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Original Article |
Department of Pediatric Endocrinology (H.V., B.I.H.-S., S.C.V.B.-O., M.J.), University Medical Center Utrecht, 3508 AB Utrecht; and Netherlands Institute of Developmental Biology (B.v.d.B.), 3584 CT Utrecht, The Netherlands
Address all correspondence and requests for reprints to: M. Jansen, Department of Pediatric Endocrinology, HP KC.03.063.0, Wilhelmina Children’s Hospital, University Medical Center Utrecht, P.O. Box 85090, 3508 AB, Utrecht, The Netherlands. E-mail: m.jansen{at}wkz.azu.nl.
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Abstract |
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Introduction |
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It is not feasible to investigate all steps in this complex signal transduction pathway in every patient. We, therefore, investigated the overall cellular response to GCs by evaluating changes in gene expression, with the objective to establish an assay for the determination of individual cellular GC sensitivity. Such an assay could help in tailoring GC treatment to the individual needs of the patient, thus minimizing GC-induced side effects. Although any gene responding to GCs might be suitable for such an assay, there are two prerequisites: First, the gene must be expressed in lymphocytes because these are the only cells that can be easily obtained by the physician; second, its expression should be unambiguously influenced by GCs. The gene encoding the 51-kDa FK506-binding protein (FKBP51) (16), which forms part of the Hsp90 steroid receptor complex, seems to meet both requirements. As reported by Baughman et al. (17), this gene is expressed in native lymphocytes and induced by GCs in the T cell line C7TK.4.
Our results show that FKBP51 mRNA strikingly increases upon GC administration in the human lymphoblastoid cell line IM-9 and native lymphocytes both ex vivo and in vivo. We have also compared the technique of real-time PCR to Northern blot analysis. Not only is an analysis using real-time PCR less time consuming, but, more importantly, it requires smaller amounts of mRNA, compared with Northern blot analysis. We thus sought to minimize the amount of blood cells required for analysis and, preserving the reliability of the determination, to make this assay applicable to small children, including premature infants.
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Materials and Methods |
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The human lymphoblastoid cell line IM-9 (no. CCL-159; American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640, supplemented with 4 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS) (Invitrogen Corp., Paisley, UK).
To compare Northern blot analysis and real-time PCR, IM-9 cells were diluted to a concentration of 0.5 x 106 cells/ml in culture medium supplemented with 10% steroid-stripped FCS (dextran-coated charcoal pretreated FCS), and aliquotted in 10 ml. In other experiments, using real-time PCR, IM-9 cells were diluted to a concentration of 0.2 x 106 cells/ml and aliquotted in 0.5 ml. Native lymphocytes were isolated using a ficoll-gradient (Amersham Biosciences, Uppsala, Sweden), diluted to 0.5 x 106 cells/ml and aliquotted in 1 ml. Following an overnight preincubation in steroid-stripped medium to remove all endogenous steroids and thus abolish any effect of GCs present in nonstripped FCS, different GCs [hydrocortisone sodium succinate (Solu-Cortef, Pharmacia & Upjohn, Peapack, NJ), dexamethasone (Sigma-Aldrich Chemie, Steinheim, Germany), dexamethasone disodium phosphate (dexamethasone-Na2P) (Decadrone, Merck Sharp \|[amp ]\| Dohme, Hoddesdon, UK), and budesonide (Pulmicort, AstraZeneca, Wilmington, DE)], the GC receptor antagonist ORG 34116 (a kind gift from Dr. M. E. de Gooyer, Organon, Oss, The Netherlands), and cycloheximide (Sigma-Aldrich Chemie) were added to the culture medium at the described concentrations and time points before isolation. In each experiment, cells were harvested simultaneously.
Total RNA was isolated using TriPure reagent according to the manufacturer’s protocol (Roche Applied Science, Mannheim, Germany).
