Mitomycin C upregulates IL-8 and MCP-1 chemokine expression via mitogen-activated protein kinases in corneal fibroblasts.
OBJECTIVE
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.
Chou
San-Fang
SF
Department of Ophthalmology, Far Eastern Memorial Hospital, Ban-Chiao, Taipei, Taiwan.
Chang
Shu-Wen
SW
Chuang
Jia-Ling
JL
D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016685:Mitomycin / Q000494:pharmacology*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013997:Time Factors; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000477:Alkylating Agents; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D016209:Interleukin-8; D016685:Mitomycin; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4