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S. Proulx, M. Gingras, A. Rufiange, S.L. Guérin, C. Salesse; Expression of the Alpha5 Integrin Subunit Gene Promoter is Altered by the Vitreous in the Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2279.
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Purpose: Proliferative vitreoretinopathy (PVR) is a disease in which normally quiescent retinal pigment epithelial (RPE) cells come in the abnormal contact of vitreous. The cells then dedifferentiate, migrate, proliferate and cause retinal detachment. Integrins, a family of cell surface adhesion proteins, have been proposed as playing a role in PVR. In the present study, we evaluated whether the vitreous might alter the transcriptional activity directed by the promoter of the α5 integrin subunit gene in primary cultured RPE cells. Methods: Both confluent and subconfluent cultures of human RPE cells were transfected with a recombinant plasmid (-954α5CAT) that bear the promoter from the human α5 gene inserted upstream of the CAT reporter gene, in either the absence or presence of different concentrations of human vitreous. Crude nuclear extracts from RPE cells grown at different confluency in the presence and absence of vitreous were collected and used as source of nuclear proteins in electrophoresis mobility shift assays (EMSA) using as labeled probes various double-stranded oligonucleotides that bear target sites for transcription factors reported to bind the α5 promoter (Sp1, AP1 and NF1). Results: A near 4-fold increase in the activity directed by the α5 promoter was observed upon addition of vitreous to 7.5% (vol/vol). No alteration was observed in the DNA binding ability of the transcription factors Sp1, AP1 and NF1 when subconfluent RPE cells were grown in the presence of vitreous. However, expression of Sp1 was substantially reduced whereas that of NF1 was increased into post-confluent RPE cells exposed to vitreous. Conclusions: These results demonstrate that the transcriptional activity directed by the α5 integrin gene is subjected to substantial alterations upon exposure of RPE cells to the vitreous. Alteration in the expression of the α5 integrin subunit as a consequence of modifications in the level of expression of transcription factors like Sp1 or NF1 may therefore contribute to the progression of PVR.
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