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J. Xu, C. Spee, S. Ryan, D. Hinton; Signaling Pathways Involved in Down-Regulation of Bmp4 by Tumor Necrosis Factor-Alpha in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1852.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous study suggested that Bone Morphogenetic Protein-4 (BMP4) may play an important role in the pathogenesis of age related macular degeneration. We showed dose- and time-dependent reduction of BMP4 expression by TNF-alpha in retinal pigment epithelial (RPE) cells. The goal of present study was to determine the signaling pathways involved in the down-regulation of BMP4 expression by TNF-alpha in RPE cells.
Confluent ARPE-19 cells were treated with cell signaling inhibitors (PD98059, SP600125, and SB203580) for 1h before TNF-alpha application. Real time RT-PCR was used to measure the mRNA level of BMP4. The effect of TNF-alpha on kinase phosphorylation was determined by western blot analysis. The three mRNA variants of BMP4 were analyzed by reverse-transcription PCR to determine the specific mRNA variant(s) transcribed in RPE cells. About 3 kb human BMP4 gene promoter region A was cloned into a luciferase reporter construct and the plasmid was transfected into ARPE-19 cells. The transcriptional activity of BMP4 promoter in response to TNF-alpha alone or together with specific pathway inhibitors was determined by evaluating the luciferase activity.
Western blot analyses showed that ERK, JNK and p38 pathways were activated in ARPE-19 cells with TNF-alpha treatment. When specific inhibitors were used, the ERK, JNK and p38 kinase phosphorylation induced by TNF-alpha were inhibited. However, only inhibition of JNK pathway could block the reduction of BMP4 mRNA expression by TNF-alpha. The effect of blocking ERK pathway was minor and inhibition of p38 pathway had negligible effect. Reverse-transcription PCR showed that BMP4 mRNA variant 1 and 2 were utilized by RPE cells and both variants were regulated under promoter A. Luciferase reporter construct containing BMP4 promoter A was active and TNF-alpha treatment decreased the BMP4 promoter activity in transfected ARPE-19 cells. Pretreatment with individual inhibitors in transfected ARPE-19 cells showed that only inhibition of JNK pathway reversed the down-regulation of BMP4 by TNF-alpha.
The regulation of BMP4 is mostly studied during developmental stage and little is known about its regulation in the adult or under disease conditions. TNF-alpha has been implicated as a key mediator in wet AMD. We have identified a novel pathway for the down-regulation of BMP4 gene expression by TNF-alpha in RPE cells.
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