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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 succinate-induced ERK1/2 and JNK signaling in RGC-5 cells. (A) Changes in ERK1/2, JNK and p38 MAPK phosphorylation (determined by western blot analysis) in RGC-5 cells transduced with LV.shScrambled or LV. shGPR91. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Effect of the JNK inhibitor, SP600125, on ERK1/2 phosphorylation in RGC-5 cells treated with succinate. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Effect of the ERK1/2 inhibitor, U0126, on JNK phosphorylation in RGC-5 cells treated with succinate. (F) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. LV.shScrambled-transduced cells.
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f3-ijmm-36-01-0130: G-protein-coupled receptor 91 (GPR91) modulates succinate-induced ERK1/2 and JNK signaling in RGC-5 cells. (A) Changes in ERK1/2, JNK and p38 MAPK phosphorylation (determined by western blot analysis) in RGC-5 cells transduced with LV.shScrambled or LV. shGPR91. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Effect of the JNK inhibitor, SP600125, on ERK1/2 phosphorylation in RGC-5 cells treated with succinate. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Effect of the ERK1/2 inhibitor, U0126, on JNK phosphorylation in RGC-5 cells treated with succinate. (F) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. LV.shScrambled-transduced cells.

Mentions: In order to assess whether succinate modulates the activation of the MAPK signaling pathway through its receptor, GPR91, we incubated the RGC-5 cells with succinate (50 µM to activate ERK1/2, 5 µM for JNK and 20 µM for p38 MAPK) for 10 min. The increase in the phosphorylation levels of ERK1/2 and JNK was significantly blocked by GPR91 shRNA (P<0.01); however, p38 MAPK phosphorylation was not affected (P>0.05). These results indicate that GPR91 mediates the activation of ERK1/2 and JNK signaling in RGC-5 cells (Fig. 3A and B).


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 succinate-induced ERK1/2 and JNK signaling in RGC-5 cells. (A) Changes in ERK1/2, JNK and p38 MAPK phosphorylation (determined by western blot analysis) in RGC-5 cells transduced with LV.shScrambled or LV. shGPR91. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Effect of the JNK inhibitor, SP600125, on ERK1/2 phosphorylation in RGC-5 cells treated with succinate. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Effect of the ERK1/2 inhibitor, U0126, on JNK phosphorylation in RGC-5 cells treated with succinate. (F) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. LV.shScrambled-transduced cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494573&req=5

f3-ijmm-36-01-0130: G-protein-coupled receptor 91 (GPR91) modulates succinate-induced ERK1/2 and JNK signaling in RGC-5 cells. (A) Changes in ERK1/2, JNK and p38 MAPK phosphorylation (determined by western blot analysis) in RGC-5 cells transduced with LV.shScrambled or LV. shGPR91. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Effect of the JNK inhibitor, SP600125, on ERK1/2 phosphorylation in RGC-5 cells treated with succinate. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Effect of the ERK1/2 inhibitor, U0126, on JNK phosphorylation in RGC-5 cells treated with succinate. (F) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). **P<0.01 vs. the untreated control. ##P<0.01 vs. LV.shScrambled-transduced cells.
Mentions: In order to assess whether succinate modulates the activation of the MAPK signaling pathway through its receptor, GPR91, we incubated the RGC-5 cells with succinate (50 µM to activate ERK1/2, 5 µM for JNK and 20 µM for p38 MAPK) for 10 min. The increase in the phosphorylation levels of ERK1/2 and JNK was significantly blocked by GPR91 shRNA (P<0.01); however, p38 MAPK phosphorylation was not affected (P>0.05). These results indicate that GPR91 mediates the activation of ERK1/2 and JNK signaling in RGC-5 cells (Fig. 3A and B).

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