Abstract
Objective
To examine whether inhibition of endogenous adrenocorticotropin (ACTH) secretion in patients with Cushing syndrome affects the expression of the ACTH receptor (ACTH-R) gene in adrenocortical adenoma and attached atrophic normal gland.
Summary Background Data
ACTH increases adrenal cell growth and steroidogenesis by means of ACTH-R. In vivo and in vitro studies have shown that expression of ACTH-R is upregulated by its own ligand ACTH in several species. In patients with Cushing syndrome resulting from adrenocortical adenoma, there is autonomous production of cortisol from the adenoma. This strongly inhibits endogenous ACTH secretion, giving rise to the speculation that the expression of the ACTH-R gene in these patients is also suppressed. However, previous studies have shown that administration of exogenous ACTH to these patients leads to a further increase in the production of cortisol, suggesting the expression of functional ACTH-R in the adenoma. The authors, therefore, examined the expression of the ACTH-R gene in these patients.
Methods
Fourteen patients with Cushing syndrome were studied. Glucocorticoid excess resulting from autonomous production from the adenomas was ascertained, and unilateral adrenalectomy was performed. The levels of ACTH-R and cytochrome P450 side chain cleavage enzyme (P450scc) mRNAs were determined by Northern blot analysis. The entire coding region of the ACTH-R gene in these patients was sequenced.
Results
ACTH-R mRNA abundance in the attached atrophic normal adrenals was suppressed and invariably less than that in the normal gland obtained from a patient with renal cancer. However, the expression of ACTH-R mRNA was not suppressed in any of the adenomas. Expression of ACTH-R mRNA in the adenomas was four- to sixfold greater than that in the attached atrophic gland. No mutation in the coding sequence of the ACTH-R gene in the adenoma was detected in any of the patients. The mRNA in the adenomas appeared to be translated into functionally active receptor because intramuscular administration of ACTH resulted in significant increases in plasma cortisol before surgery but not 3 months after surgery. In addition, there was a positive linear correlation between the expressions of ACTH-R and P450scc mRNAs in the adenoma tissue.
Conclusions
Suppressed ACTH secretion in patients with Cushing syndrome results in reduction of the ACTH-R mRNA expression in nonneoplastic adrenocortical cells. However, the regulatory mechanism of ACTH-R expression might be different in adenoma. Persistent expression in the adenoma of ACTH-R alone, even in the absence of ACTH, might result in increased basal adenyl cyclase activity, as observed in the case of thyroid-stimulating hormone receptor, and thereby might play a role in the autonomous production of cortisol.
Adrenocorticotropin (ACTH) is the major regulator of steroidogenic function of the adrenocortical zonae fascicu lata and reticularis and is a significant growth factor for normal adrenal cortical cells. 1,2 The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). ACTH-R, a 297-amino-acid protein, belongs to the seven-transmembrane domain superfamily of GTP-binding protein-coupled receptors. 3 The extracellular domain binds to the ligand. The intracellular domain interacts with the heterotrimeric Gs protein and promotes the exchange of bound GDP with a GTP moiety on the Gsα-subunit. This activates the α-subunit and results in the dissociation of the GTP-bound α-subunit from the ACTH-R complex. The activated Gsα-subunit stimulates adenyl cyclase-mediated (cAMP) production and interacts with other cell-specific intracellular effectors as well. The human ACTH-R gene is located on chromosome 18p 11.2. 4,5 It consists of two exons: the second exon contains the entire coding sequence of ACTH-R, and the first exon, encoding a 49-bp 5′-noncoding sequence, is separated from the second exon by an intron approximately 18 kb long. 6 In vivo and in vitro studies have clearly shown that ACTH-R mRNA expression is positively regulated by ACTH. 7–10 Thus, ACTH augments steroidogenesis by increasing both the steroidogenic enzymes and its own receptor.
