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O. Lorentz, J. Sahel, M. Fradot, T. Léveillard; Regulation of Smad7 Expression by USF2, a Cone Photoreceptor Specific Pathway: Involvement in Cone Function . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3526.
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Purpose: TGFß (Transforming Growth factor ß) pathway is involved in a wide array of cellular events ranging from proliferation, differentiation and apoptosis. In drosophila, TGFß homologue, DPP and its downstream effectors (MADs) have been shown to be involved in eye development and differentiation. Nevertheless, the expression and function of Smads (mouse MADs homologue) in mouse retina have never been described before. Therefore, the aim of this work consists on the study of the expression and the function of Smads in cone photoreceptor differentiation and survival. Methods: RT-PCR assays, EMSA (Electrophoretic Mobility Shift Assay) and transient transfections have been performed using standard molecular biology technics. Confocal microscopy, Y79 cell culture and retinal explants have been performed following standard cell biology protocols. Results: We demonstrate by RT-PCR all the members of the Smad family are expressed in the mouse retina. By confocal microscopy, we show that Smad7 is exclusively expressed by S cones. Futhermore, in rd7 mice retina, Smad7 expression is incresaed and is localised to S cone cells forming the rosette and to RPE cells. By transient transfection assay in Y79 cells, we show that Smad7, (i) is able to stimulate the activity of the promoter region controlling the activity of the blue opsin gene, (ii) has no effect on the bovine rhodospin promoter activity. To elucidate the retinal mechanisms involved in the regulation of Smad7 expression, we demonstrate by EMSA (Electrophoretic Mobility Shift Assay) that conditioned medium from wild type retinal explant is able to enhance Upstream Stimulatory Factor 1 and 2 (USF1 and USF2) DNA binding activity to a functional E-Box element localised in Smad7 promoter region. Furthermore, by transient transfection we demonstrate that conditioned medium from wild type retinal explant stimulates USF2 transcriptional activity whereas conditioned medium from rd1 retinal explant is not able to stimulate USF2 transcriptional activity. Conclusion: Taken together these results demonstrate that within the retina, Smad7 is exclusively expressed by S cone and regulates their differentiation. Furthermore we demonstrate that rods secrete factor(s) able to stimulate Smad7 expression by, (i) enhancing USF1 and USF2 binding to Smad7 promoter, (ii) able to stimulate USF2 transcriptional activity. This study consist of a new cone specific pathway directly involved in cone photoreceptor function and provides evidences that this pathway may be directly involved in cone survival.
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