Northern blot analysis
Northern blot analysis was essentially performed as described in Sambrook et al. (18). Briefly, total RNA (20 µg) prepared from both untreated and GC-treated IM-9 cells was size separated using gel electrophoresis on 1% agarose gels containing 6.7% formaldehyde (Merck, Darmstadt, Germany), 0.2 M 3-(N-Morpholino)propanesulfonic acid (Sigma-Aldrich Chemie), 0.01 M ethylenediaminetetra-acetic acid (Sigma-Aldrich Chemie), and 0.05 M Na-acetate (Merck). RNA was transferred to Hybond N+ membranes (Amersham Biosciences) using a vacuum blotter and UV cross-linked to the membranes.
A 459-bp FKBP51 fragment (19) was used as probe; the fragment was amplified and purified after which 25 ng was labeled with 
32P dCTP using Rediprime labeling kit, according to the manufacturer’s protocol (Amersham Biosciences). Membranes were hybridized overnight at 65 C. Increasingly stringent posthybridization washes (up to 0.1x saline sodium citrate, 0.1% SDS) were performed at 65 C to remove aspecific hybridized probe. The membranes were exposed to Biomax MR films (Eastman Kodak Co., Rochester, NY) to obtain autoradiograms and phosphor-imaging screens (Bio-Rad Laboratories, Inc., Veenendal, The Netherlands) to quantify the signals. The obtained data were processed using Molecular Analyst software (Bio-Rad Laboratories, Inc.). For normalization of the FKBP51 signals, the membranes were hybridized with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe.
Reverse transcription and real-time PCR
Total RNA was reverse transcribed using MMLV-RT RNase H Minus, Point Mutant (Promega Corp., Madison, WI), according to the manufacturer’s protocol. The synthesized cDNA was subsequently diluted 40 times in ddH2O for use in the real-time PCR. Real-time PCR experiments were performed with the LightCycler device (Roche Applied Science), using the DNA Master SYBR-green I kit (Roche Applied Science). Primers used were 5'-AAAAGGCCAAGGAGCACAAC-3' and 5'-TTGAGGAGGGGCCGAGTTC-3' for the FKBP51 PCR (TIB-Molbiol, Berlin, Germany) and 5'-CCAGCAGAGAATGGAAAGTC-3' and 5'-GATGCTGCTTACATGTCTCG-3' for the ß2-microglobulin (ß2m) PCR (20). The reaction mixture for the real-time PCR contained 5.0 µl cDNA solution, 0.5 pmol/µl each of two primers, 4 mM MgCl2 in the FKBP51 PCR or 5 mM MgCl2 in the ß2m PCR, and 10% DNA master SYBR-green I solution (Roche Applied Science). The mixture was denatured (30 sec, 95 C) and subjected to up to 40 amplification cycles (FKBP51 PCR: 15 sec, 95 C; 15 sec, 67 C; 30 sec, 72 C; and 1 sec, 82 C with a single measurement of fluorescence and ß2m PCR: 30 sec, 95 C; 10 sec, 56 C; 30 sec, 72 C; and 1 sec, 82 C with a single measurement of fluorescence).
Real-time PCR data analysis
The dye SYBR-green interacts with double-strand DNA (dsDNA), increasing its inherent fluorescence. Therefore, the fluorescence intensity is indicative for the amount of dsDNA amplified in the consecutive rounds of PCR. The LightCycler measures fluorescence intensity in each sample in each round of amplification. The crossing point is the number of PCR cycles after which the fluorescence signal in a sample rises above the background signal. This number was used to relate the initial quantity of cDNA of interest in each sample to one another (21). FKBP51 PCR results were normalized using ß2m PCR data, derived from the same cDNA samples. To verify the specificity of the amplified products, each PCR was followed by a melting curve analysis. In this analysis, samples are subjected to a gradual temperature increment from 65 C to 95 C while monitoring the fluorescence of each sample. As the dsDNA products melt, a decrease in fluorescence intensity is measured, which should be sudden, in view of the expected homogeneity of the product. The rare PCR samples that contained any byproducts were excluded.
