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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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Effects of CC-chemokine receptor 2 (CCR2) siRNA on CCR2 expression in transfected cells. Primary cultured retinal microglia were treated with specific siRNA to knock down the expression of CCR2. Representative CCR2 knockdown was identified with western blot analysis with maximum downregulation of approximately 75%. Results are statistically significant (**p<0.01 Student t test).
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f1: Effects of CC-chemokine receptor 2 (CCR2) siRNA on CCR2 expression in transfected cells. Primary cultured retinal microglia were treated with specific siRNA to knock down the expression of CCR2. Representative CCR2 knockdown was identified with western blot analysis with maximum downregulation of approximately 75%. Results are statistically significant (**p<0.01 Student t test).

Mentions: To determine the effects of CCR2 siRNA in the primary retinal microglia on CCR2 expression, western blot analysis was used to measure the amount of CCR2 protein. We downregulated CCR2, with a maximum knockdown of approximately 75% at 24 h (Figure 1).


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)

Effects of CC-chemokine receptor 2 (CCR2) siRNA on CCR2 expression in transfected cells. Primary cultured retinal microglia were treated with specific siRNA to knock down the expression of CCR2. Representative CCR2 knockdown was identified with western blot analysis with maximum downregulation of approximately 75%. Results are statistically significant (**p<0.01 Student t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016805&req=5

f1: Effects of CC-chemokine receptor 2 (CCR2) siRNA on CCR2 expression in transfected cells. Primary cultured retinal microglia were treated with specific siRNA to knock down the expression of CCR2. Representative CCR2 knockdown was identified with western blot analysis with maximum downregulation of approximately 75%. Results are statistically significant (**p<0.01 Student t test).
Mentions: To determine the effects of CCR2 siRNA in the primary retinal microglia on CCR2 expression, western blot analysis was used to measure the amount of CCR2 protein. We downregulated CCR2, with a maximum knockdown of approximately 75% at 24 h (Figure 1).

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