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Special Feature |
Division of Endocrinology and Diabetes (M.Sl., N.R., O.Z., C.M.-G., M.St., A.K., M.R., F.B.), Department of Internal Medicine II, University Hospital Freiburg, D-79106 Freiburg, Germany; and Institute of Pharmacology, Ruprecht Karls University (C.M.-G.), D-69120 Heidelberg, Germany
Address all correspondence and requests for reprints to: Martin Reincke, M.D., Medizinische Klinik Innenstedt, Ludwig-Maximilians-Universitaet Muenchen, Ziemsenstrasse 1, D-80336 Munich, Germany. E-mail: Martin.Reincke{at}med.uni-muenchen.de.
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Abstract |
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Introduction |
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An unusual feature of the ACTH receptor gene regulation is the up-regulation of ACTH receptor mRNA by its own receptor ligand (7), which involves an increase in the transcriptional rate of ACTH receptor message and a prolongation of the mRNA half-life (8). These in vitro findings might also provide the molecular basis for the clinically observed rapid decrease in adrenal responsiveness to ACTH after suppression of endogenous ACTH, which is restored within hours after exposure to exogenous ACTH (9). Several putative cAMP-responsive elements (CRE) have been identified in the promoter of the human ACTH receptor gene, suggesting transduction of the effects of ACTH on ACTH receptor gene transcription by cAMP (10). Accordingly, the effects of ACTH on ACTH receptor expression can be mimicked by treatment with cAMP analogs and forskolin (11). However, direct evidence for the role of CREs in the regulation of the ACTH receptor gene is lacking.
Although in recent years several polymorphisms in promoter regions have been described, the physiological significance of these findings is uncertain in most instances. Herein we describe a novel polymorphism within the ACTH receptor promoter transcription initiation site at position –2 bp that alters the consensus sequence from C GAG CTC ATT C to C GAG CCC ATT C (referred to as CTC and CCC). The underscore indicates that base triplets are different from the consensus sequence and polymorphism, respectively. We provide evidence for a reduced promoter activity of the CCC construct in vitro based on transfection experiments as well as in vivo as demonstrated by ACTH and CRH stimulation tests.
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Subjects and Methods |
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NCI-H295 human adrenocortical carcinoma cells were stimulated with forskolin (10–5 M) or ACTH1–24 (10–6 M) for 24 h, respectively. After RNA extraction (SV 40 RNA extraction kit; Promega Corp., Mannheim, Germany), 1 µg total RNA was used for cDNA synthesis (Superscript II, Invitrogen, Karlsruhe, Germany). Semiquantitative PCR was performed using 18S rRNA as an internal standard (QuantumRNA 18S Internal Standards Classic, Ambion, Cambridgeshire, UK). The primer/competimer ratio was 1:9. Primers for the amplification of the ACTH receptor were 5'-CAT GGG CTA TCT CAA GCC AC-3'and 5'-GAG ATC TTC CTG GTG TGG GAT C-3'. Cycle conditions were 35 cycles of denaturation (1 min at 95 C), annealing (1 min at 56 C), and extension (1 min at 72 C). To exclude amplification of genomic DNA, a control without prior RT was included (data not shown).
Transfection experiments
Mouse adrenocortical Y1 cells were grown in Ham’s F-10 medium with 7.5% horse serum and 2.5% fetal bovine serum. Full-length constructs (–1017/+40) as well as 5' deletion constructs of the ACTH receptor promoter, including the CTC and CCC sequences, were subcloned into the luciferase reporter gene vector pGL3 and transiently transfected into Y1 cells using ExGen 500 (MBI Fermentas, St. Leon-Rot, Germany). The ß-galactosidase vector cytomegalovirus-lacZ was cotransfected for normalization. Twenty-four hours after transfection, cells were stimulated with forskolin (10–5 M), and activity was measured 24 h thereafter (Luciferase Reporter Gene Assay, Roche, Mannheim, Germany). Luciferase values were normalized for galactosidase values using the Galacto-Light assay (PE Applied Biosystems, Darmstadt, Germany). Each data point represents at least four independent measurements.
Genotyping
The study was approved by the ethics committee of Freiburg; all participants of the functional tests gave written informed consent.
Blood samples from male blood donors were received from the blood bank of the University Hospital Freiburg. DNA, isolated from full blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), was used as template for PCR. 5'-GCG CGC GCA GAT CTA AGC AGG AAC TTT CTG GG-3' and 5'-CGG GGT ACC GGG ATG ACA TTT ATT CAA GG-3' were the upstream and downstream primers, binding at positions –89 and +23 (with regard to the transcription initiation site), respectively. PCR was performed with 5 U AmpliTaq (PE Applied Biosystems). Cycle conditions were 35 cycles of denaturation (30 sec at 94 C), annealing (30 sec at 56 C), and extension (1 min at 72 C). As the CCC polymorphism disrupts an existing SacI restriction site, PCR products were digested for 3 h with SacI (20 U; New England Biolabs, Frankfurt, Germany). Fragments were separated on a 2% agarose gel, stained with ethidium bromide, and photographed. Samples with the CCC/CCC genotype revealed one band (173 bp), those with CTC/CTC revealed two bands (120 and 53 bp), and those with CTC/CCC revealed three bands (173, 120, and 53 bp), respectively.
