Differentiation of Murine Embryonic Stem Cells
نویسندگان
چکیده
7 differentiation through aldosterone-stimulated activation of its alternative promoters. ABSTRACT 40 Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory 41 mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal 42 response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene 43 transcription. To examine promoter activities and their regulation during neuronal differentiation and 44 in mature neurons, we generated stably transfected recombinant murine embryonic stem (ES) cell 45 lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene 46 Green Fluorescent Protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, 47 permitted to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase 48 alkaline activity, the concomitant appearance of morphological changes (neurites) and the increase in 49 the expression of neuronal markers (nestin, β-tubulin III, MAP2) as demonstrated by 50 immunocytochemistry and qPCR. Using these cell-based models, we showed that MR expression 51 increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively 52 expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter 53 during neuronal differentiation, their activities increased by 7-and 5-fold, respectively and correlated 54 with MR expression. Finally, while progesterone and dexamethasone were ineffective, aldosterone 55 stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of 56 MR by siRNA. Concluding, we provide evidence for a tight transcriptional control of MR expression 57 during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the 58 neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and 59 psychiatric diseases.
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تاریخ انتشار 2010