A₁, A_2A, A_2B, and A₃ receptors expressed in Chinese hamster ovary cells all mediate the phosphorylation of extracellular-regulated kinase 1/2. Mol Pharmacol. 58:477–482. 2000.PubMed/NCBI
20	Yoon S and Seger R: The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors. 24:21–44. 2006. View Article : Google Scholar : PubMed/NCBI
21	Zhang W and Liu HT: MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12:9–18. 2002. View Article : Google Scholar : PubMed/NCBI
22	Raman M, Chen W and Cobb MH: Differential regulation and properties of MAPKs. Oncogene. 26:3100–3112. 2007. View Article : Google Scholar : PubMed/NCBI
23	Lee JJ, Hsiao CC, Yang IH, et al: High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5. Mol Vis. 18:838–850. 2012.PubMed/NCBI
24	Adachi T, Teramachi M, Yasuda H, Kamiya T and Hara H: Contribution of p38 MAPK, NF-kappaB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability. Arch Biochem Biophys. 520:30–35. 2012. View Article : Google Scholar : PubMed/NCBI
25	Du Y, Tang J, Li G, et al: Effects of p38 MAPK inhibition on early stages of diabetic retinopathy and sensory nerve function. Invest Ophthalmol Vis Sci. 51:2158–2164. 2010. View Article : Google Scholar : PubMed/NCBI
26	Ayalasomayajula SP, Amrite AC and Kompella UB: Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas. Eur J Pharmacol. 498:275–278. 2004. View Article : Google Scholar : PubMed/NCBI
27	Xin X, Majumder M, Girish GV, Mohindra V, Maruyama T and Lala PK: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model. Lab Invest. 92:1115–1128. 2012. View Article : Google Scholar : PubMed/NCBI
28	Zhou LH, Hu Q, Sui H, et al: Tanshinone II-a inhibits angiogenesis through down regulation of COX-2 in human colorectal cancer. Asian Pac J Cancer Prev. 13:4453–4458. 2012. View Article : Google Scholar
29	Amrite AC, Ayalasomayajula SP, Cheruvu NP and Kompella UB: Single periocular injection of celecoxib-PLGA microparticles inhibits diabetes-induced elevations in retinal PGE₂, VEGF, and vascular leakage. Invest Ophthalmol Vis Sci. 47:1149–1160. 2006. View Article : Google Scholar : PubMed/NCBI
30	Tan Y, Ichikawa T, Li J, et al: Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo. Diabetes. 60:625–633. 2011. View Article : Google Scholar : PubMed/NCBI
31	Nakabayashi H and Shimizu K: HA1077, a Rho kinase inhibitor, suppresses glioma-induced angiogenesis by targeting the Rho-ROCK and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signal pathways. Cancer Sci. 102:393–399. 2011. View Article : Google Scholar
32	Dhanasekaran DN and Reddy EP: JNK signaling in apoptosis. Oncogene. 27:6245–6251. 2008. View Article : Google Scholar : PubMed/NCBI
33	Cuadrado A and Nebreda AR: Mechanisms and functions of p38 MAPK signalling. Biochem J. 429:403–417. 2008. View Article : Google Scholar
34	Campo GM, Avenoso A, D’Ascola A, et al: 4-mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1 and in part via p38-MAPK. Curr Med Chem. 20:1162–1172. 2013. View Article : Google Scholar : PubMed/NCBI
35	Michler T, Storr M, Kramer J, et al: Activation of cannabinoid receptor 2 reduces inflammation in acute experimental pancreatitis via intra-acinar activation of p38 and MK2-dependent mechanisms. Am J Physiol Gastrointest Liver Physiol. 304:G181–G192. 2013. View Article : Google Scholar
36	Pollard PJ, Wortham NC and Tomlinson IP: The TCA cycle and tumorigenesis: the examples of fumarate hydratase and succinate dehydrogenase. Ann Med. 35:632–639. 2003. View Article : Google Scholar
37	Buzzi N, Bilbao PS, Boland R and de Boland AR: Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line. Biochim Biophys Acta. 1790:1651–1659. 2009. View Article : Google Scholar : PubMed/NCBI
38	Kern TS: Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy. Exp Diabetes Res. 2007:951032007. View Article : Google Scholar
39	Neroev VV, Zueva MV and Kalamkarov GR: Molecular mechanisms of retinal ischemia. Vestn Oftalmol. 126:59–64. 2010.In Russian. PubMed/NCBI
40	Patel N: Targeting leukostasis for the treatment of early diabetic retinopathy. Cardiovasc. Hematol Disord Drug Targets. 9:222–229. 2009. View Article : Google Scholar
41	Nagoya H, Futagami S, Shimpuku M, et al: Apurinic/apyrimidinic endonuclease-1 is associated with angiogenesis and VEGF production via upregulation of COX-2 expression in esophageal cancer tissues. Am J Physiol Gastrointest Liver Physiol. 306:G183–G190. 2014. View Article : Google Scholar
42	Yanni SE, Barnett JM, Clark ML and Penn JS: The role of PGE₂ receptor EP4 in pathologic ocular angiogenesis. Invest Ophthalmol Vis Sci. 50:5479–5486. 2009. View Article : Google Scholar : PubMed/NCBI
43	Inoue H, Takamori M, Nagata N, Nishikawa T, Oda H, Yamamoto S and Koshihara Y: An investigation of cell proliferation and soluble mediators induced by interleukin 1beta in human synovial fibroblasts: comparative response in osteoarthritis and rheumatoid arthritis. Inflamm Res. 50:65–72. 2001.PubMed/NCBI
44	Höper MM, Voelkel NF, Bates TO, Allard JD, Horan M, Shepherd D and Tuder RM: Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP. Am J Respir Cell Mol Biol. 17:748–756. 1997. View Article : Google Scholar : PubMed/NCBI
45	Cheng T, Cao W, Wen R, Steinberg RH and LaVail MM: Prostaglandin E₂ induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells. Invest Ophthalmol Vis Sci. 39:581–591. 1998.PubMed/NCBI
46	Zhong X, Wang H and Jian X: Insulin augments mechanical strain-induced ERK activation and cyclooxygenase-2 expression in MG63 cells through integrins. Exp Ther Med. 7:295–299. 2014.
47	Chuang YF, Yang HY, Ko TL, Hsu YF, Sheu JR, Ou G and Hsu MJ: Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain micro-vascular endothelial cells. Biochem Pharmacol. 88:372–383. 2014. View Article : Google Scholar : PubMed/NCBI
48	Amrite A, Pugazhenthi V, Cheruvu N and Kompella U: Delivery of celecoxib for treating diseases of the eye: influence of pigment and diabetes. Expert Opin Drug Deliv. 7:631–645. 2010. View Article : Google Scholar : PubMed/NCBI
49	Ferrara N and Davis-Smyth T: The biology of vascular endothelial growth factor. Endocr Rev. 18:4–25. 1997. View Article : Google Scholar : PubMed/NCBI
50	Li F, Xu H, Zeng Y and Yin ZQ: Overexpression offibulin-5 in retinal pigment epithelial cells inhibits cell proliferation and migration and downregulates VEGF, CXCR4, and TGFB1 expression in cocultured choroidal endothelial cells. Cur Eye Res. 37:540–548. 2012. View Article : Google Scholar