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Retinal neuronal MCP-1 induced by AGEs stimulates TNF-α expression in rat microglia via p38, ERK, and NF-κB pathways.

Dong N, Chang L, Wang B, Chu L - Mol. Vis. (2014)

Bottom Line: The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns.Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT

Purpose: Retinal microglia can be activated by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) and play a pivotal role in early retinal degeneration. The current study investigates the pathways via which retinal neuronal MCP-1 stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia.

Methods: Primary rat retinal neurons and microglia were separated and cocultured in a Transwell apparatus. The levels of TNF-α mRNA and soluble TNF-α produced by the microglia in response to advanced glycation end product (AGE)-induced retinal neuronal MCP-1 were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA). The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns. Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).

Results: Stimulation with AGEs significantly increased the expression of TNF-α mRNA and soluble TNF-α in the microglial cells. Retinal neurons that had been pretreated with AGEs and an MCP-1 antibody or microglia that were CCR2 knockdowns displayed greatly reduced TNF-α secretion. Using signaling pathway-specific inhibitors, we showed that blocking the p38, extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways significantly reduced the expression of TNF-α by retinal neuronal MCP-1-stimulated microglia.

Conclusions: This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

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Related in: MedlinePlus

Retinal neuronal monocyte chemoattractant protein-1 (MCP-1) induced by advanced glycation end products (AGEs) stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia via the p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) pathways, but not the c-Jun N-terminal kinase (JNK) pathway. A: Real-time PCR was used to measure MCP-1 mRNA expression. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble MCP-1 concentration. C, D, E: To exclude the effects of MCP-1 and/or TNF-α from the microglia exposed to AGEs in the Transwell apparatus, the primary cultured retinal neurons were pretreated with AGEs (750 μg/ml) in the culture medium for 24 h, then washed with PBS three times and removed AGEs, followed by coculture with the previously described isolated microglia in the Transwell apparatus for another 24 h. C: Real-time PCR was used to measure TNF-α mRNA expression. D: ELISA was used to measure the soluble TNF-α concentration (*p<0.05). E: The levels of phospho-p38, phospho-extracellular signal-regulated kinase (ERK), and the p50 and p65 subunits of NF-κB from the microglial cells increased. However, the levels of phospho-p38, phospho-ERK, and the p50 and p65 subunits of NF-κB from the microglial cells decreased accompanied by CC-chemokine receptor 2 (CCR2) knockdown to retinal microglia in the Transwell culture system with western blotting. Phospho-c-Jun N-terminal kinase (JNK) levels from the microglial cells remained unchanged over the entire experimental period in the retinal neuron–microglia Transwell culture system, and CCR2 knockdown did not lead to downregulation of the phospho-JNK levels.
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f7: Retinal neuronal monocyte chemoattractant protein-1 (MCP-1) induced by advanced glycation end products (AGEs) stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia via the p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) pathways, but not the c-Jun N-terminal kinase (JNK) pathway. A: Real-time PCR was used to measure MCP-1 mRNA expression. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble MCP-1 concentration. C, D, E: To exclude the effects of MCP-1 and/or TNF-α from the microglia exposed to AGEs in the Transwell apparatus, the primary cultured retinal neurons were pretreated with AGEs (750 μg/ml) in the culture medium for 24 h, then washed with PBS three times and removed AGEs, followed by coculture with the previously described isolated microglia in the Transwell apparatus for another 24 h. C: Real-time PCR was used to measure TNF-α mRNA expression. D: ELISA was used to measure the soluble TNF-α concentration (*p<0.05). E: The levels of phospho-p38, phospho-extracellular signal-regulated kinase (ERK), and the p50 and p65 subunits of NF-κB from the microglial cells increased. However, the levels of phospho-p38, phospho-ERK, and the p50 and p65 subunits of NF-κB from the microglial cells decreased accompanied by CC-chemokine receptor 2 (CCR2) knockdown to retinal microglia in the Transwell culture system with western blotting. Phospho-c-Jun N-terminal kinase (JNK) levels from the microglial cells remained unchanged over the entire experimental period in the retinal neuron–microglia Transwell culture system, and CCR2 knockdown did not lead to downregulation of the phospho-JNK levels.

Mentions: As shown in Figure 7A, retinal neuronal MCP-1 mRNA was set to 100%, when the primary cultured retinal neurons were cultured with culture medium alone for 48 h and used as a control. When cultured with AGEs for 24 h or 48 h, the retinal neurons had a markedly increased about 5.8 times or 7.6 times expression of MCP-1 mRNA relative to the control cells (Figure 7A), and had an increased effect on releasing soluble MCP-1 (Figure 7B). The expression of MCP-1 mRNA and soluble MCP-1 in the pretreated AGE 24 h and removed AGE groups were compared to the control cells.