ACTH and CRH stimulation tests
For functional tests, male subjects (age, 20–35 yr) with the appropriate genotype were contacted, and 21 suitable subjects participated in this study. Exclusion criteria were bronchial asthma, pituitary, renal or liver disease, acute or chronic stress, alcohol abuse, depressive disorders, current or former treatment with glucocorticoids, and known allergies to Synacthen or CRH-Ferring.
The applied ACTH infusion test was modified from a test described by Komindr et al. (12) and Slayden et al. (13). To suppress endogenous ACTH secretion, subjects were pretreated with dexamethasone orally: 2 mg at 2300 h the day before ACTH stimulation, and 1.5 mg at 0800, 1200, and 1600 h, respectively, on the day of the ACTH stimulation test. The doses and times of administration of dexamethasone were determined empirically in preliminary tests. Participants arrived at 1545 h in the out-patient clinic after fasting since lunch (1300 h). At 1600 h two iv lines were inserted: one for continuous ACTH infusion, doubled each hour (120–3840 ng/m2 body surface area·h), and the other for collection of blood samples for cortisol determination (3 ml each, every 20 min). The latter iv line was kept open with 0.9% sodium chloride solution (flow rate, 1.5 ml/min).
For CRH stimulation testing, subjects arrived at 1545 h in the out-patient clinic. An iv line was applied, and a bolus of 100 µg synthetic human CRH (CRH-Ferring) was administrated at 1615 h. Blood samples (serum and EDTA, 3 ml each) were drawn at –15, 0, 15, 30, 60, 90, and 120 min. Baseline hormone values were calculated from the mean of the –15 and 0 min samples. The two functional tests were separated by at least 3 d.
For measurement of glucocorticoid excretion subjects collected a single random 24-h urine sample.
Hormone assays in blood samples
When the tests were completed, all samples were centrifuged at 4000 rpm for 10 min and stored at –20 C until assayed. All cortisol samples were measured at the end of the study in one run. Serum cortisol was determined using the commercially available Advantage Chemiluminescence Assay Cortisol (Nichols Institute, Inc., San Juan Capistrano, CA). The intraassay coefficient of variation (CV) was 4.3–9.1%, and the interassay CV was 6.8–12.2%.
Plasma samples for ACTH measurement were immediately centrifuged, decanted, and kept refrigerated (4 C) until analyzed within the next day. Before starting the ACTH stimulation test, a single plasma sample was drawn to ensure baseline suppressed ACTH levels (<2.2 pmol/liter). Plasma ACTH was measured by the Nichols Advantage ACTH Assay. The interassay CV was 6.4–8.4%.
Hormone assays in urine
Urinary steroids were measured by RIAs using tritiated steroids (Amersham Pharmacia Biotech, Freiburg, Germany) and antibodies, raised and characterized in our laboratory, as described previously (14). Before RIAs, urinary free cortisol and cortisone were extracted from urine with organic solvents and chromatographically purified using Celite columns (Celite 545 AW, Sigma-Aldrich Corp., Taufkirchen, Germany) (15). Tetrahydrocortisol, 5
-tetrahydrocortisol, and tetrahydrocortisone were quantified after treatment with ß-glucuronidase (Roche) at a final dilution of 1:1200 (vol/vol). Interassay CVs were less than 15%.
Data analysis
Statistical analysis was performed with Stat View 5 (SAS Institute, Inc., Cary, NC). Time-integrated cortisol secretion was calculated by the trapezoid method from 0–360 min and expressed as nanomoles per liter x minute. Data are presented as the mean ± SEM. Statistical comparisons were analyzed by ANOVA and Fisher’s protective least significant difference test. P < 0.05 was considered statistically significant.
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Results |
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Sequencing of a BAC clone obtained from the German Resource Center and Primary Database revealed a base exchange from the consensus sequence (GenBank accession no. Y10100) at position –2 with regard to exon 1 of the human ACTH receptor promoter (CGAGCTCATTC to CGAGCCCATTC). Initial screening of a limited number of DNA samples from unrelated test persons demonstrated the existence of both sequences as a polymorphism. The human adrenocortical carcinoma cell line NCI-H295 was shown to carry the consensus sequence of the ACTH receptor promoter and revealed the expected increase in ACTH receptor mRNA upon treatment with ACTH and forskolin (Fig. 1A
). The close proximity of the polymorphic region to the transcription initiation site of the ACTH receptor (Fig. 1B) prompted us to study the functional significance of this polymorphism on transcriptional activity.
