Phenotypic change regulates monocyte chemoattractant protein-1 (MCP-1) gene expression in human retinal pigment epithelial cells.
We investigated the expression profile of monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (RPE) cells under different culture conditions and evaluated the molecular mechanism responsible for MCP-1 gene expression in RPE cells. After cellular confluence, total RNA was extracted and used for RT-PCR. Medium conditioned by RPE was used for ELISA and Western blotting. The result showed that RPE cells cultured on plastic expressed MCP-1 constitutively in the absence of any stimuli. On the other hand, growing human RPE on laminin-coated flasks instead of plastic reduced the production of MCP-1. In the RPE cells cultured on plastic, IkappaB was degraded and A20 protein increased concomitantly. MCP-1 upregulation in RPE cells on plastic was attenuated by the addition of MG-132, a proteasome inhibitor. Also, the addition of pyrolidine dithiocarbonate (PDTC) and hypoxic conditions (0.5% O2) decreased MCP-1 production in these cells. These findings suggested that the expression profile of MCP-1 is regulated by phenotypic alterations of the RPE cells. And the increased MCP-1 expression in RPE cells cultured on plastic is caused via spontaneous activation of NFkappaB induced by susceptibility to oxidative stress.
Uetama
Tomoko
T
Department of Ophthalmology and Visual Science, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.
Ohno-Matsui
Kyoko
K
Nakahama
Ken-Ichi
K
Morita
Ikuo
I
Mochizuki
Manabu
M
D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics*; D017077:Culture Media, Conditioned / Q000737:chemistry; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007797:Laminin; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D016207:Cytokines; D020672:I-kappa B Proteins; D007797:Laminin; D016328:NF-kappa B; D012333:RNA, Messenger