Localization and expression of adrenocorticotropic hormone receptor mRNA in normal and neoplastic human adrenal cortex
M Reincke, F Beuschlein, G Menig, G Hofmockel1, W Arlt, R Lehmann, M Karl2 and B Allolio
Schwerpunkt Endokrinologie, Medizinische Universitätsklinik Würzburg, 1Urologische Universitätsklinik Würzburg, Germany and 2Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland, USA
(Requests for offprints should be addressed to M Reincke, Medizinische Universitätsklinik Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany)
Abstract
The recent cloning of the ACTH receptor (ACTH-R) gene allows investigation of the tissue localization and relative abundance of ACTH-R mRNA in normal and neoplastic adrenal cortex. Using in situ hybridization (ISH) we studied the expression of ACTH-R mRNA in four adult adrenals of brain-dead patients, two cortisol- producing adenomas (CPA), three aldosterone-producing adenomas (APA), one non-functional adenoma (NFA), and three carcinomas. The results were compared with the mRNA expression of key steroidogenic enzymes and of the glucocorticoid receptor (GR) mRNA using Northern blotting. In adult adrenals, messenger RNA encoding ACTH-R was localized in all three zones of the adrenal cortex, in accordance with the stimulatory role of ACTH on mineralocorticoid, glucocorticoid and adrenal androgen secretion. In comparison, expression of side-chain cleavage enzyme (P450scc) showed a similar tissue distribution with mRNA abundance in all three zones, whereas 17- hydroxylase/17-20 lyase (P450c17) mRNA expression was only detected in the zona fasciculata and zona reticu-
laris. All CPAs and APAs expressed significant levels of ACTH-R mRNA whereas an NFA showed low expression of ACTH-R mRNA. Two of three adrenocortical carcinomas expressed ACTH-R mRNA. Northern analysis using dot blot was employed to quantify ACTH-R and GR mRNA expression and confirmed the ISH data: ACTH-R mRNA expression was high in CPAs (275 and 195% vs 100 ± 25% in adult adrenals), APAs (127, 200 and 221%) and two carcinomas (99 and 132%), but low in the NFA (7%) and in an androgen secreting carcinoma (16%). GR mRNA expression was high in the NFA (195%) and in two of three carcinomas (93, 188, 227%). We conclude that ACTH-R mRNA is up- regulated in functional adenomas by yet unidenti- fied mechanisms. The tissue distribution of ACTH-R and P450 enzyme mRNA expression is highly vari- able in neoplastic adrenals and does not allow a clear differentiation between benign and malignant tumors. Journal of Endocrinology (1998) 156, 415-423
Introduction
Adrenocorticotropic hormone (ACTH) is, besides angio- tensin II, the major peptide regulating adrenocortical steroidogenesis. ACTH stimulates, via the G-protein coupled ACTH receptor (ACTH-R), intracellular cAMP accumulation which in turn increases the transcription of the P450 side-chain cleavage (P450scc) enzyme, the initial and rate-limiting step of adrenal steroidogenesis (Miller 1988).
Recently, the ACTH-R gene has been cloned and shown to belong, together with four melanocyte- stimulating hormone (MSH) receptors, to the melanocor- tin receptor family (Mountjoy et al. 1992, Siegrist & Eberle 1995). Northern blot analysis demonstrated expression of
ACTH-R in the adrenal gland, in human skin and in rodent adipocytes, but not in a variety of other tissues (Mountjoy et al. 1992, Boston & Cone 1996, Slominski et al. 1996). In addition, high ACTH-R mRNA expres- sion was reported in functional adrenal tumors but not in non-functional adenomas (Morita et al. 1995, Arnaldi et al. 1997, Reincke et al. 1997). In situ hybridization (ISH) of ACTH-R mRNA revealed high abundance mRNA expression in the adrenal cortex of Rhesus monkeys (Mountjoy et al. 1992) and in the mid-gestational human fetal adrenal cortex (Mesiano et al. 1996). However, no ISH experiments analyzing the mRNA distribution in the adult human adrenal cortex and in adrenocortical tumors were reported. We, therefore, investigated the ACTH-R mRNA expression in normal and neoplastic human
| No. | Age (years) | Sex | Adrenal weight (g) | |
|---|---|---|---|---|
| Tissue | ||||
| Adult adrenals of brain-dead patients | 4 | 29-55 | 1 F, 3 M | <10 |
| Non-functioning adenoma | 1 | 49 | 1 F | 70 |
| Cortisol-producing adenomas | 2 | 34-53 | 2 F | 22-35 |
| Aldosterone-producing adenoma | 3 | 33-62 | 2 F 1 M | 14.5-34 |
| Carcinoma | 3 | 20-79 | 3 F | 119-1600 |
| Cushing's syndrome/virilization | 1 | |||
| Mineralocorticoid excess | 1 | |||
| Virilization | 1 |
adrenal tissue and compared the results with the expression of key steroidogenic enzymes and glucocorticoid receptor expression.
