PURPOSE: To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. METHODS: Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. RESULTS: The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. CONCLUSIONS: MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.
PURPOSE: To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. METHODS: Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. RESULTS: The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. CONCLUSIONS:MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.
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