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Using mouse adrenocortical carcinoma Y1 cells transiently transfected with the full-length as well as 5' deletion constructs of the ACTH receptor promoter, we studied the effects of the CTC and CCC sequences on ACTH receptor promoter activity. Baseline promoter activity of the CCC construct (73 ± 4%) was lower than that of the CTC construct (100 ± 5%; P = 0.02; Fig. 1C
). Similarly, forskolin-induced promoter activity was significantly lower in the CCC (143 ± 13%) than in the CTC (194 ± 15%; P = 0.0008) construct. However, the fold stimulation of promoter activity above baseline by forskolin was not affected by the polymorphism (CCC, 2.0-fold; CTC, 1.9-fold; P = 0.85). Comparable results were obtained using the 5' deletion constructs of the ACTH receptor promoter, with significantly lower baseline and forskolin-induced promoter activity in the CCC-containing constructs, whereas forskolin-induced activation over baseline was not greatly altered (–594: CCC, 2.2-fold; CTC, 1.5-fold; P = 0.32; –293: CCC, 1.5-fold; CTC, 1.3-fold; P = 0.34; –64: CCC, 1.2-fold; CTC, 1.0-fold; P = 0.63; Fig. 1D). As expected, the shortest construct (–64) lacking any CRE and AP-1 sites, showed no forskolin-induced activation.
The polymorphism is present at a significant frequency in the normal male population in Germany
Using a SacI restriction length polymorphism (Fig. 2), the frequency of the polymorphism was assessed in a large cohort of 1266 unrelated, healthy, men from a southern German population. The prevalences of the respective alleles were: CTC homozygosity, 80.2%; CTC/CCC heterozygosity, 19.0%; and CCC homozygosity, 0.8% (Table 1). As these results indicated the presence of the polymorphism in a significant proportion of the population studied, we explored the possible physiological significance of the in vitro findings on HPA axis activation in humans.
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To obtain functional data on the observed ACTH receptor promoter polymorphism in vivo, individuals with the CTC/CTC, CTC/CCC, and CCC/CCC genotypes were evaluated for clinical endocrine characteristics. Anthropometric data and baseline hormone values were not different among the groups (Table 2). No difference was seen with respect to baseline plasma ACTH and serum cortisol concentrations and free cortisol and cortisone excretion between the groups. Although the glucocorticoid excretion rate, expressed as the sum of the main glucocorticoid metabolites (tetrahydrocortisol, 5-tetrahydrocortisol, and tetrahydrocortisone), tended to be lower in homozygous CCC subjects, this difference did not reach statistical significance (Table 2).
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To study differences in ACTH responsiveness in vivo, a prolonged ACTH stimulation test was performed. Before ACTH administration, subjects were treated with oral dexamethasone to suppress endogenous ACTH secretion. Low plasma ACTH levels (
1.1 pmol/liter) in all subjects proved adequate suppression.
During hourly doubling ACTH1–24 infusion (first hour, 120 ng/m2 body surface area·h), serum cortisol concentrations in subjects with the CCC/CCC polymorphism showed lower increments than CTC/CTC subjects (Fig. 3). This difference became statistically significant 220 min after beginning the infusion (fourth hour, 960 ng/m2 body surface area/h), but was already discernible in the first 3 h. Heterozygous subjects (CTC/CCC) showed intermediate values. The integrated serum cortisol concentration was 34% lower in CCC/CCC subjects than in CTC/CTC subjects during the 220- to 360-min interval and was 26% lower during the entire period of infusion (Table 3).
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To investigate the cortisol response to endogenous ACTH stimulation, a CRH test was performed. Plasma ACTH rose significantly, and peak values were reached within 15–30 min after ovine CRH administration (100-µg iv bolus). Whereas serum cortisol levels were similar in both groups (Fig. 4B), there was a tendency for higher mean plasma ACTH concentrations in CCC/CCC subjects compared with CTC/CTC (Fig. 4A). As a parameter of ACTH sensitivity, the ACTH/cortisol ratio was calculated. In subjects with the CCC/CCC genotype, the ratio was significantly higher at 15 and 30 min after CRH administration than in the CTC/CTC group (P < 0.03; Fig. 4C). These results are in line with a lower adrenal responsiveness to ACTH in carriers of the CCC polymorphism, which is compensated by increased pituitary ACTH secretion, at least in response to a maximally stimulating dose of CRH.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Transcription initiation is a major control point of gene expression, which requires RNA polymerase II as the central enzyme of transcription as well as a number of general transcription factors to ensure promoter-specific initiation of mRNA synthesis (21). As a result of a series of isomerization steps, about 14 bp of DNA, in a region that includes the initiation start site, dissociate. This initiates RNA synthesis (22). Our in vitro data suggest that a polymorphism within the transcription initiation site of the ACTH receptor promoter results in lower basal and forskolin-stimulated promoter activity. The position of the polymorphism implicates effects on the binding of the transcriptional machinery or on melting of the DNA double helix rather than effects on the stimulation of promoter activity by transcription factors. In accordance with this idea, fold activation over baseline of ACTH receptor transcription by forskolin is not affected by the observed polymorphism. Moreover, basal and forskolin-induced gene transcription in 5' deletion constructs lacking any CRE is also significantly lower in the CCC-containing construct compared with the CTC sequence, suggesting an overall impairment of promoter activity by the polymorphic sequence. A polymorphism described in the ß2-glycoprotein I gene at position –1 of the transcription initiation site, resulting in decreased plasma levels of the gene product, bears similarities to the mechanism regulating ACTH receptor expression (23).