Materials and Methods
Tissues
Adrenal tumor tissue (n=9) was collected from patients undergoing adrenalectomy after giving written informed consent (Table 1). Human adult adrenal glands (n=4) (Table 1) were obtained after organs were removed from brain-dead patients for transplantation with the approval of the ethical committee of the University Hospital of Würzburg. After removing adjacent fat tissue the adrenals were snap-frozen and immediately stored at - 80 ℃ until processing.
In situ hybridization
Cryostat sections (12 um) were cut at approximately - 25 ℃ through the entire adrenal gland or tumor tissue and thaw-mounted onto gelatine-coated slides and dried for 30-60 s. ISH was performed as previously described (Whitfield et al. 1990, Ausubel et al. 1992) to identify areas containing mRNA for ACTH-R, P450scc enzyme, and 17-hydroxylase/17-20 lyase (P450c17) mRNA. The cryostat sections were fixed by immersion in 4% paraformaldehyde-1 × PBS for 5 min and rinsed twice in 1 × PBS. Tissue acetylation was performed by immersion in 0.25% acetic anhydride in 0.1 M TEA/NaCl. After tissue dehydration in 100% alcohol and chloroform, the slides were stored at 4 ℃ until hybridization.
cDNA probes for ACTH-R (full length cDNA gener- ated by PCR (Mountjoy et al. 1992)), human P450scc and human P450c17 (provided by Dr W L Miller, (Chung 1986, 1987, Picado-Leonhard & Miller 1987)) were labeled with 35S-CTP and 35S-ATP (Du Pont, Bad Homburg, Germany) by a Random Primed Labeling Kit (Boehringer Mannheim, Mannheim, Germany), purified by Nuc Trap Push Columns (Stratagene, Heidelberg,
Germany) and diluted with hybridization solution to 20 000 c.p.m./ul (final concentration: 500 000 c.p.m./ section). Negative control sections were digested with RNAase (20 µg/ml) for 30 min at 37 ℃ prior to tissue acetylation. After washing with 1 x SSC, formamide/ 4 × SSC and 1 × SSC the slides were subjected to film autoradiography (Kodak X-Omat) for 5 to 10 days. To localize autoradiographic silver grains, the hybridized sections were dipped into Kodak NTB-2 nuclear track emulsion. Counterstaining was performed with Mayer’s hematoxylin-eosin. The relative mRNA expression was evaluated semiquantitatively using a score from ‘negative’ to ’++’, with ’+’ corresponding to mRNA expression equivalent to that of the normal zona fasciculata.
Northern blots
Poly A-RNA was isolated from adult adrenal cortex of brain-dead patients and from tumor tissue using the guanidinium thiocyanate method (Messenger RNA Iso- lation Kit, Stratagene, Heidelberg, Germany) avoiding necrotic areas. The integrity of the RNA was checked by visualization with ethidium bromide on an agarose gel. The RNA was directly dot blotted on a nylon membrane and cross-linked by exposure to UV light. Hybridization was performed using the same probes as for ISH and a glucocorticoid receptor (GR) cDNA provided by Dr R M Evans (Hollenberg et al. 1985). We found a good corre- lation between the intensity of the major mRNA species of a conventional Northern blot (ACTH-R mRNA: 2.0 and 3.8 kb; P450scc: 2.0 kb; P450c17: 1.9 kb; and GR mRNA: 2.0 and 6.1 kb) and the dot blot experiments. Radioactive labeling was performed by means of a Ran- dom Primed Labeling Kit (Boehringer Mannheim) using 32P-dCTP. The blots were washed twice in 1 x SSC and 0.5 × SSC (each containing 0.1% SDS) at 60 ℃. After exposing for autoradiography at - 80 ℃ with intensifying screens, resulting dots were quantified by scanning densi- tometry, while the blots were stripped and rehybridized. For standardization the blots were hybridized with a mouse ß-actin cDNA probe. The steady state mRNA
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concentrations are expressed as a percentage of normal adrenals (100%). Correlation between ACTH-R mRNA and GR mRNA concentrations was determined by linear regression analysis and expressed as Pearson’s correlation coefficient. A P<0.05 was considered statistically signifi- cant. Autoradiographic images were digitized with a video camera and a Macintosh PowerMac 7100 computer-based image analysis system using the IMAGE program (NIMH, National Institutes of Health, Bethesda, MD, USA).