Retinal neuronal MCP-1 induced by AGEs stimulates TNF-α expression in rat microglia via p38, ERK, and NF-κB pathways.

Dong N, Chang L, Wang B, Chu L - Mol. Vis. (2014)

Retinal neuronal monocyte chemoattractant protein-1 (MCP-1) induced by advanced glycation end products (AGEs) stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia via the p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) pathways, but not the c-Jun N-terminal kinase (JNK) pathway. A: Real-time PCR was used to measure MCP-1 mRNA expression. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble MCP-1 concentration. C, D, E: To exclude the effects of MCP-1 and/or TNF-α from the microglia exposed to AGEs in the Transwell apparatus, the primary cultured retinal neurons were pretreated with AGEs (750 μg/ml) in the culture medium for 24 h, then washed with PBS three times and removed AGEs, followed by coculture with the previously described isolated microglia in the Transwell apparatus for another 24 h. C: Real-time PCR was used to measure TNF-α mRNA expression. D: ELISA was used to measure the soluble TNF-α concentration (*p<0.05). E: The levels of phospho-p38, phospho-extracellular signal-regulated kinase (ERK), and the p50 and p65 subunits of NF-κB from the microglial cells increased. However, the levels of phospho-p38, phospho-ERK, and the p50 and p65 subunits of NF-κB from the microglial cells decreased accompanied by CC-chemokine receptor 2 (CCR2) knockdown to retinal microglia in the Transwell culture system with western blotting. Phospho-c-Jun N-terminal kinase (JNK) levels from the microglial cells remained unchanged over the entire experimental period in the retinal neuron–microglia Transwell culture system, and CCR2 knockdown did not lead to downregulation of the phospho-JNK levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016805&req=5

f7: Retinal neuronal monocyte chemoattractant protein-1 (MCP-1) induced by advanced glycation end products (AGEs) stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia via the p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) pathways, but not the c-Jun N-terminal kinase (JNK) pathway. A: Real-time PCR was used to measure MCP-1 mRNA expression. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble MCP-1 concentration. C, D, E: To exclude the effects of MCP-1 and/or TNF-α from the microglia exposed to AGEs in the Transwell apparatus, the primary cultured retinal neurons were pretreated with AGEs (750 μg/ml) in the culture medium for 24 h, then washed with PBS three times and removed AGEs, followed by coculture with the previously described isolated microglia in the Transwell apparatus for another 24 h. C: Real-time PCR was used to measure TNF-α mRNA expression. D: ELISA was used to measure the soluble TNF-α concentration (*p<0.05). E: The levels of phospho-p38, phospho-extracellular signal-regulated kinase (ERK), and the p50 and p65 subunits of NF-κB from the microglial cells increased. However, the levels of phospho-p38, phospho-ERK, and the p50 and p65 subunits of NF-κB from the microglial cells decreased accompanied by CC-chemokine receptor 2 (CCR2) knockdown to retinal microglia in the Transwell culture system with western blotting. Phospho-c-Jun N-terminal kinase (JNK) levels from the microglial cells remained unchanged over the entire experimental period in the retinal neuron–microglia Transwell culture system, and CCR2 knockdown did not lead to downregulation of the phospho-JNK levels.
Mentions: As shown in Figure 7A, retinal neuronal MCP-1 mRNA was set to 100%, when the primary cultured retinal neurons were cultured with culture medium alone for 48 h and used as a control. When cultured with AGEs for 24 h or 48 h, the retinal neurons had a markedly increased about 5.8 times or 7.6 times expression of MCP-1 mRNA relative to the control cells (Figure 7A), and had an increased effect on releasing soluble MCP-1 (Figure 7B). The expression of MCP-1 mRNA and soluble MCP-1 in the pretreated AGE 24 h and removed AGE groups were compared to the control cells.

Bottom Line: The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns.Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT

Purpose: Retinal microglia can be activated by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) and play a pivotal role in early retinal degeneration. The current study investigates the pathways via which retinal neuronal MCP-1 stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia.

Methods: Primary rat retinal neurons and microglia were separated and cocultured in a Transwell apparatus. The levels of TNF-α mRNA and soluble TNF-α produced by the microglia in response to advanced glycation end product (AGE)-induced retinal neuronal MCP-1 were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA). The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns. Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).

Results: Stimulation with AGEs significantly increased the expression of TNF-α mRNA and soluble TNF-α in the microglial cells. Retinal neurons that had been pretreated with AGEs and an MCP-1 antibody or microglia that were CCR2 knockdowns displayed greatly reduced TNF-α secretion. Using signaling pathway-specific inhibitors, we showed that blocking the p38, extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways significantly reduced the expression of TNF-α by retinal neuronal MCP-1-stimulated microglia.

Conclusions: This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

Show MeSH
Related in: MedlinePlus