In vivo, subjects with different ACTH receptor promoter sequences were not different with respect to basal serum cortisol or plasma ACTH levels or urinary free cortisol, free cortisone, or glucocorticoid excretion rates. Several possible compensating mechanisms within the HPA axis are likely to alleviate the effects of lower ACTH receptor promoter activity in affected individuals. A compensatory increase in CRH and ACTH levels driven by lower adrenal cortisol output might not only overcome the effects of a lower number of ACTH-binding sites on the adrenocortical cell, but also increase ACTH receptor gene expression. Moreover, ACTH-independent effects mediated through other POMC-derived peptides (such as N-terminal POMC) could result in hyperplasia of the adrenal cortex. As such, the cell number, the ability of the adrenal cells to generate steroids, or normalization of ACTH receptor expression could occur under baseline conditions. However, under distinct experimental conditions, such as prolonged stimulation with ACTH, individuals with the homozygous CCC sequence showed a lower adrenal responsiveness to exogenous ACTH than subjects homozygous for the consensus sequence, and heterozygous individuals displayed an intermediate response. Significant differences in cortisol secretion during the ACTH test were first discernible when serum cortisol levels were in the range of the upper limit of normal diurnal variation (24). The major difference in cortisol response, however, was observed at later time points and at higher doses of ACTH1–24 and resulted in serum cortisol levels that are usually found only during surgery, sepsis, or other major stress (25, 26). The up-regulation of the ACTH receptor by its ligand may be involved in the observed lack of receptor down-regulation during continuous, increasing ACTH administration (7). Thus, the increase in cortisol levels over the time course of the ACTH infusion experiment, which has also been observed in the original studies (12, 13), can be explained by the increase in both ACTH dosage and duration of exposure to ACTH. We hypothesize that the lower promoter activity of the CCC polymorphism, as demonstrated in vitro, results in lower expression of the ACTH receptor in vivo, with a resulting decrease in the number of ligand-binding sites and lower sensitivity to ACTH stimulation in individuals with the CCC polymorphism. It is particularly noteworthy that despite the flexibility of the HPA axis, one base exchange in a single gene within the signal cascade of the HPA axis does result in measurable effects on adrenal steroid output, thus underscoring the importance of the ACTH receptor as a modifier of adrenal function.
In accordance with the findings of the ACTH stimulation test, subjects homozygous for the CCC polymorphism also showed a lower adrenal responsiveness to endogenous CRH-stimulated ACTH secretion, as indicated by a greater rise in plasma ACTH concentration and a higher ACTH/cortisol ratio in CCC/CCC individuals, indicative of decreased cortisol feedback inhibition of CRH-stimulated ACTH secretion. The wide range of ACTH and cortisol values achieved by CRH stimulation is a characteristic of the CRH test (27). Although a variety of factors can affect the response of the HPA axis to exogenous CRH, one might speculate that effects of the ACTH receptor promoter polymorphism contribute to the variable response and thus unfavorable test characteristics of the CRH stimulation test (27).
In conclusion, the ACTH receptor promoter polymorphism (CCC), which is homozygous in one of 125 individuals in a healthy, male population, results in lower promoter activity, as demonstrated by a luciferase assay in vitro, and is associated with lower adrenal responsiveness to ACTH stimulation in humans. Under basal conditions, impaired adrenal responsiveness may be compensated for by higher plasma ACTH concentrations (as suggested by the CRH testing), but during major stress, the reduced ACTH sensitivity of the adrenal cortex could become clinically relevant.
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Acknowledgments |
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Footnotes |
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M.Sl. and N.R. contributed equally to this work.
Abbreviations: CRE, cAMP-responsive element; CV, coefficient of variation; HPA, hypothalamus-pituitary-adrenal; PKA, protein kinase A; POMC, proopiomelanocortin.
Received November 18, 2003.
Accepted February 18, 2004.
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) and CTC/CTC (
) subjects. *, P < 0.03.