Constitutive activation of G protein or G-protein-coupled receptor has been identified as responsible for the autonomous production of hormone in thyroid and pituitary neoplasms. 11,12 Cushing syndrome resulting from adrenocortical adenoma is one of the typical disorders presenting with autonomous hormone production resulting from a functioning adenoma. In functional adrenocortical adenoma, no mutation has been identified in Gαs. 13 Lyons et al 14 reported mutations in Gαi2 in 3 of 11 adrenocortical adenomas; however, similar mutations could not be identified by two other independent studies. 13,15 Previous studies reported absence of activating mutation in the coding region of the ACTH-R gene in adenomas. 16,17 Loss of heterozygosity of the ACTH-R gene has been detected in one nonfunctional adenoma and carcinomas but not in functional adenomas. 18 In patients with Cushing adenoma, plasma ACTH, the key regulator of steroidogenesis, is completely suppressed. Thus, cortisol production in the adenoma is independent of circulating ACTH in these patients. Because activating mutations of ACTH-R have not been reported, there is a possibility that ACTH-R without activating mutation contributes to the autonomous production of cortisol in patients with Cushing syndrome.
Bertagna and Orth 19 reported that Cushing adenoma could respond to stimulation by exogenous ACTH with a further increase in cortisol secretion, even though basal tumor cortisol secretion was not dependent on endogenous ACTH. They further suggested that the source of the increased cortisol secretion is the adenoma, not the nonneoplastic adrenal tissue, because patients with a preoperative response to ACTH did not respond to ACTH within 2 weeks of surgery. These findings indicate that the adenoma tissue expresses functional ACTH-R, even in the absence of ACTH. In this report, we found similar results of cortisol response to exogenous ACTH in 14 patients with Cushing syndrome from adrenocortical adenoma. We also studied the expression of ACTH-R mRNA in adenoma and the attached adrenal gland in these patients. We observed a positive linear correlation between the expressions of ACTH-R and side chain cleavage enzyme (P450scc) mRNAs in the adenoma tissue, indicating that the receptor is functional. Our findings suggest that the ACTH-R expressed in the adrenocortical adenoma might contribute to the autonomous production of cortisol in patients with Cushing syndrome.
PATIENTS AND METHODS
Patients
Fourteen patients with Cushing syndrome were studied. All had the typical signs and symptoms of Cushing syndrome, such as moon face, buffalo hump, central obesity, hypertension, osteoporosis, hirsutism, urolithiasis, psychological disorders, and amenorrhea. Ultrasonography, computed tomography, or magnetic resonance imaging revealed a unilateral adrenal tumor in all patients. Clinical data are summarized in Table 1. The protocol for endocrinologic evaluation was approved by the Human Research Committee, Research Institute of Environmental Medicine, Nagoya University, and the Ethical Committee on Human Research, Nagoya University School of Medicine. Informed consent was obtained from each patient for the clinical evaluations and the use of surgical specimens.
Table 1. CLINICAL DATA OF THE PATIENTS
HTN, hypertension; MF, moon face (central obesity); STR, striae, AC, acne; OP, osteoporosis; DM, diabetes mellitus; HIR, hirsutism, PSY, psychological symptoms; UL, urolithiasis; AMN, amenorrhea.
Endocrinologic Evaluation
Blood was drawn at 6 am, noon, 6 pm, and midnight to determine the diurnal variations of plasma ACTH and serum cortisol. The dexamethasone suppression test was carried out by administering 0.5 mg dexamethasone (Decadron, Banyu, Tokyo, Japan) orally every 6 hours for 2 days; blood was drawn on the third morning. Dexamethasone (2 mg every 6 hours) was continued for another 2 days, blood was drawn on the fifth morning, and plasma ACTH and serum cortisol levels were determined. 20
Before adrenalectomy, ACTH-Z (Cortrosyn Z, Daiichi, Tokyo, Japan; 40 IU/2 mL) was given by intramuscular injection, and serum cortisol levels were determined before and 6 hours after the ACTH administration. In four patients included in the present study as well as a separate series of 10 patients with Cushing syndrome from adrenocortical adenoma, the ACTH administration test was performed 3 months after adrenalectomy, with informed consent obtained from the patients. Glucocorticoid supplementation had been discontinued for 7 days before the test.
Extraction of RNA and Northern Blot Analysis
After adrenalectomy, part of the resected adenoma and attached atrophic adrenal tissue were immediately frozen in liquid nitrogen and kept at −80°C until RNA extraction. As controls, an adrenocortical adenoma from a patient with a typical aldosterone-producing adenoma, a nonfunctioning adrenocortical adenoma, and a normal adrenal gland obtained during surgery for renal carcinoma were used.
