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Molecular Endocrinology 18 (12): 2866-2879
Copyright © 2004 by The Endocrine Society

Selective Recruitment of p160 Coactivators on Glucocorticoid-Regulated Promoters in Schwann Cells

Julien Grenier, Amalia Trousson, Anne Chauchereau, Larbi Amazit, Audrey Lamirand, Philippe Leclerc, Anne Guiochon-Mantel, Michael Schumacher and Charbel Massaad

Institut National de la Santé et de la Recherche Médicale (INSERM) (J.G., A.T., A.L., P.L., M.S., C.M.), Unité 488, Institut Fédératif de Recherche (IFR) 93, 94276 Le Kremlin-Bicêtre Cedex, France; Centre National de la Recherche Scientifique (A.C.), Unité Propre de Recherche 9079, 94800 Villejuif, France; INSERM (L.A., A.G.-M.), Unité 135, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris, IFR93, 94275 Le Kremlin-Bicêtre Cedex, France; and Faculté de Médecine Paris-Sud (A.G.-M., C.M.), 94276 Le Kremlin-Bicêtre Cedex, France

Address all correspondence and requests for reprints to: Charbel Massaad, Institut National de la Santé et de la Recherche Médicale Unité 488, 80 rue du Général Leclerc, 94276, Le Kremlin-Bicêtre Cedex, France. E-mail: massaad{at}kb.inserm.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the nervous system, glucocorticoid hormones play a major role during development and throughout life. We studied the mechanisms of action of the glucocorticoid receptor (GR) and its interactions with p160 coactivator family members [steroid receptor coactivator (SRC)-1 (a and e), SRC-2 and SRC-3] in mouse Schwann cells (MSC80). We found that the three p160s were expressed in MSC80 cells. We have shown by functional overexpression and RNA interference experiments that the recruitment of these coactivators by the GR is promoter dependent. A minimal promoter containing two glucocorticoid response elements, (GRE)2-TATA, recruits SRC-1 (a and e) and SRC-3, whereas SRC-2 is excluded. Within the context of the more complex mouse mammary tumor virus promoter, GR recruits SRC-1e and SRC-2, whereas SRC-1a and SRC-3 are not implicated. Furthermore, we have identified cytosolic aspartate aminotransferase as a GR target gene in MSC80 cells by microarray experiments. The GR recruits exclusively SRC-1e in the context of the cytosolic aspartate aminotransferase promoter. Because SRC-1 is the omnipresent coactivator of GR, we further investigated the interactions between GR and this coactivator in Schwann cells by reporter assays and immunocytochemistry experiments with deleted forms of SRC-1. We have shown that SRC-1 unexpectedly interacts with GR via its two nuclear receptor binding domains, thus providing a novel mechanism of GR signaling within the nervous system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOIDS REGULATE ENERGY metabolism, cell proliferation, and immune responses, but they also act on neuronal and glial genes. Thus, they play a major role in the homeostasis of the nervous system and have been reported to have beneficial effects during nerve regeneration (1, 2, 3). In most cases, their actions are mediated by their cognate nuclear receptor (NR), the glucocorticoid receptor (GR). The GR is a member of the superfamily of NRs, including steroid receptors, orphan receptors, and receptors forming heterodimers with the retinoic X receptor (4). Upon hormonal stimulation, the GR is translocated into the nucleus and binds as a homodimer to a glucocorticoid response element (GRE), present in the vicinity of glucocorticoid-modulated gene promoters. To enhance transcription activity, the GR must recruit transcription coactivators of the p160 family, which are docking platforms for other coactivators such as cAMP response element binding protein-binding protein, or its close homolog p300.

The p160 coactivator family comprises steroid receptor coactivator (SRC)-1 (nuclear receptor coactivator-1), SRC-2 (nuclear receptor coactivator-2/GR-interacting protein 1/TIF-2), and SRC-3 (p300/cAMP response element binding protein-binding protein/activator of the thyroid and retinoic acid receptor/thyroid receptor activator molecule/receptor-associated-coactivator) (5, 6, 7, 8, 9, 10). This family is characterized by the presence of several conserved functional domains: a N-terminal basic helix-loop-helix-PAS (Per/ARNT/Sim homologous domain) domain, a cAMP response element binding protein-binding protein-interacting domain (AD1), a glutamine-rich region, a C-terminal activation domain (AD2) and several LXXLL boxes involved in NR binding. SRC-1 has been shown to be implicated in numerous NR signaling pathways as it interacts with a wide variety of NRs [e.g. estrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR), retinoic X receptor]. Two functionally distinct isoforms of SRC-1 have been identified, SRC-1a and SRC-1e, issued from alternative splicing (11) and differing in their C-terminal region (12, 13, 14). The two isoforms seem to play distinct roles, SRC-1e appears to be the major isoform of SRC-1 involved in thyroid hormone signaling (13).

The expression and role of SRCs in the nervous system have begun to be established and studied only during recent years. Nevertheless, their molecular mechanisms of action and interactions in the brain and in peripheral nerves are usually extrapolated from other target tissues (liver, kidney, ovary). SRC-1 plays an important role in the nervous system, where it is thought to participate in ER signaling implicated in the defeminization of the male rat brain, but not in AR signaling leading to brain masculinization (15). Moreover, disruption of SRC-1 delays Purkinje cells development and maturation at early stages, resulting in moderate motor dysfunction in adulthood (16). In the nervous system, SRC-1a and SRC-1e are expressed at high levels in the hypothalamus, hippocampus, cerebellum, thalamus, and amygdala (17), whereas some differences in their expression were observed between brain nuclei.

SRC-2 has first been isolated as a specific GR AF-2 interactor (5). Despite its close homology with SRC-1, SRC-2 seems to have specific functions: it is involved in lipid metabolism and energy balance, appears to be essential for adhesion of Sertoli cells to germ cells in males and induces placental hypoplasia in SRC-2–/– females. SRC-2 plays an important role in reproduction because homozygous SRC-2–/– mice are sterile, whereas SRC-1–/– or SRC-3–/– are not (18). Within the brain, SRC-2 appears to be less expressed than SRC-1 but is specifically present in the anterior pituitary, contrary to SRC-1.