Statistics
Data are presented as the mean of three independent experiments ± SEM. Differences between nontreated control samples and samples treated with GC are calculated using the one-way ANOVA with the Dunnet posttest. Differences between GC-treated samples were calculated using the one-way ANOVA with the Bonferroni posttest. Statistical analyses were performed using Instat software version 3.00 (GraphPad Software, Inc., San Diego, CA).
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Results |
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To assess whether real-time PCR results employing the LightCycler and Northern blot analysis are comparable, both techniques were performed on the same samples. One FKBP51 transcript of 3.8 kb was detected on Northern blot. Figure 1
shows the relative amount of FKBP51 mRNA in IM-9 cells after 0-, 2-, 4-, 6-, 8-, and 24-h incubations with different concentrations of dexamethasone-Na2P (10-6 M, 10-7 M, 10-8 M) as detected by both techniques. IM-9 cells continuously stimulated with 10-6 M dexamethasone-Na2P showed an increase of FKBP51 mRNA within 2–4 h (P < 0.01). An approximate 10-fold increase was measured after 6–8 h, which persisted, with a possible slight decrease, until 24 h (Fig. 1A). When IM-9 cells were incubated with 10-7 M and 10-8 M dexamethasone-Na2P for 24 h (Fig. 1, B and C), an increase of approximately 10-fold (P < 0.01) and 5-fold (P < 0.01), respectively, was detected. Overall, these results show no differences between Northern blot analysis and real-time PCR data at the time points studied. To verify that the mRNAs of both housekeeping genes, GAPDH (used for normalization of the Northern blot data) and ß2m (used for normalization of the real-time PCR data), were not affected by the GC treatment, a control hybridization was performed on the Northern blots using a GAPDH probe, ß2m probe, and FKBP51 probe. Neither of the housekeeping gene mRNAs were influenced by GC treatment (data not shown).
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To verify whether the FKBP51 mRNA response to GCs is mediated through the GC receptor, IM-9 cells were incubated for 24 h with dexamethasone-Na2P in concentrations ranging from 10-10 to 10-5 M, with or without 10-6 M glucocorticoid receptor antagonist ORG 34116 (Organon) (Fig. 2). The dexamethasone-Na2P-induced FKBP51 mRNA increase appeared to be approximately 100-fold less sensitive following the addition of the antagonist (P < 0.001), indicating a GC receptor-mediated effect. At dexamethasone-Na2P concentrations exceeding 10-6 M, the antagonist-induced suppression of FKBP51 mRNA increase was overruled, showing the competitive nature between both compounds and the absence of nonspecific inhibition.
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To determine whether GCs up-regulate FKBP51 mRNA directly or indirectly, e.g. by inducing a transactivating stimulator of FKBP51 gene transcription, IM-9 cells were incubated for 0, 2, 4, and 6 h with 10-4 M cycloheximide, 10-9 M dexamethasone (an effective dose, as concluded from Fig. 4), or both. Following incubation with cycloheximide, no increase of FKBP51 mRNA was observed, but the coincubation of GC and cycloheximide still resulted in an increase of FKBP51 mRNA (Fig 3A). Efficient inhibition of protein synthesis was verified by a Tran-35S methionine incorporation into IM-9 cells, both in the presence and absence of 10-4 M cycloheximide (Fig. 3B). Together these observations strengthen the case for a direct effect of GCs on the expression of FKBP51 mRNA.