Results
Messenger RNA encoding the ACTH-R mRNA was localized in sections of the adrenals of brain-dead patients (Fig. 1). Specific staining for ACTH-R mRNA was detected in cortical cells of all three zones. The intensity of staining did not show consistent differences between zona glomerulosa, zona fasciculata or zona reticularis (Figs 1 and 2). ISH of P450scc mRNA showed a similar pat- tern with even expression in all three zones of the adrenal cortex, whereas P450c17 mRNA expression was
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| ACTH-R mRNA | P450scc mRNA | p450c17 mRNA | |
|---|---|---|---|
| Non-functional adenoma | Negative | Negative | Negative |
| Case 1 | |||
| Cortisol-producing adenoma | |||
| Case 2 | + +, inhomog. | + +, inhomog. | ++, homog. |
| Case 3 | + +, inhomog. | +, inhomog. | ++, inhomog. |
| Aldosterone-producing adenoma | |||
| Case 4 | + +, homog. | +, inhomog. | +, inhomog. |
| Case 5 | +, homog. | +, homog. | +, homog. |
| Case 6 | n.d. | +, inhomog. | +, inhomog. |
| Carcinoma | |||
| Case 7, Cushing's syndrome/virilization | +, homog. | (+), homog. | ++, homog. |
| Case 8, mineralocorticoid/hypertension | +, inhomog. | +, inhomog. | +, inhomog. |
| Case 9, virilization | n.d. | + +, homog. | +, homog. |
Homog., homogeneous mRNA expression by film autoradiography; inhomog., inhomogeneous mRNA expression; (+), weak; +, mRNA expression equivalent to the normal zona fasciculata; + +, strong expression; n.d., not determined.
(a)
ACTH-R
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undetectable in zona glomerulosa, and high in zona fasciculata and zona reticularis (Figs 1 and 2).
Specific ACTH-R mRNA was found in the majority of adrenocortical tumors (Table 2). ACTH-R mRNA abundance was noted in benign cortisol-producing adenomas (CPAs) and aldosterone-producing adenomas (APAs) (Figs 3 and 4) but not in the non-functional adenoma (NFA) (Fig. 5), whereas adrenocortical carci- nomas showed a variable ACTH-R mRNA expression. P450scc mRNA expression was found in all functional tumors although the level of expression was variable. All
APA expressed P450c17 mRNA to some degree, which is surprising, since 17-hydroxylase is not required for mineralocorticoid synthesis. The tissue distribution of ACTH-R and P450 enzyme mRNA was inhomogeneous in most of the tumors due to regressive changes in the tumor tissue (cystic areas, necrosis) (Table 2).
Quantitative assessment of mRNA expression by dot blot confirmed the ISH findings (Fig. 6 and Table 3). ACTH-R mRNA expression was high in mineralo- corticoid- and glucocorticoid-producing adenomas (CPA 195, 275%, APA 127-221%, vs 100% in adult adrenals)
(a) ACTH-R
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and low in the NFA (7%) and in one of three carcinomas. High steady-state GR mRNA levels were found in the NFA and in two of three carcinomas but not in CPAs and APAs (Fig. 6). We found a negative correlation between ACTH-R mRNA and GR mRNA expression (Fig. 7; F =- 0.73, P=0-02).