SRC-3 is implicated in the GH regulatory pathway and in the production of estrogen (19). Furthermore, it has been shown to be essential for both PR and ER activities. Indeed, disruption of SRC-3 leads to decreased mammary gland development, even after combined treatment with estrogen and progesterone. In the nervous system, SRC-3 is mainly expressed in the hippocampus and olfactory bulbs. However, the knockout of one of the three p160 coactivators leads to partial phenotypes because the disruption induces an overexpression of the other two p160 partners. Thus, the physiological functions of each p160 coactivator remain uncertain. The compensatory properties of p160 lead to the conclusion that SRC-1, SRC-2, and SRC-3 may play different but sometimes overlapping roles, especially in the nervous system.

The aim of the present work is to examine the functional interactions between the GR and its coactivators and to explore the differential recruitment and implication of SRC-1, SRC-2, and SRC-3 in GR signaling in the nervous system. We used the immortalized mouse Schwann cell line MSC80 that has retained the capacity of myelinating axons in vivo and expresses the markers of glial cells (glial fibrillary protein and S100 protein). Moreover, we have previously shown by radioligand binding studies and RT-PCR that this cell line expresses the GR and no another steroid receptor (20), thus allowing a selective study of the glucocorticoid signaling pathway. Our results show that p160 coactivator recruitment by the GR is dependent on the promoter context. Furthermore, the interaction between the GR and SRC-1 is not exclusively restricted to the C-terminal domain of SRC-1, contrasting with data previously described in other cellular models.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the Three p160 Coactivators in MSC80 Cells
We have first investigated the expression of the three p160 coactivators (SRC-1, SRC-2, and SRC-3) in MSC80 cells by RT-PCR and Western blot. As shown in Fig. 1AGo, the mRNAs coding for the three p160s [SRC-1 (a + e), SRC-2 and SRC-3] were detected on agarose gel after electrophoresis, indicating that all three coactivators are expressed in MSC80 cells. To distinguish between the two major SRC-1 isoforms, we used specific primers that recognized either the two isoforms (SRC-1 a + e) or solely the SRC-1e isoform, which contains an insertion in its mRNA 3' end. As shown in Fig. 1BGo, MSC80 cells express both SRC-1a and SRC-1e, but SRC-1a seems to be expressed at lower levels than SRC-1e. We have verified the expression of p160s proteins in total MSC80 extracts by Western blot using antibodies recognizing specifically SRC-1, SRC-2, and SRC-3. As shown in Fig. 1CGo, all three p160s proteins are present in MSC80 cells.



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  		Fig. 1. Expression of p160 Coactivators in MSC80 Cells

A, Expression of SRC-1, SRC-2, and SRC-3. Total RNA from MSC80 cells was prepared. RT-PCR experiments were performed by using primers recognizing specifically SRC-1, SRC-2, or SRC-3. PCR products were analyzed on agarose gel (1%) and visualized under UV. 18S RNA was detected by specific primers and used to normalize SRC expression levels. B, Expression of total SRC-1 (SRC1a+e), and of SRC-1e solely. The PCR was controlled using primers recognizing 18S RNA. C, Western blot was performed on 50 µg of total extracts of MSC80 cells using either anti-SRC-1, anti-SRC-2, or anti-SRC-3 antibodies to verify the expression of the three p160s in these cells. This figure is representative of three independent experiments.

 
Implication of the p160 Family in GR Signaling
To address the question of which of the p160 members is specifically recruited by endogenous GR, we studied GR-mediated transcription of three different promoters: the (GRE)2-TATA-chloramphenicol acetyltransferase (CAT) construct, composed of two GREs upstream of a TATA box, the viral mouse mammary tumor virus (MMTV)-CAT construct bearing several GREs and transcription factors binding sites, and the cytosolic aspartate aminotransferase (cAspAT-CAT), an endogenous glucocorticoid target gene promoter. We have recently shown that the cAspAT gene is a target for glucocorticoid in MSC80 cells using microarray technology (Tomkiewicz, C., J. Grenier, A. Trousson, K. Rajkowski, M. Schumacher, and C. Massaad, manuscript submitted).

To study the implication of the p160s in GR signaling, we have overexpressed Rip140 (receptor-interacting protein 140), which is a GR corepressor known to inhibit GR transactivation (21) by competing with the p160s for their binding to the GR (22, 23). In the cases of both (GRE)2-TATA-CAT and MMTV-CAT constructs, we observed a dose-dependent inhibition of GR transactivation after a 24-h treatment with dexamethasone (Dex) when increasing amounts of Rip140 expression vector were transfected (Fig. 2Go, A and B, respectively). When 2 µg of Rip140 expression vector were transfected, we obtained a maximal inhibition of GR transactivation (80%) for (GRE)2-TATA promoter, whereas a 50% inhibition was observed for MMTV promoter. In the case of the cAspAT promoter, we observed a 14-fold induction by 1 µM of Dex. Rip140 exerted a moderate but significant 40% inhibition of GR transactivation (Fig. 2CGo). We have performed similar experiments using 10 nM of Dex to avoid cross-activation of other pathways, although the MR is absent in MSC80 cells. They gave similar results (data not shown). The inhibition by Rip140 is specific to the GR signaling pathway as the basal transcriptional activity (i.e. without Dex treatment) was not affected (data not shown). Moreover, Rip140 did not elicit any inhibitory effect on GRE-less promoters [simian virus 40 (SV40)-luciferase, Rous sarcoma virus-luciferase, or TATA-CAT promoters] (data not shown). These results show the implication of p160 coactivators members in GR signaling in MSC80 cells.



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  		Fig. 2. Effect of Rip140 on GR Signaling

MSC80 cells were transiently transfected with either (GRE)2-TATA-CAT (A), MMTV-CAT (B), or cAspAT-CAT (C) plasmids and increasing amounts (0–2 µg) of Rip140. Eighteen hours after transfection, cells were incubated with Dex (10–6 M) for 24 h, and then CAT and luciferase activities were analyzed. Results are expressed as the induction over the basal activity, they represent the mean ± SEM of at least three independent experiments performed in duplicate. One-way ANOVA showed a significant effect for (GRE)2-TATA-CAT (F = 49.77; df = 14; P < 0.0001), MMTV-CAT (F = 42.53; df = 14; P < 0.0001), and cAspAT-CAT (F = 7.29; df = 14; P < 0.005). *, P < 0.05; ** P < 0.01 by post hoc Tukey’s test when compared with control.

 
Effect of Selective p160 Inhibition by RNA Interference on the GR Signaling Pathway
Because the use of Rip140 does not permit to discriminate between the p160 members involved in the endogenous GR complex, we used a RNA interference approach to confirm our previous observations and to identify which p160 is implicated in GR signaling.

We have designed three 19-bp oligonucleotides directed against either SRC-1, SRC-2, or SRC-3, inserted in pSuper short interfering (si) RNA expression vector. Each oligonucleotide recognized the mRNA 5' terminus and specifically targeted one p160 member to inhibit, respectively, the expression of SRC-1, SRC-2, or SRC-3. We were not able to make siRNA recognizing specifically SRC-1a and SRC-1e because their mRNAs only differ by their 3' ends. Thus, siSRC-1 that we have designed is able to target the expression of both SRC-1 a and e. The inhibitory efficiency of each siRNA vector was assayed by semiquantitative RT-PCR (Fig. 3AGo) and Western blot (Fig. 3BGo) using either total RNA extract or total protein extract from MSC80 cells transfected with 2 µg of empty pSuper, siSRC1, siSRC2, or siSRC3 expression vectors, respectively. Each siRNA knocked down specifically and drastically the expression of its corresponding p160 mRNA (Fig. 3AGo) and protein (Fig. 3BGo). No significant cell lethality or unspecific effect on total RNA were observed. These results demonstrate that the three siRNA specifically targeted SRC-1, SRC-2, and SRC-3, respectively, and that they were very efficient in causing specific mRNA degradation and protein extinction.



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  		Fig. 3. Effect of Selective p160 Inhibition by RNA Interference on the GR Signaling Pathway: Verification of the Efficacy of the siRNA Directed against SRC-1, SRC-2, and SRC-3

MSC80 cells were transiently transfected with siRNA expression vectors (siSRC1, siSRC2, and siSRC3). Forty-eight hours after transfection, the cells were harvested and total RNA or total protein extracts were prepared. A, Semiquantitative RT-PCR experiments were performed using primers recognizing specifically either SRC-1, SRC-2, or SRC-3. The PCR were normalized using 18S RNA. B, Western blots were performed using either anti-SRC-1, anti-SRC-2, or anti-SRC-3 antibodies to verify the efficacy and the specificity of the siRNA on protein silencing. ß-Actin was used to normalize the Western blots. These results were reproduced in three independent experiments. These figures are the representation of a typical experiment. MSC80 cells were transiently transfected with the (GRE)2-TATA-CAT (C), MMTV-CAT (D), or cAspAT-CAT (E) constructs and increasing amounts (0, 0.5, 1, 2, and 5 µg) of either one of siRNA expression vectors (siSRC1, siSRC2, or siSRC3). Eighteen hours after transfection, cells were incubated with Dex (10–6 M) for 24 h, and then CAT and luciferase activities were analyzed. 100% Corresponds to the transcriptional activation elicited by Dex alone in the absence of any transfection. In the case of (GRE)2-TATA-CAT, the induction of transactivation by Dex was 103 ± 9-fold activation of transcription over the basal activity. Results represent the mean ± SEM of at least three independent experiments performed in duplicate. One-way ANOVA showed a significant effect for siSRC1 (F = 5.32; df = 14; P < 0.01) and for siSRC3 (F = 23.34; df = 28; P < 0.0001). In the case of MMTV-CAT, the induction of transactivation by Dex was 13 ± 2-fold activation of transcription over the basal activity. One-way ANOVA showed a significant effect for siSRC1 (F = 32.93; df = 14; P < 0.0001) and for siSRC2 (F = 12.08; df = 23; P < 0.0001). Finally, in the case of cAspAT, the induction of transactivation by Dex was 13 ± 2-fold activation of transcription over the basal activity. One-way ANOVA test showed a significant effect for siSRC1 (F = 10.962; df = 14; P < 0.001). *, P < 0.05; **, P < 0.01 by post hoc Tukey’s test when compared with control.

 
We then studied the effect of the depletion of endogenous SRC-1, SRC-2, or SRC-3 on the stimulation of (GRE)2-TATA-CAT (Fig. 3CGo), MMTV-CAT (Fig. 3DGo), and cAspAT-CAT (Fig. 3EGo) reporter genes by glucocorticoids by transfecting MSC80 cells with increasing amounts of each siRNA expression vectors (0–5 µg). In the case of (GRE)2-TATA promoter, siSRC1 dose dependently inhibited the transactivation, with a maximal inhibition (50%) obtained for 2 µg of siSRC1 expression vector (Fig. 3CGo). siSRC3 also strongly inhibited GR transactivation, with a 50% inhibition for 2 µg of siSRC3 expression vector, and a total (100%) inhibition for 5 µg of expression vector. In contrast, no significant inhibition of GR transactivation was observed with siSRC2 expression vector. These results show that the GR in the context of (GRE)2-TATA promoter is able to recruit preferentially either SRC-1 or SRC-3, but not SRC-2. RNA interference experiments showed that GR recruits either SRC-1 or SRC-2 to enhance the transcriptional activity on the MMTV promoter (Fig. 3DGo). Indeed, siSRC1 and siSRC2 inhibited Dex stimulation (60% and 40% inhibition, respectively). SRC-3 knock-down had no effect on this reporter. On cAspAT promoter, siSRC-1 caused moderate but significant inhibition (20% inhibition) on Dex-mediated transcription of this natural promoter (Fig. 3EGo). This is probably because the cAspAT promoter is complex and comprises other transcription factors binding sites neighboring the GREs (i.e. CAAT enhancer binding protein factors), which could compensate for the depletion of SRC-1 by recruiting another coactivator. The other two siSRC2 and siSRC3 did not inhibit effect on GR signaling in the case of the cAspAT promoter (Fig. 3EGo). The siRNA experiments shown above, were also undertaken by using 10 nM of Dex, gave similar results than 1 µM of Dex (data not shown).

Effect of p160 Overexpression on the GR Signaling
We have challenged our results obtained by siRNA p160 knock-down with those obtained by p160 overexpressions. As shown in Fig. 4AGo, overexpression of SRC-1a potentiated GR signaling toward (GRE)2-TATA-CAT construct. The maximal effect was obtained with 500 ng of SRC-1a expression vector, leading to a 2.5-fold enhancement of GR-transactivation. Overexpression of SRC-1e also enhanced GR transactivation (Fig. 4AGo), which was maximal at 2 µg of SRC-1e. Transfection of increasing amounts of SRC-2 expression vector did not led to any significant effect (not shown). Finally, transfection of MSC80 cells with the (GRE)2-TATA-CAT construct and increasing amounts of SRC-3 expression vector, resulted in dose-dependent enhancement of GR transactivation (Fig. 4BGo), with a 2.5-fold potentiation of maximal Dex transactivation. Taken together, the siRNA and overexpression experiments showed that the GR, in the context of the (GRE)2-TATA promoter, preferentially recruits SRC-1 (a or e) or SRC-3 in the hormone-activated transcriptional complex, whereas SRC-2 seems to be excluded.



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  		Fig. 4. Effect of p160 Overexpression on GR Signaling

MSC80 cells were transiently transfected with either (GRE)2-TATA-CAT (A and B) or MMTV-CAT (C and D) or cAspAT-CAT(E) plasmids and increasing amounts (0–2 µg) of p160 expression vectors as indicated. Eighteen hours after transfection, cells were incubated with Dex (10–6 M) for 24 h, and then CAT and luciferase activities were analyzed. Results are expressed as fold activation over the basal activity; they represent the mean ± SEM of at least three independent experiments performed in duplicate. In the case of (GRE)2-TATA-CAT, one-way ANOVA test showed a significant effect for SRC-1a (F = 5.98; df = 23; P < 0.005), SRC-1e (F = 3.967; df = 14; P < 0.05) and SRC-3 (F = 10.793; df = 14; P < 0.001). In the case of MMTV-CAT, one-way ANOVA showed a significant effect for SRC-1e (F = 4.81; df = 14; P < 0.05) and SRC-2 (F = 15.32; df = 21; P < 0.0001). Finally, in the case of cAspAT, one-way ANOVA test showed a significant effect for SRC-1e (F = 7.71; df = 14; P < 0.005). *, P < 0.05; **, P < 0.01 by post hoc Tukey’s test when compared with control.

 
In contrast with the results obtained with the minimal (GRE)2-TATA promoter, overexpression of SRC-1a did not elicit any further stimulation of transactivation by glucocorticoids of the MMTV promoter even at high doses of transfected SRC-1a expression vector (Fig. 4CGo). However, SRC-1e, the truncated isoform of SRC-1a, dose-dependently stimulated MMTV transactivation by GR, showing that transactivation is mediated by SRC-1e on the MMTV promoter. SRC-2 also significantly enhanced CAT activity (Fig. 4DGo), with a maximum at 500 ng of transfected expression vector, whereas SRC-3 overexpression did not elicit any additional potentiation of GR transactivation. The Dex stimulation of cAspAT-CAT construct was only potentiated by SRC-1e overexpression (Fig. 4EGo). SRC-1a (Fig. 4EGo), SRC-2 (not shown), and SRC-3 (not shown) overexpression did not elicit any further enhancement of Dex stimulation. All of the experiments shown in Fig. 4Go were also undertaken at 10 nM of Dex. They gave similar results, although the fold induction elicited at 1 µM of Dex was higher than that observed at 10 nM (data not shown).

We have verified the potency of Rip140 in inhibiting the effect of the overexpression of the p160. We cotransfected MSC80 cells with 2 µg of Rip140 expression and one of the p160 that had a potentiating effect on GR transactivation (i.e. SRC-1a, SRC-1e, and SRC-3 for (GRE)2-TATA-CAT, SRC-1e and SRC-2 for MMTV-CAT and SRC-1e for cAspAT-CAT). As shown in Fig. 5AGo, Rip140 drastically inhibited the effect of SRC-1a, SRC-1e, and SRC-3, thus confirming the specificity of Rip140 in competing with these p160. In the cases of MMTV (Fig. 5BGo) and cAspAT (Fig. 5CGo), Rip140 inhibited moderately the potentiation of transactivation by SRC-1e and SRC-2, thus confirming our previous observations for the endogenous p160s (Fig. 2Go). This moderate inhibition observed for MMTV and cAspAT is probably because of the complexity of these promoters and the compensatory effects of other transcription factors in the vicinity of the GRE.



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  		Fig. 5. Inhibition of p160s Potentiation of Transactivation by Rip140

MSC80 cells were transiently transfected with 2 µg of Rip140 expression vector in the presence of 0.5 µg of SRC-1a, SRC-1e, or SRC-3 for (GRE)2-TATA-CAT (A), SRC-1e or SRC-2 for MMTV-CAT (B), and SRC-1e for cAspAT-CAT (C). Eighteen hours after transfection, cells were incubated with Dex (10–6 M) for 24 h, and then CAT and luciferase activities were analyzed. Results are expressed as the induction over the basal activity; they represent the mean ± SEM of at least two independent experiments performed in duplicate.

 
Together, our results show that SRC-1 is implicated in the three types of promoters that we have studied, suggesting an important role of this coactivator in the GR-regulated transcription in Schwann cells. Therefore, we next addressed the question of how the GR may interact with SRC-1, and which of the two NR binding domains (NR1 or NR2) of SRC-1 may be important for its interaction with the GR. In this prospect, we used both colocalization experiments and transactivation assays with deleted forms of SRC-1.

Nucleocytoplasmic Shuttling of SRC-1 and GR during Glucocorticoid Treatment
We first performed immunocytochemistry experiments to study the localization of both endogenous GR and SRC-1 in MSC80 cells. Cells were incubated with RU28362, which is a pure glucocorticoid agonist that does not bind to other NRs, for different periods of time (30 min to 30 h). Cells were fixed, and endogenous SRC-1 and GR were revealed by two-color confocal analysis. In the absence of hormone, endogenous GR (red) and the majority of endogenous SRC-1 (green) were localized in the cytoplasm (Fig. 6Go). A small fraction of SRC-1 was present in the nucleus. After 30 min of hormone treatment, the majority of GR and SRC-1 was recovered in the nucleus, as shown by the total colocalization of these two proteins. SRC-1 and GR colocalized in the nucleus (yellow) up to 4 h of incubation. Finally, after 30 h of treatment, the GR was still retained in the nucleus, whereas part of SRC-1 had exited into the cytoplasm. These results show that SRC-1 is expressed in MSC80 cells and that the activation by a glucocorticoid agonist provokes the interaction between GR and SRC-1, thus leading to nuclear translocation and colocalization.



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  		Fig. 6. Localization of SRC-1 and GR in MSC80 Cells

MSC80 cells were incubated in the absence (control) or in the presence of RU28362 (10–6 M) for 30 min, 4 h, or 30 h. Antibodies directed either against SRC-1 or GR were added to the cells overnight. Cells were incubated during 45 min with secondary antibodies directed either against anti-SRC-1 (Alexa 488, green) or anti-GR (Alexa 555, red). The slides were analyzed with a confocal microscope. This experiment was repeated four times, and a typical experiment is presented here. Irrelevant antibodies from the same family of Ig of either anti-SRC-1 or anti-GR were used as control for nonspecific staining (not shown). Similar results were obtained using Dex (0.1 µM) (data not shown).

 
Role of the Different LXXLL Domains of SRC-1 in the GR Pathway
To further analyze how this coactivator is recruited by the GR in MSC80 cells, we studied the GR-mediated transactivation by overexpressing different SRC-1 mutants (Fig. 7AGo). The C-terminal domain of SRC-1 has been initially identified as the region sufficient to exert a dominant-negative effect on PR-mediated transactivation (24). This NR2 region (1199–1440) has been characterized as one of the receptor binding domain which interacts exclusively with GR and PR. When increasing amounts of SRC-1 mutant corresponding to the NR2 domain were overexpressed (Mut A), we did not observe any dominant-negative effect on GR-mediated transactivation of (GRE)2-TATA-CAT plasmid (Fig. 7BGo), whereas the dominant-negative effect was still observed on PR signaling (Fig. 7CGo). Moreover, Western blot analysis showed that Mut A was as expressed as the wild-type SRC-1 in MSC80 cells (not shown). SRC-1 mutant lacking the NR2 domain (Mut D) was as effective as wild-type SRC-1 in potentiating Dex transactivation (Fig. 7BGo), indicating that the lack of NR2 domain did not alter SRC-1 transactivation potential. Thus, contrasting with two-hybrid data (25, 26, 27), our results suggested that the C-terminal NR2 region of SRC-1 is not crucial for the interaction between GR and SRC-1.



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  		Fig. 7. Role of the C-Terminal Domain of SRC-1 in the Glucocorticoid Pathway

A, Schematic representation of the wild-type SRC-1 and its mutated forms (SRC-1 mut A and D). B, MSC80 cells were transiently transfected with (GRE)2-TATA-CAT plasmid and increasing amounts of Mut A (diamond) or Mut D (triangle) expression vectors. Eighteen hours after transfection, cells were incubated with RU28362 (10–6 M) for 24 h. Results are expressed as fold induction after treatment with RU28362 over the basal activity without transfecting any expression vectors. One-way ANOVA test showed a significant effect for Mut D (F = 10.7; df = 14; P < 0.001). C, MSC80 cells were transiently transfected with (GRE)2-TATA-CAT plasmid, PRB expression vector, and increasing amounts of Mut A (diamond) expression vector or wild-type SRC-1a (square). Eighteen hours after transfection, cells were incubated with progesterone (10–6 M) for 24 h. Results are expressed as fold induction after treatment with progesterone over the basal activity without transfecting any expression vectors. One-way ANOVA test showed a significant effect for Mut A (F = 44.5; df = 14; P < 0.0001) and SRC-1a (F = 17.32; df = 21; P < 0.0001). Results are the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.05 by post hoc Tukey’s test when compared with control. PRB, Progesterone receptor B.

 
A second domain, called NR1, located in the middle of the SRC-1 protein, has been characterized as an interaction domain with NRs, most importantly for ER, AR, or peroxisome proliferator-activated receptor. This domain contains three characteristic LXXLL motifs. We have tested the respective roles of NR1 and NR2 in the GR pathway by functional transfection assays and immunolocalization in MSC80. A mutant deleted of the 1–567 N-terminal amino-acids (Mut B) exhibited the same efficiency in activating transcription as wild-type SRC-1 (Fig. 8Go, A and B). The SRC-1 mutants deleted of either the NR2 (Mut C) or the NR1 motifs (Mut E), did not affect the transcriptional activity of the (GRE)2-TATA-CAT plasmid. Interestingly, the deletion of both NR1 and NR2 motifs altered the transactivation (see Mut F). This mutant did not exert any potentiation of GR transactivation. The effects of the mutants were specific of the GRE element because no potentiation of transactivation has been observed on GRE-less promoter (like SV40 or Rous sarcoma virus promoters; data not shown). Furthermore, no effect can be attributed to differential expression levels because an equivalent expression for all mutants has been observed by Western blotting (not shown) and immunohistochemistry (Fig. 8CGo) in MSC80 cells.



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  		Fig. 8. Effect of the Mutation of the Two LXXLL-Rich Domains of SRC-1 on the Glucocorticoid Pathway

A, Schematic representation of the wild-type SRC-1 and its mutated forms (SRC-1 Mut B, C, E, and F). B, MSC80 cells were transiently transfected with (GRE)2-TATA-CAT plasmid and with increasing amounts of Mut B (diamonds), Mut C (squares), Mut E (triangles), or Mut F (X) expression vectors. Eighteen hours after transfection, cells were incubated with RU28362 (10–6 M) for 24 h. 100% Corresponds to the transcriptional activation elicited by RU28362 without transfection of any SRC-1 mutant expression vector. Results are the mean ± SEM of three independent experiments in duplicate. One-way ANOVA test showed a significant effect for Mut B (F = 93.87; df = 15; P < 0.0001), Mut C (F = 6.9; df = 14; P < 0.005), and Mut E (F = 19.42; df = 14; P < 0.0001). *, P < 0.05; **, P < 0.01 by post hoc Tukey’s test when compared with control. C, Localization of GR and SRC-1 mutants transfected in MSC80 cells. MSC80 cells were transiently transfected with pSG5-HA-wtSRC-1, or pSG5-HA-MutB, or pSG5-HA-MutC, or pSG5-HA-MutE, or pSG5-HA-Mut F expression vectors. Eighteen hours after transfection, cells were incubated with RU28362 (10–6 M) for 4 h. Antibodies directed against either HA-tag of SRC mutants or endogenous GR were added to the cells overnight. Cells were then incubated for 45 min with secondary antibodies directed either against anti-HA (Alexa 569, red) or anti-GR (Alexa 488, green). The slides were analyzed with a confocal microscope. This experiment was repeated three times, and here, a typical experiment is shown. Irrelevant antibodies from the same family of Ig of either anti-HA or anti-GR were used as control for nonspecific staining (not shown).

 
We have applied our double-staining immunofluorescence colocalization of GR and SRC-1 mutants to analysis of SRC-1 mutants. Because the N-terminal region containing the nuclear localization signal was deleted in the different mutants (28), they showed a cytoplasmic localization (Fig. 8CGo). They were able to translocate into the nucleus after hormone-induced interaction with GR that escorts the complex into the nucleus. As shown in Fig. 8CGo, all SRC-1 mutants or the wild-type version (red) were localized in the cytoplasm in the absence of hormone treatment, as was the endogenous GR (green). When RU28362 was added to the medium, all SRC-1 mutant proteins entered the nucleus, where they colocalized with GR. Only Mut F, lacking both NR domains and unable to interact with the GR, remained in the cytoplasm in the presence of hormone, whereas GR was predominantly nuclear.

Collectively, our results indicate that the presence of only one of the two NR motifs of SRC-1 is sufficient for its hormone-induced interaction with the GR in the cytoplasm. When both domains are deleted, SRC-1 can no longer interact with GR and transcription is not activated because SRC-1 cannot enter the nucleus. Our results clearly show that both NR domains of SRC-1 are able to interact with GR in glial cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this report, we have investigated the implication of the p160 coactivator family in GR signaling in Schwann cells. We have first demonstrated by RT-PCR that SRC-1, SRC-2, and SRC-3 mRNAs were all three expressed in these cells, and that the two major SRC-1 isoforms, obtained by alternative splicing (SRC-1a and SRC-1e) were also both present, SRC-1e expression being higher than that of SRC-1a. The expression and localization of SRC-1 (a and e), SRC-2, and SRC-3 in the nervous system have been studied, but the significance of their differential expression is still not understood. The different distribution of these coactivators could account for cell specificity of steroid receptor action in the nervous system as has been described for ER action in the brain (29).

The coexpression of coactivators in MSC80 cells raises the question of their selective action. In this report, we have addressed two main questions: 1) Can the GR discriminate between the three p160 family members? and 2) How does the GR interact with SRC-1 in glial cells? We have chosen three different promoters for our study: the (GRE)2-TATA promoter, the viral MMTV promoter, and a natural target gene of glucocorticoids in MSC80 cells, the cAspAT promoter. The minimal promoter formed by two GREs in tandem and a TATA box precluded any interference of the GR with other transcription factors. In contrast, the MMTV and cAspAT promoters are more complex, as they are composed of multiple GREs and other transcription factor binding sites. Thus, they correspond to more realistic models of the cross talk between the GR, other transcription factors, and the p160 family members. The implication of the p160 coactivators in the GR signaling pathway was first suggested by the inhibitory effect of Rip140 corepressor. The fact that overexpressed Rip140 repressed GR activity on the three studied promoters suggests that p160 coactivators are implicated in GR transactivation.

We have used two approaches to determine which member of the p160 family is implicated in GR signaling in MSC80 cells: one based on coactivator overexpression and the other based on specific inhibition of targeted coactivators by RNA interference. The siRNA were shown to be efficient in specific mRNA silencing and confirmed those based on overexpression studies. The minimal promoter was sensitive to SRC-1 (a and e) and SRC-3, whereas MMTV preferred to recruit SRC-1e and SRC-2. cAspAT was only sensitive to SRC-1e. Our results thus demonstrate that the recruitment of p160 family members depends on the promoter context. The GR is able to recruit all three members, but the promoter context influences the affinity of the GR for one of the p160 family members. Our results slightly differ from the hypothesis that the three p160s are interchangeable, as suggested by mouse genetic knockouts. The knockouts of each p160 family members led to moderate dysfunctions, whereas one would expect a more severe phenotype (16, 18, 19, 30, 31). As a matter of fact, infusion of antisense oligodeoxynucleotides to SRC-1 and SRC-2 into the adult rodent brain led to the blockade of female reproductive behaviors, contrasting with SRC-1 knockout animals that do not present any defect in sexual behavior. These paradoxal observations are probably because of a compensatory balance between the SRCs.

Li et al. (32) have shown that in T47D cells, using the MMTV promoter, the GR preferentially recruits SRC-2, whereas the PR interacts with SRC-1, thus demonstrating the receptor-dependent recruitment of p160s coactivators. Moreover, the type of response element could influence the interaction between the steroid receptor and SRC-1 or SRC-2 as it was described for the ER in the case of pS2 gene promoter (33). In this report, we have shown that the recruitment of the SRCs is also dependent on the promoter context. Taken together, our results and those of Li et al. (32) and Barkhem et al. (33) suggest that the interaction between a steroid receptor and a p160 family member is a multiparametric event. The cellular environment, the coexpression of several steroid receptors and coactivators, the nature of the response element, and finally the environment of the target promoters are multiple parameters that govern the interactions between the NRs and the SRCs.

We have then focused our work on the interaction between GR and SRC-1 in mouse Schwann cells, as SRC-1 was the omnipresent coactivator of the GR at the MMTV, cAspAT and (GRE)2-TATA promoters. We have first investigated their interaction in MSC80 cells upon treatment with the GR agonist RU28362. In untreated cells, the majority of GR and SRC-1 is cytoplasmic. Such a cytoplasmic localization of p160 coactivators has been described to be dependent on cell culture conditions and on their phosphorylation status (28). Thirty minutes after incubation with RU28362, GR interacts with SRC-1 and the complex enters the nucleus, as seen by the total colocalization profile. SRC-1 starts to exit the nucleus after 30 h, which was previously shown to be exportin mediated (28). SRC-2 (GR-interacting protein 1) has also been shown to exit the nucleus, to co localize with the proteasome in the cytoplasm and to be degraded (34).

We examined the implication of the three LXXLL-rich domains of SRC-1 in glucocorticoid transactivation. Previous two-hybrid screening assays had shown that the GR preferentially interacts with the C-terminal NR2 motif of SRC-1 (25, 26, 27). Therefore, overexpression of this domain should have altered glucocorticoid signaling in our model, as has been described in the case of PR for which overexpression of this NR2 domain led to a dominant-negative SRC-1 (24). In MSC80 cells, increasing amounts of NR2-domain expression vector did not inhibit glucocorticoid transactivation, and deletion of this domain had no effect. These results suggest that the C-terminal domain of SRC-1 is not crucial for the GR pathway at least in MSC80 cells. Deletion of the central NR1 box did not affect GR transactivation; nevertheless, deletion of both NR1 and NR2 domains completely abolished the potentiation of transactivation by SRC-1. These findings suggest that both domains may be involved in the potentiation of glucocorticoid transactivation by SRC-1, and that one of theses domains can be sufficient. Hence, in glial cells, these two domains seem to be redundant for GR signaling, thus differing from previous data based on in vitro studies or yeast two-hybrid (25, 26, 27). We have confirmed the requirement for NR1 or NR2 by an immunofluorescence approach. Indeed, each mutant deleted for NR1 or NR2 can be imported in the nucleus by the GR. The mutant lacking both NR1 and NR2 is still cytoplasmic as it cannot interact with the GR. In conclusion, functional as well as immunocytochemical experiments clearly show that the two NR domains of SRC-1 can bind the GR separately, and the deletion of one domain can be compensated by the presence of the other. Why does SRC-1 binding to GR differ from the classical model in MSC80 cells? One can hypothesize that SRC-1 could have a phosphorylation status in Schwann cells (35, 36) that could lead to a different conformation of the protein and as a consequence to a different interaction with GR. This would also explain the cytoplasmic localization of SRC-1 in other cells as suggested by several authors (37, 38, 39).

In conclusion, our work demonstrates that p160 recruitment by the GR is promoter sensitive and specific. Moreover, the interaction of GR with SRC-1 in Schwann cells implicated the two NR-interacting domains. Our description of a novel GR-SRC-1 coactivator complex in Schwann cells, suggests that glucocorticoids act by means of differential mechanisms in the nervous system. Finally, the fact that the GR could recruit different p160s, depending on the promoter context, should allow us to selectively target certain genes by inhibiting their promoter-specific p160s.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
The mouse Schwann cell line (MSC80) was maintained in DMEM supplemented with 10% fetal calf serum (Invitrogen Life Technologies, Carlsbad, CA), 100 U/ml penicillin, 100 µl/ml streptomycin (Invitrogen Life Technologies), and 0.5 µg/ml fungizone (Invitrogen Life Technologies).

Plasmids
Expression vectors of wild-type SRC-1 and SRC-1 mutants have been described (28, 40, 41). SRC-1 (wild type and mutants) have been subcloned in the pSG5-HA expression vector (40). SRC-2/TIF-2 expression vector was a gift from H. Gronemeyer (Ilkirch, France) and SRC-3/activator of the thyroid retinoic acid receptor was kindly provided by Ron Evans (San Diego, CA). Rip140 expression vector was a gift from V. Cavaillès (Montpellier, France) (42). The (GRE)2-TATA-CAT plasmid has been described by Chauchereau et al. (40). The cAspAT-CAT construct was a gift from R. Barouki (Paris, France). PGL2-SV40-Luciferase vector was purchased from Promega (Madison, WI).

siRNA Preparation
siRNA expression vectors directed against SRC-1, SRC-2, and SRC-3 were prepared as described in Brummelkampf et al. (43). Oligonucleotides of 64 bp of length were subcloned in BglII and XhoI sites of pSuper expression vector. Designed sequences for siRNA were (sense strand) 5'-TGGAATGTCAATTCCCCGA-3' for SRC-1, 5'-AGAGCAAACTCATCCGTTC-3' for SRC-2, and 5'-ATTCCTCCTTGACCAACTC-3' for SRC-3.

RT-PCR Experiments
The total RNA from cultured MSC-80 cells was obtained using QIAGEN Rneasy mini-kit and reverse transcribed with random primers from Biolabs (Beverly, MA) and reverse transcriptase Moloney murine leukemia virus reverse transcriptase from Finnzymes (Espoo, Finland). PCR experiments were performed using Taq DNA polymerase purchased from Biolabs and primers specific to each SRCs from Proligo (Boulder, CO), sequences are shown below:

SRC-1: F, 5'-AGGAACAATGGGAAACAAC-3'
SRC-1: R, 5'-CCATCTGCGTCTGTTTG-3'
SRC-1e: 5'-GTCACCACAGAGAAGAAC-3'
SRC-1a+e: 5'-GCCTACCAGATTCACTGT-3'
SRC-2 sense: 5'-GACAGATCGTGCCAGTAACACAA-3'
SRC-2 antisense: 5'-TTCAGCTGTGAGTTGCATGAGG-3'
SRC-3 sense: 5'-GCAGATGAGTGGAGCTAGGTATG-3'
SRC-3 antisense: 5'-CACGATTACGAGGAGAAATCATG-3'

Antibodies
The antibodies against GR (rabbit polyclonal PA1–510A) were purchased from ABR (Golden, CO), SRC-1 (mouse monoclonal) IgGk from Upstate (Lake Placid, NY), SRC-2/TIF2 (mouse monoclonal ref: 610985) and SRC-3/AIB-1 (mouse monoclonal ref: 611105) from BD Transduction Laboratories (Lexington, KY). Fluorescent antibodies were purchased from Molecular Probes (Eugene, OR): Alexa 488 (mouse), Alexa 555 (rabbit), and Alexa 568 (mouse).

Western Blot
Aliquots of 50 µg of total MSC80 extracts transfected or not with 2 µg of either siSRC1, siSRC2, or siSRC3 expression vectors were used or each sample. The Western blot analysis was performed as described by Chauchereau et al. (40). The antibodies were used at the following dilutions: SRC-1 (1/1000), SRC-2, and SRC-3 (1/250).

Transient Transfections
MSC80 cells were transiently transfected using the polyethylenimine reagent (Sigma, St. Louis, MO). One day before the transfection, MSC80 cells (4.105 cells/6-cm dish) were seeded into 6-cm dishes and incubated in the DMEM culture medium containing 10% fetal calf serum. The (GRE)2-TATA-CAT plasmid (3 µg), the pGL2-SV40-Luciferase expression vector (1 µg), and the coactivator expression vectors or mock vector at the concentrations indicated in the figure legends were mixed with a solution containing polyethylenimine (0.85 mg/ml) in serum-free DMEM. The mixture was then added on the cells for 5 h. One day after the transfection, the medium was replaced by DMEM containing 10% charcoal-treated fetal calf serum containing or not glucocorticoid agonist RU28362 (10–6 M) or Dex (10–6 M).

Luciferase and CAT Assays
Luciferase activity was used to normalize the transfection efficiency in all culture dishes. The assay was performed as described by Massaad et al. (44). The CAT activity was determined using the two-phase assay described by Massaad et al. (45).

Immunocytochemistry
MSC80 cells were seeded at the density of 2 x 105 cells in 4 cm2 glass Lab-Tek wells (Nunc, Roskilde, Denmark). RU28362 was added 18 h later. The cells were incubated with RU28362 (10–6 M) from 30 min to 30 h (as indicated in the figure legends). The cells were then washed and fixed in paraformaldehyde (1/9) for 15 min. After three washes, cells were permeabilized at –80 C for 30 min. Then, cells were incubated with primary antibodies (dilution 1:1000) overnight at 4 C and labeled with conjugated antibodies (dilution 1:400) for 45 min at room temperature. The slides were imaged using a confocal microscope LSM410 (Carl Zeiss Inc., Le Pecq, France) with a x40 (numerical aperture 1.2) lens and sequential excitation with laser lines 488 nm (Ar.Ion laser) and 543 nm (HeNe laser).


   ACKNOWLEDGMENTS
 
We are very grateful to Dr. Ulrike Krummrei for helpful discussion and the experiments of immunoprecipitation that she had undertaken, and Olivier Trassard for the confocal microscopy. We also thank Dr. Cosima Fonté for her critical reading of the manuscript.


   FOOTNOTES
 
This article has been prepared with financial support from the Commission of the European Communities, specifically, Research Technology Development program "Quality of Life and Management of Living Resources," QLK6-CT-2000-00179, "The role of neurosteroids in healthy ageing: therapeutical perspectives," by the Myelin Project (Dunn Loring, VA); and by the Projet Myéline (Nancy, France).

Present address for L.A.: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.

Abbreviations: AR, Androgen receptor; cAspAT, cytosolic aspartate aminotransferase; CAT, chloramphenicol acetyltransferase; Dex, dexamethasone; ER, estrogen receptor; GR, glucocorticoid receptor; GRE, glucocorticoid response element; MMTV, mouse mammary tumor virus; NR, nuclear receptor; PR, progesterone receptor; Rip140, receptor-interacting protein 140; si, short interfering; SRC, steroid receptor coactivator; SV40, simian virus 40; TIF, transcriptional intermediary factor.

Received for publication June 14, 2004. Accepted for publication August 17, 2004.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   GR
Coregulators:   RIP140  |  SRC-1  |  GRIP1  |  AIB1
Ligands:   Dexamethasone  |  Progesterone



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