Total RNA was isolated by the method described by Chomczynski and Sacchi. 21 Fifteen micrograms total RNA was subjected to Northern blot analysis, as previously described. 22 The probes used for hybridization were human ACTH-R cDNA, 7 bovine P450scc cDNA, 20 and human glyceryldehyde-3-phosphate dehydrogenase (GAPDH) cDNA 7 labeled with [α-32P]dCTP (New England Nuclear, Boston, MA) using a Random Primed DNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany). Radioactivity of the probes hybridizing to each mRNA was determined by a Bio-Rad GS-363 Molecular Imager System (Bio-Rad Laboratories, Hercules, CA).
Sequencing
One microgram total RNA, isolated from each adenoma tissue, was reverse-transcribed with (dT)15 primer. The single-stranded cDNAs were used as templates for subsequent polymerase chain reaction (PCR) by using a specific primer pair: sense: 5′-CCCGCCTTAACCACAAGCAGGAG-3′ and antisense: 5′-GGATTCTAAAACCAGGGATCAGC-3′. The sense primer was located at position −24 when the translation start site was designated as +1. The antisense primer was located at position +921. The amplified DNA contained the entire coding sequence of human ACTH-R. The PCR fragments were cloned into PCR2.1 vector (Invitrogen, San Diego, CA). The recombinant DNAs were used to transform Epicurian Coli XL1-Blue competent cells (Stratagene, La Jolla, CA). At least four clones were picked up in each patient, and the plasmids were purified using QIAprep Miniprep kit (Qiagen, Hilden, Germany). DNAs were sequenced using the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin Elmer, Foster City, CA).
RESULTS
In all patients, plasma ACTH levels were completely suppressed (<5 pg/mL) throughout the day. Serum cortisol did not show diurnal variations (Fig. 1). When the ACTH level was determined before and after dexamethasone administration, the levels were less than 5 pg/mL. Serum cortisol was not suppressed after 2-mg or 8-mg dexamethasone administration (Fig. 2).
Figure 1. Absence of diurnal variations of serum cortisol in patients with Cushing syndrome. In 13 patients with Cushing syndrome, diurnal variations of serum cortisol were determined. The numbers on the right correspond to the patient numbers in Table 1. The dotted region represents normal diurnal variations of serum cortisol.
Figure 2. Serum cortisol is not suppressed by administration of dexamethasone in patients with Cushing syndrome. In 14 patients with Cushing syndrome, the dexamethasone suppression test was carried out. The numbers on the right correspond to the patient numbers in Table 1. The dotted region represents the normal response to the dexamethasone suppression test.
In 11 patients examined, serum cortisol levels were increased after intramuscular injection of ACTH (Fig. 3). In 8 of these 11 patients, serum cortisol was increased more than 40 μg/dL. However, 4 patients included in the present study as well as a separate series of 10 patients with Cushing syndrome from adrenocortical adenoma showed either no response or responded much less than the preoperative response at 3 months after adrenalectomy. These results indicate that in patients with a preoperative cortisol response to ACTH, the adenoma, not the nontumorous adrenal tissue, including the contralateral normal gland, was the source of the increased cortisol secretion. The results also suggest the expression of functional ACTH-R in the adenoma.
Figure 3. Serum cortisol increases after intramuscular injection of adrenocorticotropin (ACTH) before surgery but not 3 months after surgery. The ACTH stimulation test was performed before adrenalectomy in (A) 11 patients with Cushing syndrome and 3 months after adrenalectomy in (B) 4 patients in this study and (C) another 10 patients with Cushing syndrome. The numbers on the right correspond to the patient numbers in Table 1. The dotted region represents the normal response to the ACTH stimulation test.
