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Departments of Internal Medicine (H.R., P.S., E.L.T.v.d.A., E.F.C.T.v.R., F.H.d.J., S.W.J.L., J.W.K.) and Reproduction and Development (A.O.B), Erasmus MC, University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Henk Russcher, Erasmus Medical Center, Department of Internal Medicine, Room Ee593, Dr. Molewaterplein 40, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: h.russcher{at}erasmusmc.nl.
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Abstract |
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Objective and Design: We examined the basis of this altered GC sensitivity by expressing GR(N363S) and GR(ER22/23EK) in COS-1 cells and investigating their transactivating and transrepressing capacities using a GC response element-luciferase reporter and a p65-activated nuclear factor 
B-luciferase reporter, respectively. Furthermore, we evaluated the transactivating and transrepressing capacities of the GR in peripheral blood mononuclear lymphocytes of homozygous and heterozygous carriers of these polymorphisms by determining the maximum effect of dexamethasone on transactivation of the GC-induced leucine-zipper and transinhibition of the IL-2 gene by means of real-time RT-PCR.
Results: The effects of the polymorphisms in the GR gene previously observed in population studies were also detected at the level of gene expression. The ER22/23EK polymorphism resulted in a significant reduction of transactivating capacity, in both transfection experiments (–14 ± 5%, P < 0.05) and peripheral blood mononuclear lymphocytes of carriers of this polymorphism (homozygous: –48 ± 6%, P < 0.01, n = 1; heterozygous: –21 ± 4%, P = 0.08, n = 3). The N363S polymorphism, associated with increased GC sensitivity, resulted in a significantly increased transactivating capacity, both in vitro (8 ± 3%; P < 0.02) and ex vivo (homozygous: 204 ± 19%, P < 0.0001, n = 1; heterozygous: 124 ± 8%, P = 0.05, n = 3). Neither the ER22/23EK nor the N363S polymorphism seemed to influence the transrepressing capacity of the GR.
Conclusion: The presence of these and other GC sensitivity-modulating polymorphisms may have consequences for the use of GCs in a clinical setting.
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Introduction |
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Within the healthy population, considerable interindividual variation in GC sensitivity exists, as is demonstrated by a variable suppressive response to 0.25 mg dexamethasone (DEX) (3). This implies that each subject, when treated with GCs, needs an individually optimized dose to maintain a balance between beneficial and adverse effects (e.g. diabetes mellitus, peptic ulcer, osteoporosis, skin atrophy, psychosis, glaucoma, and many others) associated with GC treatment (4). Within an individual person, however, GC sensitivity is rather stable (3), and the response to cortisol is correlated with that of several other corticosteroids (5). This implies that a set point for GC sensitivity with respect to the feedback system exists, which might be genetically determined.
One of the genetic factors involved is the occurrence of polymorphisms, generally defined as common variations at the DNA level with a frequency of more than 1% in the normal population. GC action is mediated by the GC receptor (GR). Two polymorphisms in the open reading frame of the GR gene have been described to be associated with altered sensitivity to GCs and may contribute to the interindividual differences (6, 7).
The most intriguing polymorphism is ER22/23EK present in exon 2, consisting of two linked single nucleotide mutations in codons 22 and 23: GAG AGG (GluArg) 
GAA AAG (GluLys) (rs6189 and rs6190) (8). This polymorphism reduces sensitivity to GCs and results in a phenotype that can be summarized as a more favorable metabolic profile, resulting in an increased survival rate for carriers of the ER22/23EK polymorphism (7, 9, 10). The polymorphism probably alters the secondary structure of the mRNA of the GR, resulting in a higher expression of the GR-A (94 kDa) at the expense of the GR-B (91 kDa) isoform, of which the latter has more transactivating capacity. The shift in GR-A to GR-B expression ratio leads to an overall decrease in transcriptional activity (11).
Further downstream in exon 2, a polymorphism was identified that changes codon 363 from AAT to AGT (rs6195), resulting in a serine for asparagine substitution (8). This polymorphism increases sensitivity to GCs, whereas an increased insulin response to DEX and a tendency toward lower bone mineral density have also been observed. Some studies also found an association with increased body mass index (12, 13), but others did not (14, 15). The molecular mechanism through which the N363S polymorphism exerts its effects is unknown. It has been postulated that the polymorphism contributes a new serine residue for phosphorylation, whereby protein interactions with transcription cofactors might be altered (16).