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Hydrocortisone sodium succinate, dexamethasone-Na2P, and budesonide are commonly used GCs in clinical practice. They were compared with the in in vitro experiments frequently used free dexamethasone in their potency to induce FKBP51 mRNA in IM-9 cells. Figure 4
shows 24-h incubations with glucocorticoid concentrations ranging from 10-11 M to 10-5 M. The relative potency of each GC was estimated by determining the distance between the parallel, linear sections of the lines that match the average increase in FKBP51 mRNA. Dexamethasone-Na2P appeared to be approximately 5- to 10-fold more potent to induce FKBP51 mRNA, compared with hydrocortisone in this in vitro system. The increase of FKBP51 mRNA following incubations with free dexamethasone and budesonide differed but not significantly. Both were approximately 100 times more potent than hydrocortisone, and both showed a reduced increase of FKBP51 mRNA levels at concentrations exceeding 10-6 M.
FKBP51 mRNA is induced by GCs both ex vivo and in vivo
To exploit the possibility that the response of FKBP51 mRNA to GCs can be applied to clinical research, an ex vivo experiment using native lymphocytes was performed. Incubations for 24 h with concentrations of 10-9 M, 10-8 M, and 10-7 M dexamethasone-Na2P, or dexamethasone showed not only a dose-dependent increase of FKBP51 mRNA, but also, as in IM-9 cells, there appears to be a significant, approximately 100-fold difference in potency between free dexamethasone and dexamethasone-Na2P in native lymphocytes (P < 0.001) (Fig. 5A).
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). Twenty-four hours after dexamethasone intake, FKBP51 mRNA levels had returned to normal. Application of the method to a patient with partial GC insensitivity
To further investigate the clinical applicability of these measurements, we applied our method to the peripheral blood lymphocytes of a 6-month-old girl with apparent GC insensitivity. This patient was diagnosed shortly after birth with congenital adrenal hyperplasia because of 21-hydroxylase deficiency, based on virilization of the external genitalia (Prader stage III-IV); elevated serum levels of 17OH-progesterone (470 nmol/liter), androstenedione (138 nmol/liter), and testosterone (27 nmol/liter) (normal levels on d 1 of life are 13 ± 5.2 nmol/liter, 6.1 ± 2.6 nmol/liter, and 1.6 ± 0.5 nmol/liter, respectively); echographic presence of a uterus; and a normal 46, XX karyotype in lymphocytes and skin fibroblasts. The diagnosis was confirmed later by sequence analysis of the CYP21-locus demonstrating compound heterozygosity for several known mutations (Ile172Asn on one allele and a cluster of four previously described mutations, among which a nonsense mutation, on the other allele). Treatment with hydrocortisone was initiated on d 2 at a dose of 40 mg/m2 per day. On this rather high dose of hydrocortisone, however, the adrenal androgen levels were insufficiently suppressed. A 2-d dexamethasone suppression test at a high dose (4 x 1.6 mg/m2 per day) failed to sufficiently suppress the adrenal steroids as well, but when continued for 2 months at a dose of 2 x 3 mg/m2 per day, her biochemistry was normalized (Fig. 6A).
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shows the dexamethasone-induced increase of FKBP51 mRNA in native blood lymphocytes of the patient and an age-matched control. In accordance with the approximately 10-fold higher-than-usual dose of glucocorticoids to maintain adequate adrenal suppression, an approximate 10-fold reduction in GC sensitivity could be demonstrated. |
Discussion |
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We found an increase of FKBP51 mRNA in IM-9 lymphoblasts within 2–4 h following addition of various concentrations of dexamethasone-Na2P to the cell culture medium. This time scale suggests a classical direct genomic effect mediated through the glucocorticoid receptor. The maximal FKBP51 mRNA response for the time points and lowest concentrations tested was found after 24 h of incubation, and this incubation time was used in all our subsequent experiments. The data of the time range experiment were obtained using Northern blot analysis as well as real-time PCR. The results demonstrated that both Northern blot analysis, showing one FKBP51 transcript of 3.8 kb (17), and real-time PCR detected FKBP51 mRNA with equal sensitivity and with a comparable relative increase. However, real-time PCR has several advantages over Northern blot analysis: Less mRNA is required to make an analysis, the procedure itself is less time consuming, and real-time PCR offers the possibility to perform a melting curve analysis. This analysis allows validation of the specificity of the PCR, whereas the specificity of a Northern blot hybridization, when using a probe homologous to other mRNAs with a comparable length, is more difficult to verify.