We therefore determined the expression of ACTH-R mRNA in the adenoma and in the attached atrophic normal gland. Northern blot analysis revealed two ACTH-R mRNAs of 2 and 4 kb in size (Fig. 4), which is in accordance with the previous report. 3 ACTH-R mRNA expression in cortisol-producing adenomas was not suppressed; in some patients it even increased compared with that in the normal adrenal obtained from a patient with renal cancer. Abundance of ACTH-R mRNA in cortisol-producing adenomas was similar to that in aldosterone-producing adenoma and nonfunctioning adrenal adenoma. ACTH-R mRNA expression in the attached atrophic adrenals with cortisol-producing adenomas was invariably less than that in the normal gland obtained from a patient with renal cancer. In the attached adrenals with aldosterone-producing adenoma and nonfunctioning adrenal adenoma, ACTH-R mRNA abundance was not suppressed compared with that in the normal gland obtained from a patient with renal cancer. In all the patients with Cushing syndrome, ACTH-R mRNA expression in the adenoma was greater than in the attached atrophic gland. The mean value of ACTH-R mRNA, corrected by GAPDH mRNA, was four- to sixfold greater in the adenoma than in the adjacent atrophic gland (Table 2).
Figure 4. Expression of adrenocorticotropin receptor (ACTH-R) and cytochrome P450 side chain cleavage enzyme (P450scc) mRNAs is suppressed in adjacent atrophic adrenal but not in the adenoma. Expression of ACTH-R, P450scc, and glyceryldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs was determined by Northern blot analysis in the adrenal gland from patients with Cushing syndrome (1–14), normal adrenal from a patient with renal cancer (RC), aldosterone-producing adenoma (APA), and nonfunctioning adrenocortical adenoma (NFA). T, adenoma; N, attached atrophic normal gland.
Table 2. EXPRESSION OF ACTH-R MRNA IN ADRENAL ADENOMA AND ATTACHED ATROPHIC NORMAL GLAND
Expression of ACTH-R mRNA was normalized by that of GAPDH mRNA and the data were represented as mean ± SE. Statistical analysis was carried out using one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) analysis.
In correlation with the ACTH-R mRNA expression, the expression of P450scc mRNA was also not suppressed in the adenoma tissue. The abundance of P450scc mRNA in the adenoma was significantly greater than in the attached atrophic gland. The expression of P450scc mRNA showed a significant positive linear correlation (r = 0.846, P < .001) with the expression of ACTH-R mRNA both in the adenoma and the attached adrenal (Fig. 5), indicating that ACTH-R in the adenoma is functional.
Figure 5. Expression of adrenocorticotropin receptor (ACTH-R) and cytochrome P450 side chain cleavage enzyme (P450scc) mRNAs shows a significant positive linear correlation. Expression of ACTH-R and P450scc mRNAs was normalized by that of glyceryldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The numbers adjacent to the open squares or filled diamonds correspond to the patient numbers in Table 1. r = 0.846;P < .001.
Indeed, sequencing of the entire coding region of human ACTH-R revealed no mutation in all the patients with Cushing adenoma (data not shown).
DISCUSSION
Cushing adenoma responds to exogenous ACTH. As Bertagna and Orth 19 reported, the serum cortisol level was increased after ACTH administration. The administration of exogenous ACTH increased the plasma cortisol concentrations in 22 dogs with hyperadrenocorticism caused by adrenocortical neoplasia. 23 The attached adrenal glands are severely atrophic and the steroidogenesis in the attached atrophic adrenal is markedly decreased in the patient with Cushing syndrome, indicating that the increased steroids after ACTH administration are produced from the adrenocortical adenoma and not from the attached or contralateral atrophic adrenals. The failure of serum cortisol levels to increase after ACTH administration 3 months after adrenalectomy in patients with Cushing syndrome (see Fig. 3) indicated that the atrophic adrenal glands had not been able to produce cortisol under excess ACTH administration. Doherty et al 24 reported that patients with Cushing syndrome from adrenocortical adenoma required a median of 15 months (range 9–22) to recover a normal response to the ACTH stimulation test. Although Cushing adenomas are independent of endogenous ACTH control, these results indicate that the functional ACTH-R is expressed in the adrenocortical adenoma and adenyl cyclase pathways are retained, which are responsible for stimulating adrenocortical steroid production in these patients. Indeed, the expression of ACTH-R mRNA was not suppressed, or indeed it even increased in the adrenocortical adenoma.