In this study, we set out to establish the effects of these polymorphisms in functional bioassays. Previous reports showed that transient transfection assays by calcium phosphate precipitation did not show significant differences in activation or repression of gene expression driven from various promoters (17, 18). However, improvement in transfection methods (e.g. cationic liposome-mediated transfection) and possibilities to correct for transfection efficiency (e.g. renilla luciferase) encouraged us to reinvestigate the transactivating and transrepressing capacities of GR(ER22/23EK) and GR(N363S) from a GC response element (GRE)-driven or a p65-activated nuclear factor-B (NF-B) luciferase reporter, respectively.
In addition, we investigated the effects of these polymorphisms on the regulation of two GC-sensitive genes: the GC-induced leucine-zipper (GILZ), which is up-regulated (19, 20), and IL-2, which is down-regulated by GCs (21, 22, 23).
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Materials and Methods |
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Dexamethasone was purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Oligonucleotide primers for mutagenesis and quantitative PCR (Q-PCR) were synthesized by Biosource Europe S.A. (Nivelles, Belgium).
The pcDNA3 and pRL-cytomegalovirus (CMV) vectors were purchased from Invitrogen (Breda, The Netherlands) and Promega (Leiden, The Netherlands), respectively. The pRShGR
expression plasmid, the GRE-luciferase (LUC) and NF-B-LUC reporter plasmid, p65 plasmid, and pTZ plasmid were described previously (17, 24).
Peripheral blood from three heterozygous and one homozygous ER22/23EK carriers and three heterozygous and one homozygous N363S carriers was used to study the transactivating and transrepressing capacities of the GR variants. Peripheral blood of 10 volunteers, all noncarriers of both polymorphisms, was used as control material. All subjects were healthy, and none were using exogenous GCs. From all subjects, informed consent was obtained and the Medical Ethics Committee of Erasmus MC, The Netherlands, approved this study.
Plasmid construction
pcDNA3hGR was generated by digesting pRShGR with KpnI and XhoI and cloning the resulting fragment into the KpnI and XhoI sites of pcDNA3. The ER22/23EK and N363S polymorphisms were introduced independently into pcDNA3hGR by using a QuickChange site-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands) according the manufacturer’s guidelines.
Cell culture
Monkey kidney (COS-1) cells were maintained in a 5% CO2 humidified incubator at 37 C in DMEM tissue culture medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/liter penicillin, 100 mg/liter streptomycin, and 1.25 mg/liter Fungizone and passaged every 3–4 d.
Transfections
For transcription regulation studies and Western immunoblot analysis, COS-1 cells (6.0 x 105/ml) were plated at 3.0 x 105 cells/well (2.8 cm2) and grown for 24 h. Cells were transfected using FuGENE6 reagent (Roche Diagnostics, Almere, The Netherlands). Per well, 0.7 µl of reagent was diluted in 100 µl serum-free medium and mixed with 215 ng plasmid DNA. For the GRE-LUC measurements, this pool of plasmid DNA contained the indicated human (h)GR expression plasmids (7.5 ng), GRE-LUC reporter (50 ng), CMV-renilla expression (2 ng/well), and pTZ carrier plasmid and for the NF-B-LUC measurements, the indicated hGR expression plasmid (4.0 ng), NF-B-LUC reporter (50 ng), p65 expression (10 ng), CMV-renilla expression (2 ng), and pTZ carrier plasmid. After an incubation period of 30 min at room temperature, the mixture was added to the cells. Cells were subsequently returned to the incubator until the reporter luciferase assay or Western immunoblot analysis.
Reporter luciferase assay
Five hours after transfection, the indicated concentrations of DEX were added. Twenty hours later, cells were lysed in 100 µl lysis buffer [25 mM trisphosphate (pH 7.8), 15% glycerol, 1% Triton X-100, 1 mM dithiothreitol, and 8 mM MgCl2]. Luciferase activity was measured in 25 µl in a TOPCOUNT luminometer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), using the Dual-Glo luciferase assay system (Promega). By using the Stop&Glo reagents, luminescence was also measured from the pCMV-renilla expression plasmid to correct for transfection efficiency.
Western immunoblot analysis
Cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 0.1% sodium dodecyl sulfate, 150 mM NaCl, 5 mM dithiothreitol, and protease inhibitors (Complete, Roche Diagnostics). After a 10-min high-speed (100,000 rpm) spin to remove cellular debris, protein concentrations were determined by a Bradford assay. Equal amounts (30 µg) of protein were separated on SDS-PAGE (8%) (25) and transferred to nitrocellulose membranes. The membranes were washed in TBS-T (Tris-buffered saline with 0.1% Tween 20) and blocked in TBS-T with 5% nonfatty milk powder for 1 h at room temperature. Blots were incubated overnight at 4 C in block buffer, supplemented with the anti-hGR 57 (1:2500) (10P’s, Breda, The Netherlands) or anti-hGR (P-20) (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). Thereafter, blots were washed in TBS-T and subsequently incubated with horseradish peroxidase goat antirabbit IgG secondary antibody (1:10,000) (DakoCytomation, Glostrup, Denmark) for 2 h at room temperature. After washing the blots in TBS-T, the proteins were visualized by enhanced chemoluminescence (Amersham Pharmacia Biotech, Roosendaal, The Netherlands).