To gain a better understanding of the mechanism by which GCs increase FKBP51 mRNA levels, we blocked the GC receptor using the GC receptor antagonist ORG 34116 (22). This showed the FKBP51 mRNA increase to be GC receptor mediated. The possibility remains, however, that FKBP51 mRNA is not regulated directly by GCs but by an intermediate transactivator. The time course of the FKBP51 response in our experiments appears to argue against such a mechanism, and the increase in FKBP51 mRNA following addition of dexamethasone after inhibition of protein synthesis with cycloheximide further strengthens the case for a direct transcriptional effect of GCs.
Although the FKBP51 response to GC thus seems to be receptor mediated and direct, we were unable to find a canonical glucocorticoid response element (GRE) (5'-GGTACAnnnTGTTCT-3') in the promoter region of the FKBP51 gene, using homology searches on chromosome 6 sequences (23). However, a perfect GRE is present in the 21.7-kb intron between exons 4 and 5. Possibly this intronic GRE is responsible for the mRNA induction, which would be unusual but not without precedent (24). Alternatively there might be an unrecognized, imperfect GRE located in the promoter region, which regulates FKBP51 transcription.
Individual GC (in)sensitivity can be measured by several methods. A common clinical procedure is to test for suppression of the hypothalamic-pituitary-adrenal axis by graded doses of dexamethasone (25, 26). Despite several modifications and refinements, it is doubtful whether this test can ever be made sensitive enough to discriminate between different levels of GC sensitivity or insensitivity (27). Another method to assess GC sensitivity is to measure the effect of GCs on mitogen-stimulated lymphocytes. Various parameters can be measured, e.g. inhibition of proliferation (28, 29, 30) or inhibition of cytokine production (31, 32). The artificial immunostimulation, however, might induce changes in the number of GC receptors or in the expression of other genes that might interfere with GC sensitivity. To adhere to the normal, in vivo situation as close as possible, we preferred to use the response of nonstimulated lymphocytes as a basis for our GC sensitivity assay. Our results show that the induction of FKBP51 mRNA by GCs in native lymphocytes ex vivo might very well be suitable to assess different levels of individual GC hypo- or hypersensitivity. Validation of this supposition, however, requires extensive clinical studies, which we are now able to perform.
As in IM-9 the time course of the GC-induced increase in lymphocytic FKBP51 mRNA in vivo is compatible with a classic genomic response to glucocorticoids. This response might be used in a bioassay to determine GC bioavailability in the circulation, enabling, for instance, the monitoring of patient compliance with the steroid medication prescribed in, among others, inflammatory bowel disease (33).
Besides for the determination of individual GC sensitivity or GC bioavailability, an FKBP51 mRNA-based assay could be applied to a comparative assessment of the pharmacological potency of newly developed GCs. Presently the most commonly used test to discriminate among steroid potencies is the skin-blanching assay. This assay appears to be rather difficult to interpret, however, because it relies on the subjective assessment by a trained observer using a visual score (34). Moreover, the skin-blanching assay appears to be influenced by the positioning of the tested limb (35) and the position at which the GC is applied on the limb (36). The induction of FKBP51 mRNA by GC in a standardized cell line might circumvent most of these problems. Therefore, we wanted to determine whether different GCs used in the clinic display different potencies with respect to the induction of FKBP51 mRNA in IM-9 cells. Four GC preparations were compared using concentrations ranging from 10-11 M to 10-5 M. Dexamethasone-Na2P was only 5–10 times more potent than hydrocortisone in inducing FKBP51 mRNA levels in this in vitro system, in which we, based on the literature, expected dexamethasone-Na2P to be 30–100 times more potent than hydrocortisone (37). It may be that the disodium phosphate group, which renders the dexamethasone molecule water soluble, interferes with GC diffusion across cell membranes, resulting in a diminished induction of FKBP51 mRNA. Indeed, the free, nonwater-soluble form of dexamethasone, had the expected potency of approximately 100 times that of hydrocortisone in both IM-9 cells and native lymphocytes. The potency of budesonide at least equals that of free dexamethasone in inducing FKBP51 mRNA expression.
The immunophilin FKBP51 forms part of the Hsp90 steroid receptor complex (38) and has been shown to reduce the affinity of both the squirrel monkey and human GC receptor for its ligand when overexpressed (39, 40). It is interesting to find that FKBP51 mRNA is up-regulated by GCs in both IM-9 cells and native lymphocytes in our study because it might point to a role of FKBP51 in a short-loop negative feedback regulatory mechanism following GC administration. Although the potency to reduce GC receptor affinity in humans is less than in squirrel monkeys (40), it is conceivable that constitutively elevated FKBP51 levels would induce a certain degree of GC insensitivity, but mutations in this gene, attenuating a supposed feedback mechanism, might be the cause of sustained GC hypersensitivity. This might explain the diminution in FKBP51 mRNA increments that we observed in the comparison of GC potencies at concentrations of dexamethasone and budesonide exceeding 10-6 M. On the other hand, it has been reported that GCs induce down-regulation of GC receptor mRNA itself in IM-9 cells (41). It is entirely possible, therefore, that both a decrease in GC receptor numbers and desensitization of the GC receptor signal transduction mechanism contribute to a reduced increase in FKBP51 mRNA.
The defect underlying the apparent glucocorticoid insensitivity of the patient we described in our report has not yet been identified. Partial insensitivity to glucocorticoids can be caused by several mechanisms and has been described in many other patients (42). Our patient appears to be unique in that she presented with biochemical evidence of congenital adrenal hyperplasia as well. Analysis of the 21-hydroxylase locus in our patient has confirmed this diagnosis. Thus, although the precise etiology of her diminished glucocorticoid sensitivity remains elusive at present, it is interesting to see how well the biochemical findings correlate with the ex vivo data regarding her lymphocytic sensitivity to GCs using our methodology: They both point to an approximately 10-fold reduced sensitivity at the time of evaluation.
In conclusion, the response of FKBP51 mRNA to GCs appears to be suitable for application in a bioassay to establish GC potency using a standardized cell line in vitro and measure individual cellular GC sensitivity of patient-derived native lymphocytes ex vivo and for the in vivo determination of GC bioavailability. Real-time PCR appears to be a valid method to measure the relative increase of FKBP51 mRNA, which should allow determinations of individual GC-sensitivity and GC bioavailability to be performed, even in premature infants.
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Acknowledgments |
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Footnotes |
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Abbreviations: ß2m, ß2-Microglobulin; dsDNA, double-strand DNA; FCS, fetal calf serum; FKBP51, 51-kDa FK506-binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC, glucocorticoid; GRE, glucocorticoid response element.
Received March 6, 2002.
Accepted September 19, 2002.
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) and real-time PCR (
). Maximal FKBP51 levels are obtained following 24 h of incubation, and both techniques yield similar results. Representative Northern blots hybridized with the FKBP51 probe and the corresponding GAPDH probe are shown at the bottom.
), or budesonide (
) show distinct potencies to induce FKBP51 mRNA in the lymphoblast cell line IM-9. Budesonide and dexamethasone appear equally potent, 50- to 100-fold more potent than hydrocortisone, and 10-fold more potent than dexamethasone-Na2P.
), but coincubation of 100 µM cycloheximide with 10-9 M dexamethasone (
) does not appear to influence FKBP51 transcription, compared with 10-9 M dexamethasone alone (