Discussion
In mammals, ACTH acutely regulates glucocorticoid secretion of the adrenal gland by binding to its receptor and stimulation of cAMP production (Miller 1988). More chronically, ACTH is necessary for the normal develop- ment of the adrenal cortex and increases adrenal cortex size (Lebrethon et al. 1994, Albrecht et al. 1996, Mesiano et al. 1996). Binding studies using Tyr-23 labeled ACTH have demonstrated the presence of specific adrenocorticotropin receptors in adrenal cells of several species (Buckley & Ramachandran 1981). The ACTH-R gene was cloned in 1992 (Mountjoy et al. 1992). Its sequence of 297 amino acids is highly homologous to the sequence of several MSH receptors (MSH-R) which, therefore, are sum- marized as the melanocortin receptor family (Siegrist & Eberle 1995). Northern blot experiments demonstrated that the ACTH-R is mainly expressed in the adrenal cortex, whereas MSH-R mRNAs are distributed widely throughout the organism. An intriguing finding is upregulation of ACTH-R mRNA by its own ligand, ACTH, which has been demonstrated in human and bovine fasciculata-reticularis cells (Lebrethon et al. 1994, Penhoat et al. 1994) and in adrenocortical cancer cell lines (Mountjoy et al. 1994).
Localization and relative abundance of the ACTH-R in normal and neoplastic human adrenals and comparison
with the regional distribution of steroidogenic enzyme mRNA such as P450scc or P450c17 have not been studied previously. The adrenal cortex is composed of three distinct zones, the subcapsular zona glomerulosa which secrets mainly mineralocorticoids, the glucocorticoid- producing zona fasciculata and the androgen-producing zona reticularis, the innermost cortical zone (Neville & O’Hare 1985). The physiology of steroid secretion suggests expression of ACTH receptors in all three zones of the adrenal cortex, since acute administration of ACTH results in enhanced mineralocorticoid (Kater et al. 1989), gluco- corticoid and adrenal androgen secretion (Rosenfeld et al. 1975). Our data are in accordance with this notion, with localization of ACTH receptor mRNA by ISH in all three adrenocortical zones. In mice, ACTH-R mRNA expres- sion was found mainly in zona glomerulosa and fasciculata (Xia & Wikberg 1996). Zona reticularis cells expressed ACTH-R mRNA to a lesser degree than zona glomeru- losa and fasciculata cells, and a few scattered cells expressed ACTH-R mRNA in the mouse adrenal medulla. The different pattern of ACTH-R mRNA distribution in human and mouse zona reticularis can represent an inter- species difference. However, since we used adrenal tissue from brain-dead patients, major physical stress with acti- vation of the hypothalamic-pituitary-adrenal axis could have changed the ACTH-R mRNA distribution in our adrenals.
P450scc initiates steroidogenesis in the adrenal and the gonads by conversion of cholesterol to pregnenolone. It is the rate-limiting step of steroid hormone biosynthesis and is essential for production of mineralocorticoids and glucocorticoids, as well as sex hormones, which explains the even distribution of P450scc mRNA in the three zones of the adrenal cortex (Miller 1988). P450c17 mRNA expression was not detected in the zona
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P450scc
.
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+RNAase
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glomerulosa, but was high in the zona fasciculata and zona reticularis. P450c17 is the key enzyme responsible for glucocorticoid and adrenal androgen production and encodes for P450c17a-hydroxylase responsible for conver- sion of pregnenolone to 17-hydroxy-progesterone as well as for 17-20 lyase activity converting 17-hydroxy- progesterone to dihydroepiandrosterone (Chung et al. 1987). P450c17 activity is not required for mineralo- corticoid production, which, hence, explains its missing expression in the zona glomerulosa.
Data on in situ localization of ACTH-R mRNA, P450scc and P450c17 enzymes in the fetal adrenal gland have been reported (Mesiano et al. 1993, 1996). The developing fetal adrenal cortex consists, similar to the adult cortex, of three zones, the definitive zone which corre-
sponds to the zona glomerulosa in the adult adrenal, the innermost fetal zone corresponding to zona reticularis in the adult and the transitional zone between fetal and definitive zones. In adrenal tissue from midgestational human fetuses (16-24 weeks) messenger RNA encoding the ACTH-R was localized in cells from all cortical zones, with higher abundance in the definitive zone compared with the fetal zone (Mesiano et al. 1996). P450scc and P450c17 mRNA were only detected in the fetal zone and transitional zone cells but not in definitive zone cells (Mesiano et al. 1993). In the baboon fetal adrenal gland, a biphasic developmental expression of ACTH-R mRNA was detected, with minimal expression in early gestation, high expression in midgestation characterized by a 13-fold increase, and a decline of ACTH-R mRNA expression by
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GR
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70% in late gestation, apparently mainly in the fetal zone, reflecting diminished synthesis of adrenal androgens (Albrecht et al. 1996).