Blood cell preparations
Peripheral blood was collected by venepuncture in heparinized tubes, and peripheral blood mononuclear lymphocytes (PBMLs) were obtained after density centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden) as previously described (26). PBMLs were resuspended in RPMI 1640 medium with L-glutamine (300 mg/liter) (Invitrogen) supplemented with 100 U/liter penicillin, 100 mg/liter streptomycin, and 10% fetal bovine serum. Cells were incubated for 1 h at 37 C in a shaking water bath to remove endogenous cortisol. Afterward medium was replaced, and 2 x 106 cells/well were precultured overnight (5% CO2, 37 C) in a 48-well plate at a density of 4 x 106 cells/ml. The next day PBMLs were incubated for 4 h with increasing DEX concentrations together with 10 µg/ml phytohemagglutinin (PHA).
RNA isolation, RT reaction, and Q-PCR
PBMLs were washed with 0.15 M NaCl, and total RNA was isolated using a high pure RNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol.
cDNA was synthesized in a reverse transcription reaction with Taqman reverse transcriptase reagents (Applied Biosystems) as described previously (5).
GILZ, IL-2, and GR mRNA expression levels were determined in a Q-PCR, by using primers and probes (Biosource International, Camarillo, CA), that were designed by using the Primer Express software (Applied Biosystems, Foster City, CA). Correction for assay variability was performed using the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) of which expression levels are stable and not influenced by GCs in this cell system (data not shown). The primer sequences used are presented in Table 1
. The reaction to determine GILZ, IL-2, and HPRT mRNA expression levels, with a total volume of 25 µl, contained 2.5 µl cDNA template (corresponding to 10 ng total RNA in the RT-PCR), 12.5 µl universal master mix (Roche, Branchburg, NJ), 0.3 pmol/µl forward and reverse primers (0.5 pmol/µl for HPRT), and 0.1 pmol/µl probe (0.2 pmol/µl for HPRT), whereas the reaction to determine GR levels contained 2.5 µl cDNA template, 7.5 pmol/µl of each primer, 5 pmol/µl probe in a Q-PCR-core kit (Eurogentec, Liege, Belgium). Standard PCR conditions, as supplied by the manufacturer, were used for analysis on an ABI 7700 sequence detector system (Applied Biosystems). The expression levels of GILZ, IL-2, GR, and HPRT were calculated using the comparative threshold method, according to the manufacturer’s guidelines.
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Data were analyzed statistically using Instat software version 2.01 (GraphPad Software, Inc., San Diego, CA). The differences in transcriptional activity of GR variants measured in vitro were determined using one-way ANOVA. Bonferroni post hoc tests were used to test for differences between each GR variant and to correct for multiple comparisons. The differences in total response of DEX-induced up- and down-regulation of GILZ and IL-2 mRNA levels in carriers of the indicated GR polymorphisms, compared with noncarriers, were analyzed by the Student’s t test using the area under the curve. Data were expressed as mean ± SEM. P < 0.05 was considered as significant.
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Results |
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The GR(ER22/23EK) and GR(N363S) variants were expressed in COS-1 cells, a system known to be devoid of endogenous GR (17). To examine overall receptor expression, immunoblot analysis was performed with GR antibody 57, which is raised against amino acid 346–367 (27). Figure 1A shows that all GR constructs were expressed. The 94-kDa band is the full-length GR (amino acids 1–777), also called GR-A (Met-1), whereas the 91-kDa band represents the translation variant GR-B (Met-27). The 82-kDa band was thought to be a degradation product of GR-B (28); however, recently additional translation variants have been reported, and the 82-kDa band has been named GR-C (Met-86) (29). We have previously shown that the ER22/23EK polymorphism leads to a modest shift in translation in favor of GR-A over GR-B, but this difference is difficult to detect by semiquantitative Western blotting (11). Densitometric scanning indicates that the GR-B band of GR(ER22/23EK) is slightly weaker than the band of GR[wild type (WT)]; this difference, however, did not reach statistical significance. Because the epitope of GR antibody 57 contains codon 363, proper expression of GR(N363S) was further confirmed by using an antibody (GR antibody P20) directed against a C-terminal epitope (Fig. 1B).
