Transcription factors essential for pluripotency modulate superoxide dismutase 1 and glutathione reductase gene expression in mouse embryonic stem cells.

CAMILA VÁZQUEZ ECHEGARAY; MARCOS GABRIEL FRANCIA; GABRIELA CHAUFAN; SANTIAGO MIRIUKA; MARIA VICTORIA PETRONE; ARIEL WAISMAN; CLAUDIA SOLARI; PABLO EVELSON; MARÍA SOLEDAD COSENTINO; LINO BARAÑAO; TIMOTEO MARCHINI; MARIA DEL CARMEN RIOS DE MOLINA; ALEJANDRA GUBERMAN

Ciudad de Buenos

Congreso; Reunión Conjunta de Biociencias 2017; 2017

Our aim was to study Superoxide dismutase 1 (Sod1) and Glutathione reductase (Gsr) gene expression in embryonic stem cells (ESCs) in order to unravel the relationship between redox homeostasis and pluripotency. For this purpose, R1 ESCs were differentiated with a non-directing differentiation protocol, culturing them in absence of the cytokine LIF for 4 days. RNA was extracted from undifferentiated and differentiated cells, and the expression profile of Sod1 and Gsr genes was analyzed by RT-qPCR. We found that these genes were expressed in the undifferentiated state and they were modulated in opposite ways when ESCs were induced to differentiate. While Sod1 was downregulated during differentiation, Gsr expression was upregulated (randomized block design ANOVA and Tukey test). Next, we performed a shRNA approach to downregulate Oct4, Nanog and Sox2. Sod1 and Gsr mRNA levels were analyzed in ESCs transfected with vectors encoding each shRNA. In accordance with the expression profile found along differentiation, we found a reduction in Sod1 mRNA levels when Oct4, Nanog and Sox2 were downreguated. Moreover, Gsr expression was increased when Nanog was downregulated (randomized block design ANOVA and Tukey test). We also performed a trans-activation assay with reporter vectors to study Sod1 and Gsr promoter responsiveness. We found that Oct4, Sox2 and Nanog induced luciferase expression in Sod1 reporter vector, but we did not find a clear response with the Gsr reporter vector studied (randomized block design ANOVA and Tukey test). Finally, we analyzed Sod activity and we found that it decreased along differentiation (Student test).Our results suggest that Sod1 gene expression is induced by the transcription factors Oct4, Nanog and Sox2, and that Gsr gene expression could be repressed by Nanog in ESCs. These results evidence a role of the crucial pluripotency transcription factors in preservation of redox homeostasis in stem cells