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

Hu J, Li T, Du S, Chen Y, Wang S, Xiong F, Wu Q - Int. J. Mol. Med. (2015)

Bottom Line: The knockdown of GPR91 inhibited ERK1/2 and JNK activity, but did not inhibit the activation of the p38 MAPK pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

G-protein-coupled receptor 91 (GPR91) modulates the succinate-induced activation of the ERK1/2(JNK)/COX-2/PGE2 pathway in RGC-5 cells. (A) Western blot analysis of COX-2 protein expression in RGC-5 cells treated with succinate for 24 h. The cells were transduced with LV.shGPR91 or pre-treated with U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or NS-398 (COX-2 inhibitor). (B) Quantitative analysis of the band density of COX-2/β-actin. (C) Changes in COX-2 mRNA levels (determined by RT-qPCR) in RGC-5 cells from each group. (D) Changes in PGE2 release (determined by ELISA) by RGC-5 cells from each group. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. succinate-treated cells.
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f4-ijmm-36-01-0130: G-protein-coupled receptor 91 (GPR91) modulates the succinate-induced activation of the ERK1/2(JNK)/COX-2/PGE2 pathway in RGC-5 cells. (A) Western blot analysis of COX-2 protein expression in RGC-5 cells treated with succinate for 24 h. The cells were transduced with LV.shGPR91 or pre-treated with U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or NS-398 (COX-2 inhibitor). (B) Quantitative analysis of the band density of COX-2/β-actin. (C) Changes in COX-2 mRNA levels (determined by RT-qPCR) in RGC-5 cells from each group. (D) Changes in PGE2 release (determined by ELISA) by RGC-5 cells from each group. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. succinate-treated cells.

Mentions: To examine whether COX-2 expression remains elevated following incubation with succinate, we exposed the RGC-5 cells to 50 µM succinate for 24 h. COX-2 expression increased significantly (P<0.01; Fig. 4A and C). PGE2 levels were also measured as the production of PGE2 denotes COX-2 activity; the PGE2 levels were also markedly increased in the succinate-stimulated RGC-5 cells (P<0.01; Fig. 4D). However, the upregulation of COX-2 expression and activity were markedly decreased following the knockdown of GPR91 (P<0.01; Fig. 4A and D). Furthermore, pre-treatment with 10 µM U0126 (ERK1/2 inhibitor), 10 µM SP600125 (JNK inhibitor) or 50 µM NS-398 (COX-2 inhibitor) significantly blocked the upregulation of COX-2 and PGE2 expression in the RGC-5 cells (P<0.01). These findings indicate that the ERK1/2 and JNK pathway is upstream of the COX-2 and PGE2 pathway.


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

Hu J, Li T, Du S, Chen Y, Wang S, Xiong F, Wu Q - Int. J. Mol. Med. (2015)

G-protein-coupled receptor 91 (GPR91) modulates the succinate-induced activation of the ERK1/2(JNK)/COX-2/PGE2 pathway in RGC-5 cells. (A) Western blot analysis of COX-2 protein expression in RGC-5 cells treated with succinate for 24 h. The cells were transduced with LV.shGPR91 or pre-treated with U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or NS-398 (COX-2 inhibitor). (B) Quantitative analysis of the band density of COX-2/β-actin. (C) Changes in COX-2 mRNA levels (determined by RT-qPCR) in RGC-5 cells from each group. (D) Changes in PGE2 release (determined by ELISA) by RGC-5 cells from each group. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. succinate-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4-ijmm-36-01-0130: G-protein-coupled receptor 91 (GPR91) modulates the succinate-induced activation of the ERK1/2(JNK)/COX-2/PGE2 pathway in RGC-5 cells. (A) Western blot analysis of COX-2 protein expression in RGC-5 cells treated with succinate for 24 h. The cells were transduced with LV.shGPR91 or pre-treated with U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or NS-398 (COX-2 inhibitor). (B) Quantitative analysis of the band density of COX-2/β-actin. (C) Changes in COX-2 mRNA levels (determined by RT-qPCR) in RGC-5 cells from each group. (D) Changes in PGE2 release (determined by ELISA) by RGC-5 cells from each group. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. succinate-treated cells.
Mentions: To examine whether COX-2 expression remains elevated following incubation with succinate, we exposed the RGC-5 cells to 50 µM succinate for 24 h. COX-2 expression increased significantly (P<0.01; Fig. 4A and C). PGE2 levels were also measured as the production of PGE2 denotes COX-2 activity; the PGE2 levels were also markedly increased in the succinate-stimulated RGC-5 cells (P<0.01; Fig. 4D). However, the upregulation of COX-2 expression and activity were markedly decreased following the knockdown of GPR91 (P<0.01; Fig. 4A and D). Furthermore, pre-treatment with 10 µM U0126 (ERK1/2 inhibitor), 10 µM SP600125 (JNK inhibitor) or 50 µM NS-398 (COX-2 inhibitor) significantly blocked the upregulation of COX-2 and PGE2 expression in the RGC-5 cells (P<0.01). These findings indicate that the ERK1/2 and JNK pathway is upstream of the COX-2 and PGE2 pathway.

Bottom Line: The knockdown of GPR91 inhibited ERK1/2 and JNK activity, but did not inhibit the activation of the p38 MAPK pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus