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Unité Mixte de Recherche 788, Institut National de la Santé et de la Recherche Médicale and University Paris-Sud 11, 94276 Le Kremlin-Bicêtre, France
Address all correspondence and requests for reprints to: Dr. F. Cadepond, Institut National de la Santé et de la Recherche Médicale, Unité 788, 80, rue du Général Leclerc, 94276 Le Kremlin-Bicêtre, France. E-mail: cadepond{at}kb.inserm.fr.
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Abstract |
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and ß) receptors in rat SC cultured under proliferative (in the presence of insulin and forskolin, which induces a high intracellular cAMP content) and quiescent conditions. We found no or very low expression and activity of the sex steroid receptors, as shown by mRNA concentrations determined with real-time PCR and transcriptional activities using transient expression of reporter plasmids in SC. These data and binding studies in SC lines demonstrated that the levels of the sex steroid receptors were the limiting factors. GR was clearly expressed (
8000 sequences/ng total RNA) and functional. No significant modification in GR mRNA levels was observed, but an increase in transcriptional efficiency was recorded in proliferating cells compared with quiescent cells. MR was also significantly expressed at the mRNA level (450 sequences/ng total RNA) under the two culture conditions. No MR transcriptional activity was observed in SC, but a low specific binding of aldosterone was detected in SC lines. 11ß-Hydroxysteroid-dehydrogenase type 2 (HSD2), an enzyme that inactivates glucocorticoids, was strongly expressed and active in quiescent SC, although in proliferating cells, HSD2 exhibited a strong decrease in activity and mRNA concentration. These data support a physiological role for HSD2 regulation of glucocorticosteroid concentrations in nerve SC. |
Introduction |
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The ubiquitous glucocorticoid receptor (GR) is expected to exert its pleiotropic control on a large set of genes depending on the cell context. In SC, the role of GR is poorly documented. For the mineralocorticosteroid receptor (MR), which also binds glucocorticosteroids, it is not known whether it is expressed and mediates specific effects in SC. Progesterone (P) and its metabolites may be synthesized and are biologically active in the nervous system (11, 12), thus belonging to the class of neurosteroids (13, 14). The presence of progesterone receptor (PR) in cultured SC has been detected by steroid binding and immunocytology (11, 15). On the contrary, Chan et al. (16), who detected PR mRNA and PR protein in DRG neurons cocultured with SC by RT-PCR and immunocytology, did not detect clear PR message and protein in SC. Other sex steroids may exert important functions in the nervous system. In SC, previous reports have described trophic effects of estrogens in the presence of cAMP (17), acting via the estrogen receptor (ER) and the stimulation of the expression of myelin protein P0 by androgens in the absence of the androgen receptor (AR) (11). In cultured segments of rat sciatic nerve, estradiol (E2) stimulated SC proliferation in males and P did so in females, but in this report cAMP acted as an inhibitor (18). Although these studies show that SC contain steroid receptor message and protein, they do not provide information on the amounts of receptors and their functional state.
Steroids can regulate target gene expression using different mechanisms. They can activate a nuclear steroid receptor leading to stimulation or inhibition of the expression of a target gene by direct genomic action on specific regulatory elements located in its promoter. In some cases, their cross-binding to another steroid receptor type may occur, leading to agonistic (19) or antagonistic effects (20). In addition, activated steroid receptors can also modulate the activity of other transcription factors on responsive promoters and even interfere with upstream signaling pathways, thus mediating nongenomic effects (21, 22, 23). Such effects can also result from the action of membrane receptors: classical steroid receptors with membrane location (23, 24), novel membrane steroid-binding proteins or G protein-coupled receptors, and neuromediator receptors (25, 26, 27, 28). Therefore, it appears very important to know the pattern of expression and the functional state of the various classical steroid receptors in SC to determine whether they can be involved in the mechanisms of steroid action.
After a brief report dealing with steroid receptors in several glial cell lines of the central nervous system (CNS) and PNS (29), the present work investigates the expression of various steroid receptors in primary cultures of purified SC under proliferative and quiescent conditions by real-time PCR. The functional state of the receptor proteins was checked by transcriptional measurements from exogenous synthetic promoters controlling reporter proteins, introduced by transfection. Complementary steroid binding studies were performed in SC lines (rat CR3a1 and CR1b4 cells and mouse MSC80 cells) (30, 31). These SC lines display some characteristics of primary cultured SC, i.e. partial differentiation and myelin protein expression in the presence of neurons. Moreover, to investigate a possible regulation of GR and MR activity in purified SC, the expression of 11ß-hydroxysteroid-dehydrogenase type 2 (HSD2), an enzyme able to inactivate glucocorticosteroids by converting corticosterone (CS) into 11ß-dehydrocorticosterone, was examined. The possibility of coordinated regulation between steroid receptors and steroid metabolic enzymes, leading to invalidation of the GR pathway, is discussed with respect to nerve regeneration.
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Materials and Methods |
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-dehydrotestosterone (DHT), and Aldo) or NEN Life Science Products (R1881 and R5020). The GR agonist 17-1-propynyl-11ß,17ß-dihydroxy-6-methyl-androsta-1,4,6-triene-3-one (RU28362) were gifts from HMR (Romainville, France; now Aventis). Mouse antirat GR antibody (BuGR) was purchased from Affinity Bioreagents (Golden, CO). Measurements of cAMP were performed using the [3H]cAMP Biotrak assay system (TRK432) from GE Healthcare.
Cell culture
SC from the sciatic nerves of 3- to 4-d-old Sprague Dawley rats were purified and propagated as previously described (4). They were 3 wk old at the end of the purification steps and were cultured at most another 4 wk. They were used after a 48-h incubation with DMEM containing 10% charcoal/dextran-treated serum (32) (to eliminate the steroids present in the serum), 4 mM glutamine, and a 1/200 mixture of penicillin, streptomycin, and fungizone (1000 U/ml, 10 mg/ml, and 1 mg/ml, respectively).
MSC80 (mouse) and CR3a1 and CR1b4 (rat) cell lines were cultured as previously described (30, 31). They were maintained in 10% charcoal/dextran-treated serum for 48–72 h.
To investigate ER binding and ER mRNA content, cells were cultured for at least 5 d in medium containing phenol-red-depleted DMEM with 10% fetal calf serum before the 48-h terminal incubation with steroid-depleted serum.
Total RNA preparation and RT
Total RNA was prepared using the RNeasy kit from QIAGEN (Courtaboeuf, France) including an additional DNase step (RNase-free DNase set kit). A mean yield of 1 µg RNA was obtained from approximately 4 x 105 quiescent SC or from 3.6 x 105 SC cultured in proliferative medium or approximately 2 x 105 cells from the different cell lines. This correspondence allowed conversion of mRNA concentrations per nanogram RNA into mRNA concentrations per cell. RT was carried out with Superscript II RNase H-Reverse Transcriptase (Invitrogen Life Technologies, Paisley, UK) with 1 µg total RNA in 20 µl, using hexanucleotide primers (250 ng) in the presence of the ribonuclease inhibitor RNAsin (0.5 µl; Promega, Charbonnières, France). A mean of 14% incorporation of [32P]dCTP into cDNA was determined by trichloroacetic acid precipitation, a value used to estimate sequence number per nanogram original RNA (i.e. retrotranscribed with a 100% efficiency).
PCR experiments
PCR experiments were carried out using a real-time PCR apparatus, ABI PRISM 7000 (Applied Biosystems, Courtaboeuf, France). Assays were performed in 20 µl according to Applied Biosystems’ recommendations with the cDNA from 20 ng total RNA. We used TaqMan methodology for rat GR, MR, PR, AR, and ER and -ß (gene expression assays nos. Rn01405583, Rn00565562, Rn00575662, Rn00560747, Rn00562166, and Rn00562610, respectively) and SYBR Green technology for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HSD (type 1 and 2). For MSC80 mRNAs, except for ER, we performed estimations using the above-mentioned rat gene expression assays. GAPDH primers were TGC ACC ACC AAC TGC TTA GC (sense) and GGC ATG GAC TGT GGT CAT GAG (antisense). Primers for HSD2 sequences were chosen in exons 2 and 3 (sense, TTG GCA AGG AGA CAG CTA AG, and antisense, CCA GAA CAC GGC TGA TAT CCT), whereas for HSD1 they were GGA GCC CAT GTG GTA TTG A (sense) and AGT GCC GGC AAT GTA GTG A (antisense). For mouse ER mRNA, an adaptation of the classical method was performed using a robocycler (Stratagene, La Jolla, CA) and common mouse/rat primers: sense, CCCCTACTACCTGGAGAAC; antisense, GGTTTGTAGCTGGACACATG. Accurate presentation of gene expression data required normalization and quantification steps that are described in detail below.
PCR data normalization with GAPDH
Amplification of an endogenous control was necessary to standardize the amount of cDNA in a PCR, and we followed the expression of the housekeeping gene GAPDH in each sample using a standard GAPDH curve obtained with a bulk preparation of kidney retro-transcribed RNA. This curve was very close to that obtained with SC preparations.
Validation of GAPDH as a reference gene for RNA samples of SC and related cell lines was performed by analyzing several housekeeping genes with geNorm software (33). We found that GAPDH was one of the most stably expressed control genes. Furthermore, by exploiting the cycle threshold (Ct) values obtained from several SC preparations, cultured in parallel either with or without insulin and forskolin, the low mean Ct variation (
Ct = 0.16) observed between the two culture conditions was taken into account for the expression of the results in Fig. 1A
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and -ß) and SC (when possible), showing no differences in amplification efficiencies between cDNA and DNA in most experiments, allowed this quantification of mRNA sequences.
For mouse ER, we quantified the amount of 294-bp amplicon after gel electrophoresis in the presence of ethidium bromide. Absorbance of ethidium bromide-stained bands was measured at several successive cycles with a Bio-Rad (Marne-la-Coquette, France) image analysis system using molecular analysis software. After subtracting background and plotting the logarithm of intensities as a function of cycle numbers, an absorbance value at a given cycle was read in the linear part of the curve. The same experiment was carried out with the GAPDH gene for normalization and with increased quantities of plasmid DNA for the estimation of sequence numbers.
Relative quantitation of HSD1 and HSD2 mRNA concentrations was performed by real-time PCR using SYBR Green and GAPDH as reference gene.
Cell cytosol preparation and steroid binding measurements
The binding activities for various steroids were determined in cytosol extracts from cell lines. Cells were cultured with charcoal/dextran-treated serum 48–72 h before harvesting and then collected by scraping and centrifugation. Cell pellets were suspended in the GTED-Mo homogenization buffer [13% glycerol (vol/vol), 20 mM Tris-Cl, 1 mM EDTA, and 1 mM dithiothreitol (pH 7.4) at 4 C, containing 5 µg/ml antipain, 0.2 mM phenylmethylsulfonyl fluoride and 15 mM Na2MO4; 2 ml/g cell pellet] and homogenized with a motor-driven glass/glass homogenizer. Low-salt cytosol extract was obtained after 1 h centrifugation at 105,000 x g. Aliquots were removed for protein concentration measurements. The 10% glycerol and 1 mg/ml ovalbumin were then added to prevent inactivation during incubation and freezing/thawing steps. For each steroid receptor tested, standard incubations (50 µl) contained 20 nM tritiated steroid (TA, Aldo, R5020, R1881, and E2) with and without a 1000-fold molar excess of corresponding cold competitor steroid (RU28362, Aldo, R5020, R1881, and E2, respectively). To eliminate possible cross-reactivity of a given steroid with another steroid receptor, parallel incubations were also performed with additional steroids (20 µM RU28362 for MR, 10 µM CS and 15 nM DHT for PR, 50 µM TA for AR and 15 nM DHT for ER). When performing titration experiments, various radioactive steroid concentrations (from 0.5–20 nM) were used with and without a 1000-fold molar excess of the corresponding cold steroid and in the presence of complementary steroid when necessary (RU28362 for Aldo binding to MR).
Separation of bound and free steroid was performed according to the hydroxyapatite method (34). Briefly, 500 µl of hydroxyapatite slurry (10%) was pelleted by centrifugation in a 5-ml glass scintillation vial. After supernatant removal, aliquots of 20 µl cytosol were layered on it (two vials per sample). Ten minutes later, four rapid washings of the pellet were performed with 1.5 ml buffer, before the addition of 5 ml picofluor 15 and counting. Total radioactivity was also measured from two 2-µl aliquot supernatants. Titration data were analyzed by the nonlinear regression analysis included in PRISM 2 software.
Detection of GR by Western blotting
Cytosol samples (30 µg protein) were mixed with concentrated Laemmli’s sample buffer and boiled for 5 min before loading on 7% SDS-polyacrylamide gel for electrophoretic separation. Proteins were electrophoretically transferred to a polyvinylidene difluoride membrane using Towbin transfer buffer (35). Membranes were blocked and incubated overnight at 4 C with BuGR antibody (0.025 µg/ml). After rinsing, they were incubated 1 h at room temperature with 3.125 ng/ml horseradish-peroxidase-conjugated secondary antibody (goat antimouse antibody from Pierce Biotechnology, Brebières, France) and processed for chemiluminescence detection (Supersignal West Femto; Pierce).
Cell transfection and transcriptional activity measurements
SC were collected by trypsination (2 x 106 to 5 x 106 cells) and transfected according to Nucleofector Technology (Amaxa GmbH, Köln, Germany) using the astrocyte kit and the V20 program with 3 µg MMTV-luciferase (36) or overERE-MTV-CAT (37) reporter plasmid. These promoters displayed a stronger response to steroids than promoters containing simple glucocorticoid-responsive elements or estrogen-responsive elements (ERE). After transfection, the bulk of cells were quickly distributed in six-well polylysine-coated dishes and, 3 h later, treated with different steroids (10 nM or 1 µM for RU28362, R5020, RU1881, and E2 and 0.1 or 10 nM for Aldo). Cell lysis was performed after 18–24 h incubation with steroids. Measurements of chloramphenicol acetyl transferase (CAT) activity using [3H]chloramphenicol in a liquid-phase method and luciferase activity by luminescence with a luciferin substrate were performed as previously described (38, 39).
Batch transfection allowed a stock of cells with a unique transfection efficiency to be made available for a series of steroid incubation conditions. The efficiency of transfection was determined by including a small proportion of green fluorescent protein-expressing plasmid (0.5 µg) and by counting green fluorescent cells.
For exogenous steroid receptor expression, 1 µg of each plasmid containing rat GR, MR, PR, AR, and ER sequences (pC7GR, MR-cDNA3, PR6B-pcDNA3.1/Zeo, pSG5-rAR or pCMVratAR, and ER-pEGFP, respectively; see Acknowledgments) were cotransfected with the appropriate reporter plasmid.
Measurements of HSD2 activity
For measurements of 11ß-dehydrogenase activity, cells maintained 48 h with steroid-depleted serum in 35-mm-diameter dishes were used at subconfluence with or without insulin and forskolin. After 2 h in medium containing 2% steroid-depleted serum, 10 nM [3H]CS was added (the Km of HSD2 is in the nanomolar range) (40). At different times, the supernatant of each well (1 ml) was removed and extracted with 9 ml methanol containing 1 mg 3-ter-butyl-4-hydroxy-anisol, 5 nmol CS and 5 nmol 11-dehydrocorticosterone, vortexed and left overnight at room temperature. After centrifugation at 4500 rpm for 10 min, the supernatant and 0.5-ml pellet were concentrated to 50–150 µl and layered on thin-layer chromatography plates. Two discrimination systems were used: chloroform/methanol/water (100:5:0.1) or chloroform/ethyl acetate (4:1). Cold steroids (markers and carriers) were detected with UV light and the radioactivity profile obtained with a Tracemaster 20 apparatus (Berthold, Thoiry, France) using Chroma software. The radioactivity comigrating with the cold dehydrocorticosterone peak was expressed as a percentage of total slot radioactivity and the amount normalized to the initial CS concentration. Spontaneous CS conversion was very low and subtracted from the experimental values. After careful rinsing, cell layers were lysed and used for total protein determination according to the Pierce BCA kit.
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Results |
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, A and B). GR mRNA (8500 ± 2460 and 7500 ± 2070 sequences/ng total RNA, mean ± SEM; n = 10) and MR mRNA (398 ± 98 and 511 ± 85 sequences/ng; mean ± SEM; n = 8 and 10) were found in cells cultured in proliferative and quiescent medium, respectively. GR and MR expression did not significantly differ in SC cultured in the absence and in the presence of insulin and forskolin, which allow cell proliferation. These values corresponded approximately to 20 GR and one MR mRNA molecule/SC. In cell lines, the GR mRNA sequence number per nanogram was of the same order of magnitude as in SC, except Msc80, which exhibited a lower content. On the contrary, the MR mRNA level was higher in CR1b4 and MSC80 lines (Fig. 1B).
A lower content of PR mRNA was detected in SC: 125 ± 46 (mean ± SEM; n = 10) and 112 ± 33 (mean ± SEM; n = 10) sequences/ng in the absence or in the presence of insulin and forskolin, respectively (Fig. 1A). These values corresponded to approximately 0.3 sequences per SC. PR mRNA content was very low in the three SC lines (Fig. 1B).
In all cells, no significant AR mRNA was detected either by TaqMan or by classical methodology (not shown). Extremely low concentrations of ER mRNA were measured (around 25 sequences/ng in the absence of insulin and forskolin and 15 sequences/ng in their presence). ERß mRNA levels were still lower (<10 sequences/ng; not shown). The same results were obtained with cell lines.
Although some differences in PR and MR mRNA contents were observed between SC and cell lines, we performed complementary steroid binding studies using cytosols from SC lines. In addition, to check for the synthesis of efficient receptors, we investigated the transcriptional responses of SC exposed to different steroid treatments.
Significant high-affinity glucocorticosteroid and low mineralocorticoid binding activities but no significant sex steroid binding in SC lines
Binding studies were performed on cytosol extracts and thus concerned the intracellular content of nuclear steroid receptors. Cytosol preparation requires the availability of sufficient cell material; therefore, steroid binding activities were studied in cell lines instead of SC. Determination of the GR binding in CR3a1, CR1b4, and MSC80 cells was performed after cytosol incubation with 20 nM [3H]TA with or without an excess of cold RU28362. This last glucocorticosteroid agonist was used rather than TA because it displayed no cross-reactivity with other steroid receptors. The mean concentration of steroid binding sites ranged from 630–890 fmol/mg cytosol protein for the three cell lines. The number of GR binding sites per cell was calculated from the cell numbers taken for each cytosol preparation. They were 11,000 ± 3,280 (mean ± SEM; n = 4) for MSC80, 5370 ± 1345 (mean ± SEM; n = 3) for CR3a1, and 4340 ± 1263 (mean ± SEM; n = 3) for CR1b4. Titration curves (as shown for CR3a1 in Fig. 2A, curve with filled circles) gave a KD value in the nanomolar range with a very low nonspecific background (slope = 0.0027, curve with open circles).
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, curves with filled and empty squares, respectively). Both these facts led to increased variability of the measurements. In addition, a pseudo binding was observed with buffer alone (not shown). For these reasons, no clear-cut conclusion whether PR protein was present in any significant amount could be drawn directly from these binding measurements.
The specific binding of Aldo was measured in the presence of the glucocorticoid RU28362. This steroid, which does not bind to MR, was introduced to saturate GR binding sites, thus eliminating any Aldo binding to GR. MR binding activity was very low but significant in the three cell lines studied (10–15 fmol/mg cytosol protein) as shown for MSC80 cytosols (Fig. 2B). These values were precise because the nonspecific slope was very low (0.00125) and [3H]Aldo did not generate a mock titration curve as observed for the progestin R5020.
No specific binding to ER was observed with CR3a1 and CR1b4 cell cytosols (Fig. 3A). Exploitation of estradiol binding experiments with MSC80 extracts (Fig. 3B) led to detection of a binding component with a low capacity (40 fmol/mg cytosol protein and moderate affinity Kd of 7 nM) above an important nonspecific binding (slope 0.0125). No mock titration was detected with estradiol, but an increased variability of the measurements was observed at high steroid concentrations that impeded correct estimation of the plateau level. No specific binding to AR was observed in the three cell lines studied (not shown).
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Glucocorticosteroids, but not the other steroids, activate the transcription of reporter genes driven by synthetic promoters containing their corresponding responsive elements
Transient transfection experiments using reporter plasmids containing steroid-responsive promoters were performed to test the functional state of the various steroid receptors in cultured SC isolated from sciatic nerves. Cells were transfected using Amaxa technology with the reporter plasmid MMTV-luciferase, which can be activated by GR, MR, PR, and AR. As shown in Fig. 4, only glucocorticosteroids increased the transcriptional activity of reporter plasmid. All the other steroids, mineralocorticosteroids (Aldo), progestagens (R5020), and androgens (R1881) did not activate transcription. Moreover, the transcriptional activity of glucocorticosteroid-stimulated GR and the basal activity were significantly higher when cells were cultured in proliferation medium (Fig. 4, filled bars).
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). Similar results were obtained with lower steroid concentrations (10–10 M Aldo, 10–8 M R5020, and 10–8 M R1881; not shown). Exogenous GR expression was accompanied by increased transcriptional response to RU28362 (10–6 and 10–8 M). These results indicate that exogenous receptors were active and that the transcriptional machinery necessary for the steroid response was functional but that the endogenous contents of PR, MR, and AR were too low to sustain the transcription from exogenous promoters in SC. All transcriptional responses in the presence of exogenous receptor were significantly higher when cells were proliferating (compare Fig. 5, A and B), and this was unrelated to a difference in transfection efficiency.
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MTV-CAT to analyze the estrogen (10–7 M E2) response in SC. As above, no endogenous activity was recorded, but clear transcriptional activity was measured in the presence of E2 after cotransfection of the ER expression plasmid (Fig. 6). Insulin and forskolin moderately increased transcription mediated by exogenous ER. Surprisingly, even after 9 d of phenol red depletion, exogenous ER exhibited E2-independent activity (30–50% in SC and 80% in cell lines).
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, investigated by transient transfection of reporter plasmids, consistent with very low expression of these receptors. That Aldo failed to activate the transcription of the reporter gene is in contradiction with the observation that SC contain significant amounts of MR mRNA and that low binding was observed in cell lines. Therefore, to investigate a potential MR function that requires Aldo to access MR in the presence of glucocorticosteroid excess, we analyzed the activity of HSD2, an enzyme that inactivates glucocorticosteroids and has been found in tissues expressing both corticoid receptors and able to respond to mineralocorticosteroid via the MR (41).
High levels of HSD2 activity in quiescent SC but a strong decrease in SC cultured in proliferation medium
We checked for the expression and activity of the HSD2 protein by determining the rates of transformation of [3H]CS into [3H]11ß-dehydrocorticosterone. Figure 7A shows that quiescent SC displayed the higher conversion efficiency (28 pmol/h·mg protein) compared with proliferating SC. This activity fell to no more than 1 pmol/h·mg protein in the presence of insulin and forskolin. After 48 h withdrawal of insulin and forskolin, the concentration of cAMP was 1.9 pmol/106 SC, whereas it increased to 23 and 9.9 pmol/106 cells 30 min and 4 h after 10 µM forskolin introduction, respectively. CR3a1 exhibited lower rates of conversion (0.14 pmol/h·mg protein) than proliferative SC (0.4 pmol/h·mg in the same experiment), whereas CR1b4 and MSC80 activities were extremely low (0.028 and 0.012 pmol/h·mg protein) (Fig. 7B). The importance of the changes in HSD2 activity in SC prompted us to investigate the levels of HSD2 RNAs under both culture conditions and in SC lines to check for the occurrence of this regulation at the mRNA level.
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), corresponding to a HSD2/HSD1 ratio close to 250. The accumulation of HSD2 in SC (normalized with GAPDH) was maximal in the absence of insulin and forskolin and decreased by a factor of approximately 9 in their presence (Fig. 8B). In additional experiments, if both insulin and forskolin were removed from cell cultures for 24 h and then only forskolin reintroduced, it was found that HSD2 mRNA decreased in a time-dependent manner (Fig. 8C). In cell lines, HSD2 levels were always low. HSD1 mRNA exhibited variable levels according to cell line and SC culture conditions, but its concentration was always 10- to 100-fold lower than that of HSD2 mRNA. These data demonstrate that HSD2 is the main HSD isoform expressed in SC, that the intracellular cAMP level is involved in HSD2 regulation, and that this regulation occurs at the mRNA level (synthesis and/or stability).
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Discussion |
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No sex steroid receptor activity in SC
In our experimental conditions, purified SC did not express significant levels of functional sex steroid receptors, as shown by mRNA concentrations, transcriptional activities, and binding studies in SC lines. Demonstration that the receptors were limiting factors was provided by the positive transcriptional responses obtained when exogenous receptors were transiently expressed.
Our results showing that AR mRNA was barely detectable and that protein functions were lacking in SC agree with two previous studies concluding that AR expression is absent from SC (11, 43). The hypothesis of a PR-mediated androgen effect (11) is not in agreement with the absence of functional PR.
For ER and ERß, our data with SC indicate the absence of expression in our culture conditions. No binding was found in CR3a1 and CR1b4 in concordance with the very low level of mRNA and lack of transcriptional activity, whereas in the MSC80 cell line, a binding component, which differed from ER, was found (lack of mRNA and of transcriptional activity and affinity lower than usually reported for ER). Furthermore, in SC, exogenous ER displayed ligand-independent activity (44, 45).
Our present results indicating that the low level of PR protein, if any, is not sufficient to trigger biological activity agree with the study by Chan et al. (16), who did not detect significant PR expression in SC cultured alone or cocultured with DRG neurons and who proposed the hypothesis of P action via the PR expressed in the neighboring neurons. The discrepancy with previous observations in SC may result from the difficulty to precisely determine very low concentrations using methods with restricted sensitivity and with small-sized biological samples. Even with the availability of more sensitive methods, i.e. RT-PCR, we cannot clearly conclude that there is PR gene expression in purified Schwann cells.
Moreover, several reports indicate that P may act via nonsteroid receptor pathways. The reported P effect on the myelin protein P0 promoter and on one of the two PMP22 promoters devoid of a known glucocorticoid-responsive element/P-responsive element and the agonistic effect of RU486 in SC suggest the intervention of nonclassical effects (8). In addition, the effect of P derivative tetrahydroprogesterone on PMP22 expression, unable to bind PR, may involve membrane receptors such as the GABAA receptor (11). More recently, data on rapid and transient induction in SC of several transcription factors were reported, which required further investigation (46, 47). Recently described nonclassical progestin receptors (48, 49, 50), some of which have a membrane location and are detected in the nervous system (51), may also be involved in P responsiveness.
Finally, it can be hypothesized that stimuli such as diffusible factors (steroids, neurotrophins, cytokines, and neuromediators) or direct contacts with axons that occur in the in vivo environment may influence the expression of sex steroid receptors in SC. Nevertheless, studies performed with cocultured SC and DRG neurons by Chan et al. (16) did not provide evidence for such an effect with respect to PR. Current work is in progress to analyze receptor mRNA contents of the nerve.
Glucocorticosteroid receptor expression and activity in purified SC cultures
The presence of functional GR in SC and SC lines agrees with the large distribution of GR in all cells of higher organisms. Its level of expression in SC is similar to that reported in specialized cells such as hepatocytes or lymphocytes (52, 53). Lower binding capacities have been reported in total brain (52) or brain subregions (54) and even in astrocyte cultures (55).
A better transcriptional efficiency was observed for endogenous GR in the presence of insulin and forskolin. The stimulatory effect of cAMP on GR-mediated transcription has previously been reported (56). Because the same observation was made with other transiently expressed steroid receptors, this increased efficiency could result from cell physiological context such as modulation of signaling pathways (25, 56) or expression of a given set of transcriptional cofactors (57, 58, 59).
Glucocorticosteroids display complex activities on the nervous system either deleterious or favorable, depending on the type of cell (neuron or glia), their state of development, the physiological process studied, and the dose of steroid present. In cultured SC, glucocorticosteroids may enhance the potency of mitogens, probably via a GR-mediated effect (10). GR was also present in astrocytes, oligodendrocytes, and diverse neuronal populations of the CNS (60, 61, 62), where glucocorticosteroids generally exert antiproliferative and differentiating effects as shown on neonatal external granule cells of the cerebellum and on oligodendrocytes (63, 64).
The effects of glucocorticosteroids on myelination also differ according to the cell type. Chari et al. (64) stressed, in their model of experimental demyelination of the spinal cord, that remyelination via the resident oligodendrocytes was delayed by glucocorticosteroids, although SC-mediated repair was almost unaffected. Accordingly, in SC, glucocorticosteroids were shown to mediate an increase in the rate of myelin formation (7) and in the activity of P0 and PMP22 promoters (9). Few data are available on other glucocorticosteroid target genes. In the CNS, the effects of glucocorticosteroids have been demonstrated on glycerol phosphate dehydrogenase (65), glutamine synthetase (66), and some growth factors (67, 68). In the PNS, glutamine synthetase also appears to be positively regulated (our unpublished results in cultured SC).
Finally, the influence of glucocorticosteroids on neurodegeneration, neuroprotective, and regenerative processes seems to be largely dependent on their concentration (69, 70, 71), indicating the importance of both the GR/MR equilibrium (69) and enzymatic regulation of glucocorticosteroid levels (via HSD2) in cells expressing both corticosteroid receptors.
Low levels of mineralocorticosteroid receptor
This report is the first to deal with the expression of MR in SC and related cell lines. We found that MR mRNA was present in SC primary cultures and CR3a1 cells at levels representing 5–10% of the GR content but higher levels for other cell lines (30% in CR1b4 and 60% in Msc80 cells). The proportion of MR relative to GR mRNAs is similar to that reported in CNS astrocytes (72) and most brain subregions (57, 73). Binding studies in cell lines showed that the concentration of MR was low (240–450 sites per cell), corresponding to approximately 1/25 the observed GR site number. Such a concentration of MR binding sites in SC was lower than reported for astrocyte cultures (55) and most specialized brain subregions (54). In addition, no transcriptional activity could be detected in cultured SC. However, low levels of binding sites and low biological activity of endogenous MR have also been reported for well-known Aldo target cells such as renal cortical collecting ducts (CCD) (cell lines and primary cultures) (74, 75, 76, 77), and thus they do not necessarily imply the in vivo absence of a functional receptor.
In the myelinating SC, the expression, localization, and clustering of various types of ion channels near the node of Ranvier and in specialized junctions of paranodal loops show analogies with polarized Aldo target cells such as renal CCD or distal colon (78, 79). In these cells, MR-mediated genomic and nongenomic effects have been characterized. Na+/K+ ATPase activity, which is regulated by mineralocorticosteroids in both kidney cells and in the hippocampus (80, 81), is expressed in the nerve and undergoes regulation of subunit expression upon lesion (82, 83). Therefore, a possible switch in MR transcriptional activity in SC, resulting from extracellular signals and axonal contact and acting on MR mRNA content, translational efficiency, or protein stability should be investigated.
In the CNS, the posttranscriptional mechanisms have been shown to differentially regulate the levels of GR and MR in different brain subregions and during development and in response to stress (54, 73, 84, 85). Furthermore, a recent report indicates that MR expression is increased after injury in rat hippocampus neurons both in primary cultures and in vivo and that an increased MR expression is correlated with neuronal survival (86). Such a role of MR and identification of its target genes remain to be explored in SC.
Regulation of HSD2 expression and activity in primary SC cells indicates a local inactivation of glucocorticoids when intracellular cAMP levels are low
Regulation of GR activity and GR/MR equilibrium could occur at a prereceptor level by local inactivation of glucocorticosteroids by HSD2 (80, 87). HSD2 activity in quiescent SC was similar to that measured in CCD and osteoblastic cell lines, only six times lower than the activity recorded in rat primary CCD cells (76, 88) and remarkably down-regulated by cAMP. It should be noticed that after a peripheral nerve injury, intracellular cAMP levels are strongly decreased (to approximately one tenth of the level observed in the intact nerve) as a consequence of a simultaneous decrease in adenylyl cyclase and an increase in phosphodiesterase activity (89, 90). Such a cAMP starvation should induce strong HSD2 activity, leading to a decrease in active glucocorticosteroid concentration and reduction in GR transcriptional activity. Indeed, our results for the regenerating nerve after cryolesion show that HSD2 expression is increased (unpublished results).
Therefore, the following scenario can be advanced. After a peripheral nerve injury, the levels of glucocorticosteroids should increase in the circulation. A high HSD2 activity would limit their intracellular accumulation in the lesioned nerve and their potential deleterious effect on cellular processes such as dedifferentiation and proliferation (63, 64) during the first period of regeneration. Because glucocorticosteroids have been shown to enhance myelin protein synthesis by Schwann cells (7, 9), they would have a beneficial effect during the following remyelination period at the time of axonal contact establishment, when HSD2 expression returned to basal levels.
Interestingly, in the adult brain, the major reaction is the reactivation of inactive 11-ketoglucocorticoids driven by HSD1 (91). Thus, our data demonstrating the extent of HSD2 activity and its regulation by cAMP in SC may constitute a very important new aspect of nerve physiology. Further investigation of MR involvement and a possible functional balance between GR and MR, already described in the CNS (69, 70, 71), is required in the PNS, where data on steroid receptor expression during development, in the mature state, and during regeneration still have to be collected.
In summary, this work is the first to present a comprehensive study of corticosteroid and sex steroid receptors in SC at the level of expression (mRNA and protein) and activity. We clearly show the expression and function of GR on synthetic responsive genes, an observation that can be extended to several endogenous genes (work in progress) and the regulation of its intracellular activity by HSD2. A possible role of low levels of MR remains to be elucidated. Other steroid nuclear receptors (PR, ER and -ß, and AR) are poorly expressed in SC and are not expected to mediate the sex steroid effects. Because steroids such as P and CS and their metabolites may regulate GR, MR, and HSD2 activity (92, 93, 94), we are now investigating how steroid biosynthetic and metabolic enzymes as well as the steroids produced locally or accumulated from the periphery could influence the function of corticosteroid receptors after peripheral nerve injury and during the regeneration process.
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Acknowledgments |
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MTV-CAT, Didier Picard for rat GR-PC7, Stanley Watson for MR cDNA sequence, Marie-Edith Rafestin-Oblin for introduction of MR sequences in pcDNA3, Benita Katzenellenbogen for pratPR6B and Stephane Savary for introduction of PR sequence in pcDNA3.1/Zeo, Jorma Palvimo for pSG5-rAR, Elisabeth Wilson for pCMVratAR and pGEMrAR1, Catherine Pasqualini for rat ERpEGFP plasmid, and HMR (now Aventis) for providing us with RU28486 and RU28362. CR3a1 and CR1b4 cells were kindly given by Julien Ghislain and Patrick Charnay and MSC80 cells by Jean-Jacques Hauw. |
Footnotes |
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Disclosure statement: all authors have nothing to declare.
First Published Online June 8, 2006
Abbreviations: AR, Androgen receptor; CAT, chloramphenicol acetyl transferase; CCD, cortical collecting ducts; CNS, central nervous system; CS, corticosterone; Ct, cycle threshold; DHT, 5-dehydrotestosterone; DRG, dorsal root ganglia; E2, estradiol; ER, estrogen receptor; ERE, estrogen-responsive elements; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticosteroid receptor; HSD2, 11ß-hydroxysteroid-dehydrogenase type 2; MR, mineralocorticosteroid receptor; P, progesterone; PR, progesterone receptor; PNS, peripheral nervous system; SC, Schwann cells; TA, triamcinolone acetonide.
Received December 20, 2005.
Accepted for publication May 31, 2006.
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-aminobutyric acid type A receptor. Proc Natl Acad Sci USA 92:3769–3773,5-Tetrahydroprogesterone (allopregnanolone) and -aminobutyric acid: autocrine/paracrine interactions in the control of neonatal PSA-NCAM+ progenitor proliferation. J Neurosci Res 78:770–783[CrossRef][Medline]3-subunit mRNA expression in rat hippocampus. Am J Physiol 266:C423–C428This article has been cited by other articles:
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  C. Girard, S. Liu, F. Cadepond, D. Adams, C. Lacroix, M. Verleye, J.-M. Gillardin, E.-E. Baulieu, M. Schumacher, and G. Schweizer-Groyer Etifoxine improves peripheral nerve regeneration and functional recovery PNAS, December 23, 2008; 105(51): 20505 - 20510. [Abstract] [Full Text] [PDF]
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 M. Schumacher, R. Guennoun, A. Ghoumari, C. Massaad, F. Robert, M. El-Etr, Y. Akwa, K. Rajkowski, and E.-E. Baulieu Novel Perspectives for Progesterone in Hormone Replacement Therapy, with Special Reference to the Nervous System Endocr. Rev., June 1, 2007; 28(4): 387 - 439. [Abstract] [Full Text] [PDF]
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and 
, total binding) or with a 1000-fold molar excess of the cold competitor steroid (RU28362 for GR and R5020 for PR; 
and 
, respectively) corresponding to nonspecific binding. Binding parameters for GR receptor (TA binding) were Bmax of 0.82 pmol/ml of cytosol and Kd = 0.4 nM. The slope of nonspecific binding was 0.0027. For PR (R5020 binding), the binding parameters obtained were Bmax of 0.130 pmol/ml of cytosol and Kd =7 nM with a nonspecific binding slope of 0.0086. For MR (Aldo binding), aliquots of cytosol (3.4 mg protein/ml) of MSC80 were incubated with increasing concentrations of [3H]Aldo (
