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J. Xu, D. Zhu, S. He, C. Spee, S. Ryan, D. Hinton; Bmp4 Expression Is Down-Regulated by Tumor Necrosis Factor-Alpha in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):475.
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© ARVO (1962-2015); The Authors (2016-present)
Tumor necrosis factor (TNF)-alpha has been shown to regulate expression of many growth factors secreted by retinal pigment epithelial (RPE) cells. We have previously suggested that Bone Morphogenetic Protein-4 (BMP4) may play an important role in the pathogenesis of age related macular degeneration. The goal of the current study was to determine whether TNF-alpha regulates BMP4 expression in RPE cells in vitro, and establish which signaling pathway is involved.
Confluent ARPE-19 cells or fetal human RPE cells were treated with TNF-alpha in serum free growth medium (0,1,2,5,10ng/ml TNF-alpha for 24h or 10ng/ml TNF-alpha for 0,3,6,12, 24 h). Real time RT-PCR was used to measure the mRNA level of BMP4 using GAPDH gene as an internal control. Western blot was used to determine NF-kB activation by TNF-alpha in RPE cells. Different lengths of human BMP4 gene promoter region A were cloned into a PGL3-enhancer vector that drives expression of a luciferase reporter and the plasmids were transfected into ARPE-19 cells. The transcriptional activity of BMP4 promoter fragments in response to TNF-alpha alone or together with NF-kB pathway inhibitors was determined by the luciferase activity normalized to the control (beta-galactosidase activity).
The relative BMP4 mRNA level was reduced by 80% with 10ng/ml TNF-alpha treatment for 24h in confluent ARPE cells, compared with no treatment control. The down regulation of BMP4 expression by TNF-alpha was early: there was 60% decrease in BMP4 mRNA level as soon as 3h after TNF-alpha treatment (10ng/ml). Similar results of dose-and time-dependent reduction of BMP4 expression by TNF-alpha were obtained in early passage human RPE cells. TNF-alpha activates the NF-kB pathway in RPE cells. Luciferase reporter constructs containing BMP4 promoter were active in transfected ARPE-19 cells. TNF-alpha treatment decreased the BMP4 promoter activity in transfected ARPE-19 cells and co-transfection of dominant-negative IKB reversed the down-regulation of BMP4 by TNF-alpha.
Treatment of ARPE-19 cells and early passage human RPE cells with TNF-alpha resulted in a rapid and prominent decrease in BMP4 expression. The intracellular pathway mediating down-regulation of BMP4 gene expression by TNF-alpha in RPE cells involves NF-kB activation. Promoter analysis will further our understanding of the specific BMP4 promoter region regulated by TNF-alpha and NF-kB signaling in RPE cells.
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