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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

Succinate-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in RGC-5 cells. (A) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells incubated with various concentrations of succinate for 10 min. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells treated with 10 µM of succinate for different periods of time. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Immunofluorescence images showing ERK1/2, JNK and p38 MAPK in the cytoplasm of RGC-5 cells treated with 10 µM succinate for 10 min. **P<0.01 vs. the untreated control. Scale bar, 50 µm.
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f2-ijmm-36-01-0130: Succinate-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in RGC-5 cells. (A) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells incubated with various concentrations of succinate for 10 min. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells treated with 10 µM of succinate for different periods of time. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Immunofluorescence images showing ERK1/2, JNK and p38 MAPK in the cytoplasm of RGC-5 cells treated with 10 µM succinate for 10 min. **P<0.01 vs. the untreated control. Scale bar, 50 µm.

Mentions: Next, we attempted to clarify the signaling mechanisms of action of GPR91 in the succinate-treated RGC-5 cells. As shown in Fig. 2A–D, the results revealed that treatment with succinate activated ERK1/2, JNK and p38 MAPK signaling. The cells incubated with various concentrations of succinate for 10 min exhibited a dose-dependent increase in MAPK signaling, and a time-dependent trend was also observed when the cells were incubated with 10 µM succinate. Succinate induced a >2-fold increase in the p-ERK1/2, p-JNK and p-p38 MAPK levels in the RGC-5 cells (Fig. 2A–D). The results from immunofluorescence staining indicated that p-ERK1/2, p-JNK and p-p38 became notably more visible in the cytoplasm following incubation with 10 µM succinate for 10 min (Fig. 2E).


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)

Succinate-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in RGC-5 cells. (A) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells incubated with various concentrations of succinate for 10 min. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells treated with 10 µM of succinate for different periods of time. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Immunofluorescence images showing ERK1/2, JNK and p38 MAPK in the cytoplasm of RGC-5 cells treated with 10 µM succinate for 10 min. **P<0.01 vs. the untreated control. Scale bar, 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijmm-36-01-0130: Succinate-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in RGC-5 cells. (A) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells incubated with various concentrations of succinate for 10 min. (B) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (C) Western blot analysis of ERK1/2, JNK and p38 MAPK phosphorylation in RGC-5 cells treated with 10 µM of succinate for different periods of time. (D) Quantitative analysis of the band density. Each column represents the mean ± SD (n=3). (E) Immunofluorescence images showing ERK1/2, JNK and p38 MAPK in the cytoplasm of RGC-5 cells treated with 10 µM succinate for 10 min. **P<0.01 vs. the untreated control. Scale bar, 50 µm.
Mentions: Next, we attempted to clarify the signaling mechanisms of action of GPR91 in the succinate-treated RGC-5 cells. As shown in Fig. 2A–D, the results revealed that treatment with succinate activated ERK1/2, JNK and p38 MAPK signaling. The cells incubated with various concentrations of succinate for 10 min exhibited a dose-dependent increase in MAPK signaling, and a time-dependent trend was also observed when the cells were incubated with 10 µM succinate. Succinate induced a >2-fold increase in the p-ERK1/2, p-JNK and p-p38 MAPK levels in the RGC-5 cells (Fig. 2A–D). The results from immunofluorescence staining indicated that p-ERK1/2, p-JNK and p-p38 became notably more visible in the cytoplasm following incubation with 10 µM succinate for 10 min (Fig. 2E).

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