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The expressed GR(ER22/23EK) and GR(N363S) proteins were activated by increasing amounts of DEX, whereafter their capacity to transactivate GRE-driven transcription was investigated. The maximal response of the GR(WT) to activate transcription was set to 100%, and Fig. 2A shows that the N363S polymorphism increased maximal transactivation by 8.0 ± 3% (P < 0.02), whereas a reduction of the maximal response by 14 ± 5% (P < 0.05) occurred when the ER22/23EK polymorphism was present. No significant differences in EC50 were found.
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B transcriptional repression by the GR variants
Transfection of the 5 x NF-B response element-LUC reporter gene in COS-1 cells established a basal luciferase activity that could not be repressed by GR(WT) in the presence of DEX (data not shown). After cotransfecting a plasmid expressing the p65-subunit, luciferase expression was increased 5-fold, which was maximally repressed to 57 ± 8% by GR(WT). (Fig. 2B
). Figure 2B also shows that the ER22/23EK and N363S receptor protein variants seemed to repress the p65-dimer activity to a similar extent, as did the wild type. However, the variability in these experiments was higher than in the transactivation experiments, and any effects might be obscured by noise.
Transactivating and transrepressing capacities in PBMLs of homozygous and heterozygous GR variant carriers
GILZ and IL-2 are GC-responsive genes in PBMLs. GILZ expression is strongly up-regulated by GCs, whereas IL-2 is down-regulated (5). PHA was necessary to induce transcription of the IL-2 gene but did not affect expression of GILZ or GR mRNA levels (data not shown). The effects of GILZ and IL-2 are GC specific because they can be abrogated by addition of RU38486 and are not evoked by other steroids (e.g. progesterone, estradiol, etc.) (5). The response to DEX in expression of these genes is therefore a measure for the transactivating and transrepressing capacities of the GR variants.
PBMLs from carriers of the polymorphisms or noncarrier controls were stimulated with PHA and the indicated doses of DEX. The changes in mRNA levels for GILZ and IL-2 (relative to the values in the absence of DEX) are shown in Fig. 3. No systematic differences between genotypes were observed for the values in the absence of DEX (data not shown).
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Overall expression levels of the GR were not significantly different between controls and heterozygous or homozygous carriers of the two polymorphisms as measured by [3H]DEX binding capacity of the cells [n (receptors/cell) = 5547 ± 991 in controls; n = 5844 ± 640 and 5213 in, respectively, hetero- and homozygous N363S carriers; n = 6258 ± 300 and 5785 in, respectively, hetero- and homozygous ER22/23EK carriers] and quantitation of the GR mRNA by real-time RT-PCR (data not shown). On the indicated DEX treatment, GR mRNA levels decreased with 21 ± 6% (P < 0.02), and this decrease was equal in PBMLs of controls, ER22/23EK, and N363S carriers (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-transactivating domain but is not within its core region, which is variably defined as amino acid 77–262 (33) or 98–305 (34). This study shows that the polymorphism influences the transactivating capacity of the GR: in transfected cells we found a reduced capacity for GRE-driven LUC activation (Fig. 2A), whereas in PBMLs from hetero- and homozygous carriers of this polymorphism treated with increasing concentrations of DEX, a significant reduction in the activation of GILZ transcription was observed (Fig. 3A). This was not mediated through differences in the regulation of the GR expression because the decrease in GR mRNA levels during the 4 h DEX treatment was not different from that in the controls. We could not detect differences in the transrepression of NF-B activity (Fig. 2), and although variability in these experiments was rather high, this result was supported in the experiments in PBMLs, in which transrepression of IL-2 was equal to that in the control group (Fig. 3D).
We have recently shown that the ER22/23EK polymorphism leads to a modest shift in translation in favor of the translation variant GR-A over GR-B (11), of which the latter had a stronger transactivating effect in transient transfection experiments (28). This possibly explains the decreased GC sensitivity in GR(ER22/23EK) carriers. GR-A and GR-B are equally potent in inhibiting NF-B activity (28), explaining the unchanged transrepression.
With respect to the N363S polymorphism, we found that it increased the transactivating capacity of the GR. In transfection experiments, the N363S polymorphism increased transcription from the GRE-LUC reporter (Fig. 2A), whereas in PBMLs, expression of GILZ mRNA was increased in cells from both heterozygous and homozygous carriers of this polymorphism (Fig. 3A). GR(N363S) down-regulation during DEX treatment was not different from that in controls.
