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L. Blanchon, F. Chiambaretta, C. Prat, B. Yue, V. Sapin; Regulation of Myocilin by Nuclear Receptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3635.
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© ARVO (1962-2015); The Authors (2016-present)
The excessive presence of the myocilin and/or mutated forms of this protein in the extracellular compartment leads to an increase of the Intra-Ocular Pression, a principal cause for the appearance of the glaucoma called Primary Open Angle Glaucoma. To better understand the exact role and importance of this protein in this pathology, the precise regulation of the myocilin gene expression had to be clarified. To achieve that, the first part of our work was to conduct an in silico myocilin promoter study focused on nuclear receptors responsive elements. The second part of our work was to confirm in primary trabecular meshwork cells and TM5 cells (generous gift from Dr. Allan R. Shepard, Alcon Research, USA) the presence of nuclear receptors. Then, transfection experiments were conducted in trabecular meshwork TM5 cells in order to find functional nuclear receptors binding sites in the myocilin promoter.
In silico promoter study was done with Genomatix© software. Total RNAs and proteins were extracted from primary TM cells and TM5 cells, and subjected to RT-PCR and Western-blot experiments. TM5 cells were co-transfected with various myocilin promoter constructs and by expression plasmids for nuclear receptors.
In silico study of the myocilin promoter has permitted the identification of potential binding site for nuclear receptors like RARs, RXRs, PPARs and LXRs. Western-blot experiments confirmed the presence of RAR, RXR and PPARγ in the primary TM cell line. Furthermore, we demonstrated the presence of RAR, β, γ; RXR, β; LXR, β and PPARγ in TM5 cells by RT-PCR. Finally, transfection studies leads to the identification of important functional RAR/RXR (RARE) and LXR/RXR (LXRE) binding sites in the promoter.
Expression and regulation of the myocilin gene in TM cells seems to be under the control of transcription factors like RARs, RXRs and LXRs. These results are an important step to understand the fine myocilin regulation in order to better prevent its dependant glaucoma pathology
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