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L. Blanchon, C. Prat, F. Chiambaretta, B. Yue, V. Sapin; Myocilin Transcription is Under the Control of Vitamin A and Its Active Derivatives. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6126.
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A better understanding of the myocilin regulation is essential due to important role of this protein in intra-ocular pressure (IOP) control. Indeed, a deregulation of IOP could lead to primary open angle glaucoma (POAG). We established by an in-silico study of the myocilin gene promoter that the regulation of its transcription is probably not only under the influence of glucocorticoids but also dependant on the retinoid nuclear receptors RARs and RXRs. Our first hypothesis was confirmed by preliminary co-transfection experiments combining promoter constructs and expression plasmids for both RAR and RXR. To complete these first results, cell treatments were conducted to confirm influence of retinoids on endogenous myocilin expression at the mRNA and protein levels. We also precisely determined the retinoids nuclear receptors binding sites by in-vitro transfection in TM5 cells.
TM5 cells (from Dr. A.R. Shepard, Alcon Res®, USA) were co-transfected with various myocilin promoter reporter gene constructs and by expression plasmids for nuclear receptors. The cells were then treated with ligands for nuclear receptors RAR and RXR. ChIP assays were done to demonstrate fixation of the heterodimer RAR/RXR on the promoter. qPCR, Immunocytochemistry and western-blot assays were used to confirm the retinoids influence on the endogenous myocilin level.
Transfection studies localised a promoter zone containing functional RAR/RXR binding sites. Furthermore, site directed mutagenesis permited their identification. Both previous results were confirmed by ChIP assays. The regulation of the endogenous myocilin gene transcription by retinoids was demonstrated by qPCR, immunocytochemistry and western-blot after (48 hours) retinoids treatments.
RAR and RXR are important for the retinoids transcriptional regulation of the myocilin gene. Since the discovery of the glucocorticoïd implication, these results suggest a new research direction to develop medical strategies in the prevention of the myocilin linked POAG pathology.
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