The effects of the N363S polymorphism on transrepression are more difficult to interpret. This polymorphism did not significantly interfere with the NF-B-driven transcription of the LUC reporter gene (Fig. 2B) in transfection experiments, although the variability in these experiments was rather high, possibly obscuring any effects. However, the response of PBMLs from heterozygous carriers with respect to IL-2 transcription also did not differ from that of normal controls (Fig. 3C), but cells from the homozygous carrier showed a reduced response to DEX, suggesting a decreased sensitivity. The GR can interfere in at least two ways with IL-2 expression: by direct inhibition of the activator function of NF-B and up-regulation of inhibitory-B (21, 22, 23). We assume that, like in the transfection assay, direct inhibition of NF-B is not affected by the N363S polymorphism. Therefore, the observed effect on IL-2 may be due to GR-induced up-regulation of inhibitory-B. However, it is also possible that GILZ (35, 36) or other aspects of the signaling network are affected. Finally, it is possible that this effect is due to the homozygous presence of the polymorphism or rather to the absence of the wild-type allele. The results also differ from those previously observed in nine heterozygous carriers whose PBMLs were tested in a mitogen-stimulated proliferation assay (6). There we found a tendency to increased sensitivity (lower IC50 values) for the carriers. However, whereas IL-2 production certainly plays a role in that assay, it is carried out over a much longer time scale (4 d, rather than 4 h in the current assays), and the outcome is formed by the integration of many processes, including apoptosis.
The exact molecular mechanism underlying the increased sensitivity in N363S carriers is still unknown. The GR has been shown to be poly-phosphorylated on serine and threonine residues in the N-terminal domain of the protein (35, 36), and it has been suggested that the N363S variant introduces a new phosphorylation site, possibly altering interactions with other transcription factors (16). However, this serine residue is not in one of the presently known consensus phosphorylation sites (36).
The allele frequencies of the ER22/23EK (2.5%) and N363S (4.5%) polymorphisms are relatively low (30). In the (Caucasian) population that we studied, approximately one of 1000 subjects is homozygous for the ER22/23EK or N363S polymorphism (6, 8, 30). Therefore, the possibility to investigate the transactivating and transrepressing properties ex vivo is rather exceptional. This can partly be overcome by also performing transfection experiments. Clearly these assays show less sensitivity and more variation than the experiments with PBMLs from carriers. On the other hand, in the transfection experiments in COS cells, the polymorphisms are the only variable, whereas in PBMLs of our subjects, variables other than the GR polymorphisms might also influence GC-mediated gene regulation. Overall, both types of experiments showed similar results.
In conclusion, the ER22/23EK and N363S polymorphisms in the GR gene can partly explain interindividual variation in sensitivity to GCs. These polymorphisms alter sensitivity to GCs, which is mainly caused by alterations in the transactivating capacity of the receptor. In our population-based studies, carriers of the ER22/23EK or N363S polymorphisms showed impressive effects on cardiovascular and metabolic profiles (30). Recent studies suggested that GC excess in utero is linked to cardiovascular and metabolic diseases in adulthood due to changes in fetal programming of the hypothalamus-pituitary-adrenal axis (37, 38). This programming might also be influenced by the GR polymorphisms predisposing for the described cardiovascular and metabolic profiles. The relatively small direct effects of the polymorphisms reported in this study, to which carriers are indeed exposed for life, could add up to these programmed effects, together causing the dramatic phenotypes observed.
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Acknowledgments |
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Footnotes |
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First Published Online July 19, 2005
Abbreviations: CMV, Cytomegalovirus; DEX, dexamethasone; GC, glucocorticoid; GILZ, GC-induced leucine-zipper; GR, GC receptor; GRE, GC response element; h, human; HPRT, hypoxanthine phosphoribosyltransferase; LUC, luciferase; NF-B, nuclear factor-B; PBML, peripheral blood mononuclear lymphocyte; PHA, phytohemagglutinin; Q-PCR, quantitative PCR; TBS-T, Tris-buffered saline with Tween 20; WT, wild type.
Received March 24, 2005.
Accepted July 13, 2005.
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B or activator protein-1: molecular mechanisms for gene repression. Endocr Rev 24:488–522B mediated transcriptional activation: therapeutic potential in autoimmune diseases and structural diversity. Curr Med Chem 9:219–227[Medline]
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), GR(ER22/23EK) (•), and GR(N363S) (
). COS-1 cells were cotransfected with a GRE-LUC reporter construct (A) or a p65-activated NF-