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T Uetama; Dedifferentiation of Retinal Pigment Epithelial Cells Induces Monocyte Chemoattractant Protein-1 (mcp-1) Gene Expression Through Nf-kb Transcription Factor Activation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):690.
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Purpose: Differentiation of retinal pigment epithelial (RPE) cells characterizes a marked pathologic feature of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Monocyte chemoattractant protein-1 (MCP-1), a key molecule in macrophage recruitment, has also been known to play important roles in the expression of angiogenic factors and in the activation of matrix metalloproteinases. Therefore, this study was undertaken to determine the regulation of MCP-1 expression in dedifferentiated RPE cells. Methods: Human RPE cells cultured on plastic flasks were used as dedifferentiated cells. RPE cells seeded on laminin-coated flasks were used as differentiated counterparts. Some flasks were treated with TNF-α. RNA was extracted and used for reverse transcriptase-polymerase chain reaction using MCP-1 specific primers. The conditioned media were used to measure MCP-1 protein by enzyme-linked immunosorbent assay. Nuclear factor (NF)-kB activation was examined by Western blot analysis using antibody against inhibitor of NF-kB (I-kB) and A20, and by immunofluorescent detection of NF-kB nuclear translocation. Results: TNF-α induced MCP-1 secretion both in dedifferentiated and differentiated RPE cells, while,the dedifferentiated RPE cells expressed MCP-1 constitutively in the absence of stimuli. In the dedifferentiated RPE cells, I-kB was degrtaded and concomitantly A20 protein increased, and NF-kB translocated to nuclei. MCP-1 upregulation in dedifferentiated RPE cells was abrogated adding a proteasome inhibitor, MG132. Conclusion: Dedifferentiation of RPE cells upregulated expression via spontaneous activation of NF-kB, which presumably resulting in augmentation of inflammation and CNV. The inhibition of MCP-1 may be a valid therapeutic option against CNV associated with AMD.
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