In rats, ACTH increases ACTH-R mRNA. 7 It has also been shown in vitro that ACTH positively regulates ACTH-R mRNA in primary cultures of bovine and human adrenocortical cells. 8,9 ACTH-R mRNA abundance in the attached atrophic adrenals in patients with Cushing syndrome was far less than in the normal gland obtained from a patient with renal cancer (see Fig. 4). ACTH-R mRNA abundance in the attached adrenals with aldosterone-producing adenoma and nonfunctioning adrenal adenoma was not suppressed compared with that in the normal gland obtained from a patient with renal cancer. These results indicate that ACTH positively regulates its own receptor mRNA in human adrenal gland as well. The enhanced glucocorticoid secretion observed in humans after repeated or long-term treatment with ACTH, as well as in patients with Cushing disease or ectopic ACTH syndrome, is probably caused not only by the positive effect of ACTH on the expression of the gene encoding the enzymes of the steroidogenic pathway, 25 but also by the positive effects of the hormone on its own receptors. Adrenal steroidogenic cytochrome P450s are strongly expressed in the adrenocortical adenoma in patients with Cushing syndrome, 20 and the increased expression of ACTH-R may contribute to the increased expression of P450s in the adrenocortical adenoma in these patients. This notion is supported by our present finding that the expression of ACTH-R and P450scc mRNAs shows a significant positive linear correlation. Reincke et al 26 also reported that ACTH-R mRNA expression was correlated closely with the expression of P450scc mRNA in adrenocortical adenoma. P450scc is the rate-limiting step of steroidogenesis, and P450scc mRNA expression is increased by ACTH administration. 27 It is likely that increased ACTH-R expression followed by constitutive activation of adenyl cyclase and elevated intracellular cAMP levels results in increased expression of P450scc, thus stimulating adrenocortical steroidogenesis. However, the observation that the plasma ACTH level is suppressed in these patients raises a question of how ACTH-R can be activated in the adenoma in the absence of its ligand.
Activating mutation of ACTH-R in the adrenocortical adenoma in the patients with Cushing’s syndrome is one of the possible causes of the autonomous cortisol production from the adenoma. Activating mutations of the thyroid-stimulating hormone (TSH) receptor in the functioning thyroid nodules are the typical model of this kind of disorder. 12 Mutation of specific residues within the carboxy-terminal portion of the third intracytoplasmic loop of the α1b adrenergic receptor results in constitutive activation of phospholipase C, and this segment is probably critical for the activation of G proteins in the gene family. 28 Constitutively activating mutations of the human luteinizing hormone receptor causing familial male precocious puberty were found in the sixth transmembrane domain, 29 and three different, naturally occurring activating mutations were reported in the second transmembrane domain and first intracellular loop of the mouse skin melanocyte-stimulating hormone receptor, producing altered coat colors. 30 However, in concordance with previous reports, 16,17 we found no mutation in the coding region of ACTH-R in the adrenal adenomas from patients with Cushing syndrome. When the TSH receptor of normal sequence was transfected into COS-7 cells, intracellular cAMP production was increased without ligand addition, whereas TSH administration exaggerated the cAMP production. 31 Increased cAMP production by basal expression of normal TSH receptor in COS-7 cells suggests that an activating mutation is not always necessary, but expression of G-protein-coupled receptor itself is important to cause stimulation of adenyl cyclase pathways. The pathogenesis of adrenocortical tumorigenesis and nonsuppressed expression of ACTH-R in Cushing adenoma is not due to an activating mutation of ACTH-R. However, the expression of ACTH-R itself may be responsible for tumorigenesis or stimulating steroidogenesis.
It remains to be studied how ligand-independent ACTH-R gene expression occurs in the adenoma. The promoter region of the human ACTH-R gene has been cloned. 6 Abnormalities in the promoter region of the ACTH-R gene or in the factors regulating its expression might be one of the causative factors for the expression of ACTH-R in Cushing adenoma. Other possibilities could be increased mRNA stability or decreased degradation of ACTH-R mRNA. Studying the regulation of ACTH-R expression in the adrenocortical adenoma may provide better insight into the pathogenesis of Cushing syndrome.
Acknowledgments
The authors thank Dr. Samuel Refetoff and Dr. Roy Weiss, University of Chicago, for their critical comments and suggestions.
Footnotes
The first two authors contributed equally to this work.
Supported in part by Grants-In-Aid (06671197) for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
Correspondence: Hisao Seo, MD, PhD, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
E-mail: hseo@riem.nagoya-u.ac.jp
Accepted for publication December 13, 2000.
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