Aldosterone- and glucocorticoid-producing adreno- cortical adenomas are responsive to ACTH in vivo and in vitro, suggesting the expression of functional ACTH-Rs (Ishizuka et al. 1988, Kajitsu et al. 1994). In accordance with this notion, we found high ACTH-R mRNA expression in CPAs and APAs by means of ISH but not in an NFA. Since plasma ACTH is suppressed in patients with CPA via the negative feedback of glucocorticoids, ACTH can not be the main factor explaining upregulation of ACTH-R mRNA expression in these tumors. Our data on ISH are in accordance with previous data from our laboratory showing high ACTH-R mRNA expression by Northern blotting in a large series of APA and CPA, but
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low expression in non-functional adenomas (Reincke et al. 1997). In that series we also observed a significant positive correlation between ACTH-R mRNA and P450scc mRNA expression in adenomas. Regulation by similar factors is supported by the fact that cAMP response elements and steroidogenic factor-1 binding sites are found in both the ACTH-R and P450 steroidogenic enzyme promoters (Naville et al. 1997).
Using ISH on a limited number of tissues in this series, tumors with significant ACTH-R mRNA expression also had P450scc mRNA expression. However, the abun- dance of ACTH-R mRNA and the association with P450scc expression was variable. This may in part be due to inter-experiment variations, such as the efficiency of the
| ACTH-R (%) | P450scc (%) | P450c17 (%) | GR (%) | |
|---|---|---|---|---|
| Normal adrenals of brain-dead patients (n=4) | 100 ± 25 | 100±7 | 100± 22 | 100± 29 |
| Non-functional adenoma | ||||
| Case 1 | 7 | 30 | 8 | 195 |
| Cortisol-producing adenoma | ||||
| Case 2 | 275 | 151 | 120 | 20 |
| Case 3 | 195 | 133 | 112 | 105 |
| Aldosterone-producing adenoma | ||||
| Case 4 | 221 | 129 | 128 | 102 |
| Case 5 | 200 | 63 | 36 | 80 |
| Case 6 | 127 | 67 | 68 | 85 |
| Carcinoma | ||||
| Case 7, Cushing's syndrome/virilization | 99 | 24 | 60 | 93 |
| Case 8, mineralocorticoid/hypertension | 132 | 78 | 52 | 188 |
| Case 9, virilization | 16 | 82 | 68 | 227 |
422 M REINCKE and others . ACTH receptor localization in adrenal tissue
labeling reaction and differences in the autoradiographic exposure time, which are a general problem of ISH. The dot blot procedure which represents a single experiment and is normalized for ß-actin expression is a more reliable tool for inter-tumor comparisons. Nevertheless, based on the findings of this study a distinction between a benign and malignant tumor phenotype using the abundance of ACTH-R and P450 mRNA seems not to be possible.
All APA in this study expressed P450c17 mRNA. This has been described previously by other investigators (Racz et al. 1993), demonstrating zona fasciculata-like steroido- genesis in some of the APAs. In vitro studies using dispersed cells from adrenocortical adenomas showed that ACTH and phorbol ester can induce cortisol secretion in some APAs similar to CPAs (Ishizuka et al. 1988, Kajitsu et al. 1994). Whether this indicates that these APAs are derived from the P450c17-expressing zona fasciculata has to be determined.
An interesting aspect of adrenal tumorigenesis is the variable expression of GR mRNA in our tumor material. Using dot blotting, we found upregulation of GR mRNA in the NFA and in two of three carcinomas, but not in CPAs and APAs. One explanation for this finding could be the existence of a glucocorticoid ultrashort intra-adrenal feedback that, under physiological circumstances, inhibits GR mRNA expression in adrenocortical cells. In this notion, missing or ineffective glucocorticoid secretion, such as those of NFAs or carcinomas, can result in upregulation of GR mRNA in the tumor tissue. In accordance with this hypothesis, we observed that, in the glucocorticoid-producing NCI-h295 adrenocortical carcinoma cell line, adrenostatic treatment with amino- glutethimide, a potent inhibitor of the side-chain cleavage enzyme, resulted in upregulation of GR mRNA, whereas stimulation of glucocorticoid synthesis by ACTH and forskolin lowered GR mRNA expression (M Reincke, unpublished observations).
Acknowledgements
This study was supported by the Deutsche Forschungs- gemeinschaft (Re 752/5-1).
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Received 16 April 1997 Revised manuscript received 4 August 1997 Accepted 24 September 1997