Abstract
Purpose::
To investigate the expression of chemokines and their signaling pathways following application of mitomycin C (MMC) to corneal fibroblasts.
Methods::
Primary porcine and human corneal fibroblasts from passages 3-6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2mg/mL for 1, 2, 5, or 10 minutes. The relative expressions of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific anti-phosphokinase 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 expressions of IL-8 and MCP-1 were upregulated after MMC treatment in both treatment time- and concentration-dependent manners. Furthermore, the upregulated expressions 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 expressions were inhibited by both a p38-specific inhibitor (SB203580) and an ERK-specific 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 MAPKs in corneal fibroblasts.
Keywords: cornea: stroma and keratocytes • gene/expression • wound healing