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. 2010 May;151(5):2244-54.
doi: 10.1210/en.2009-0753. Epub 2010 Mar 5.

Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells

Mathilde Munier  1 , Geri Meduri, Say Viengchareun, Philippe Leclerc, Damien Le Menuet, Marc Lombès
Affiliations

Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells

Mathilde Munier et al. Endocrinology. 2010 May.

Abstract

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

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Figures

Figure 1
Figure 1. Generation of hMR promoter-GFP expressing stable ES cell lines
A) Schematic representation of the two P1-GFP and P2-GFP constructs. The 5′-flanking regions of hMR gene contains the two first untranslated exons 1α and 1β (solid boxes). The Hind III-Ava II (−969, +239) P1 fragment and SspI-SspI (−1682, +123) P2 fragment were used to generate P1-GFP and P2-GFP plasmids, respectively. The GFP (hatched box) was used as a reporter gene. As described in the “Materials and Methods” section the pGL3-Enhancer Vector contains the SV40 Enhancer (black box). B) PCR genotyping of undifferentiated resistant cell lines (numbered 1 to 7). Rapsn was used as internal genomic control. C) RT-PCR analyses of GFP, MR, β-actin (used as internal control) mRNA expression in 7 day embryoid bodies (from 1.106 cells in suspension in 15% FCS supplemented medium). D) GFP protein expression was observed in 7 day embryoid bodies (from 1.106 cells in suspension in 15% FCS supplemented medium). Original magnification: x20
Figure 2
Figure 2. Neuronal differentiation of ES cell lines
A) Alkaline Phosphatase activity of ES cells and neurons. Alkaline phosphatase activity was measured using the colorimetric Alkaline Phosphatase Detection assay kit. Original magnification: x10 B, C) Relative Nestin and MAP2 mRNA expression levels were determined using qPCR at different stages of differentiation: undifferentiated ES cells, embryoid bodies (EB) and neurons. Results are means ± SEM of six independent experiments of three samples performed in duplicate for each developmental stage, and represent the relative expression compared with basal levels of ES (arbitrarily set at 1). *** P<0.001. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section). D) Immunocytochemical detection of Nestin, β-tubulin III, GFAP and APC in neurons. Original magnification: x40.
Figure 3
Figure 3. MR expression during neuronal differentiation
Relative MR mRNA expression levels were determined using qPCR at different stages of differentiation: undifferentiated ES cells, embryoid bodies (EB) and neurons. Results are means ± SEM of six independent experiments of three samples performed in duplicate for each developmental stage and represent the relative expression compared with basal levels of ES (arbitrarily set at 1). *** P<0.001. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section).
Figure 4
Figure 4. MR is exclusively expressed in neurons
Double immunolabelling analyses of neurons with antibodies against MR (red) (left panel), Nestin (green), β-tubulin III (green), and GFAP (green) (middle panel), and merge (right panel). Original magnification: x40.
Figure 5
Figure 5. Comparison of P1 and P2 activity during neuronal differentiation
A) Relative GFP mRNA expression levels, which represent P1 and P2 activity, were determined using qPCR at different stages of differentiation: embryoid bodies (EB) and neurons. Results are means ± SEM of at least four independent experiments of three samples performed in duplicate and represent the relative expression compared with basal levels in embryoid bodies (arbitrarily set at 1). ** P<0.01. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section). B) Relative GFP mRNA expression levels were determined using qPCR at different stages of differentiation: ES cells and neurons. Results are means ± SEM of at least four independent experiments of three samples performed in duplicate and represent the relative P2 activity compared with basal levels of P1 activity (arbitrarily set at 1). ** P<0.01. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section). C) Double immunolabelling analyses of neurons with antibodies against MR (red) (left panel), and GFP (green) (middle panel), and merge (right panel). Original magnification: x40.
Figure 6
Figure 6. Hormonal regulation of P1 and P2 activity and MR expression
Neurons were exposed to 100 nM aldosterone (ALDO), dexamethasone (DXM) or progesterone (PROG) (A,C,E) or to increasing concentrations of aldosterone (1 to 100 nM) (B,D,F). A, B, C, D) Relative GFP mRNA expression levels were determined using qPCR. Results are means ± SEM of four independent experiments of six samples performed in duplicate and represent the relative expression compared with basal levels of control untreated cells (C) (arbitrarily set at 1). *** P<0.001 ** P<0.01 * P<0.05. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section). E, F) Relative MR mRNA expression levels were determined using qPCR. Results are means ± SEM of four independent experiments of six samples performed in duplicate and represent the relative expression compared with basal levels of control untreated cells (C) (arbitrarily set at 1). *** P<0.001. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section).
Figure 7
Figure 7. MR down-regulation inhibits aldosterone-induced P2 promoter activity
Neurons were transfected with either the control scrambled siRNA (scr MR) or by two unrelated MR siRNA (si1 MR, si2 MR). After transfection, neurons were incubated or not with 100 nM aldosterone (ALDO). A) Relative MR mRNA expression levels were determined using qPCR. Results are means ± SEM of six samples performed in duplicate and represent the relative expression compared with basal levels of control scrambled siRNA transfected (scr MR) (arbitrarily set at 1). ** P<0.01. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section). B) Relative GFP mRNA expression levels were determined using qPCR. Results are means ± SEM of six samples performed in duplicate and represent the relative expression compared with basal levels of control untreated cells (C) (arbitrarily set at 1). *** P<0.001 ** P<0.01. Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods section).
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