Retinal neuronal MCP-1 induced by AGEs stimulates TNF-α expression in rat microglia via p38, ERK, and NF-κB pathways.
OBJECTIVE
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.
Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.
Ning
N
Dong
Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.
Libin
L
Chang
Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.
Bingsong
B
Wang
Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.
Liqun
L
Chu
D000818:Animals; D018932:Chemokine CCL2 / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D054390:Receptors, CCR2 / Q000378:metabolism; D055351:Retinal Neurons / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
C515336:Ccr2 protein, rat; D018932:Chemokine CCL2; D017127:Glycation End Products, Advanced; D016328:NF-kappa B; D034741:RNA, Small Interfering; D054390:Receptors, CCR2; D014409:Tumor Necrosis Factor-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases