Personne : Masson-Gadais, Bénédicte
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Centre de recherche en endocrinologie, métabolisme et inflammation, CHU de Québec, Université Laval
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- PublicationAccès libreDifferential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human α5 integrin gene dictates its transcriptional activity(Association for Research in Vision and Ophthalmology, 2009-01-01) Masson-Gadais, Bénédicte; Guérin, Sylvain; Germain, Lucie; Drouin, Régen; Zaniolo, Karine; Gingras, Marie-Ève; Leclerc, SteevePurpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the α5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the α5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress α5 promoter activity in a cell density–dependent manner by differentially altering the ratio of these TFs. Conclusions. These results suggest that α5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) α5 transcription in corneal epithelial cells.
- PublicationRestreintThe feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3(Wiley, 2005-10-21) Masson-Gadais, Bénédicte; Guérin, Sylvain; Germain, Lucie; Paquet, Claudie; Fugère, Claudia.; Lefort, Natalie; Leclerc, SteevePrimary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell–cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle.
- PublicationRestreintRescue of the transcription factors Sp1 and NFI in human skin keratinocytes through a feeder-layer-dependent suppression of the proteasome activity(Academic Press, 2012-03-12) Masson-Gadais, Bénédicte; Guérin, Sylvain; Germain, Lucie; Rochette, Patrick J.; Duval, Céline; Gaudreault, Manon.; Vigneault, François; Touzel-Deschênes, LydiaCo-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.