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I. Pascual, I. M. Larrayoz, I. R. Rodriguez; All-Trans Retinoic Acid Increases the Transcriptional Activity of Both Methionine Sulfoxide Reductase a (MSRA) Promoters. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4440. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
MSRA is an important anti-oxidative enzyme highly expressed in the macular RPE. The transcriptional control of MSRA is not well understood but is dependent on two distinct promoters, 1 and 2. The purpose of this study is to characterize both MSRA promoters in human RPE-derived cells (D407) and to determine which factors affect its transcriptional regulation in these cells.
Various lengths of MSRA promoters were amplified from human genomic DNA and cloned into a luciferase reporter vector. Coding region of retinoic acid receptors were amplified from human retina and cloned into pcDNA 3.1/V5-His-Topo expression vector. D407 cells were transfected with the different promoter constructs and luciferase activity was measured 48h after transfection. Renilla luciferase was used as internal transfection control. RPE cells were seeded in 6 well plates and were treated with All-trans retinoic acid for 24 and 48 hr before RNA isolation. Real-time qRT-PCR was performed with SYBR Green using an ABI 7500 instrument. Electrophoretic mobility shift assay (EMSA) was performed with biotinylated probes and chemiluminescent EMSA Kit.
Both MSRA promoters show high luciferase activity in D407 cells but promoter 2 is less active in other cell types. In promoter 2 a 65 bp sequence between positions -693 and -628 demonstrated very high activity and presented potential retinoic acid response elements. EMSA analysis demonstrated interactions with RAR and RXR in this region. Treatments with non-toxic concentrations of all-trans retinoic acid and transient overexpression of the retinoic acid receptors increased the activity of both promoters. Retinoic acid treatment also increased the mRNA expression of all MSRAs (from promoters 1 and 2) in a dose dependent manner.
Both MSRA promoters respond to retinoic acid. Promoter 2 contains a 65 bp enhancer region which is highly responsive to retinoic acid and may be important in sustaining high expression of MSRA in RPE cells.
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