Neuro -endocrinology
Neuroendocrinology 2005;82:274-281 DOI: 10.1159/000093126
Corticotropin-Releasing Hormone Receptor Expression on Normal and Tumorous Human Adrenocortical Cells
Holger S. Willenberga Matthias Haaseª Claudia Papewalisa Matthias Schottª Werner A. Scherbauma Stefan R. Bornsteinb
aDepartment of Endocrinology, Diabetes and Rheumatology, University of Düsseldorf, Düsseldorf, and b Department of Endocrinology, Diabetes and Metabolism, University of Dresden, Dresden, Germany
Key Words
Adrenal gland · Corticotropin-releasing hormone receptors . Steroidogenesis . Adrenal steroids · Pheochromocytoma . Cushing’s disease . Neuroendocrine tumors . Clinical neuroendocrinology
Abstract
Corticotropin-releasing hormone (CRH) is not only the principal regulator of the central hypothalamic-pituitary- adrenal (HPA) axis but also exerts direct actions on pe- ripheral tissues. We analyzed the expression of CRH re- ceptors in microdissected preparations of normal human adrenal glands and in adrenocortical and adrenomedul- lary tumors, employing immunohistochemistry, quanti- tative RT-PCR of microdissected adrenal tissues, and in situ hybridization. The effect of CRH on adrenal steroido- genesis was tested in adrenal cells. Immunoreactive CRH1R was found primarily within the zona reticularis. In addition, we found a higher expression of CRH type-1 and 2 receptors mRNAs in preparations of adrenal corti- ces as compared to pheochromocytomas, a 6-fold in- crease in preparations of clinically unapparent adreno- cortical adenomas, and a 10- to 60-fold increase in cortisol-producing adrenal adenomas. Stimulation of
This work was supported by a grant from the University of Düs- seldorf to H.S.W.
the adrenal tumor cell line NCI-H295R with CRH elicited a 1.4-fold increase in DHEA secretion. This result could be reproduced in a culture of primary human adrenocor- tical cells. We conclude that adrenocortical cells exhibit a higher expression of functional CRH receptors than chromaffin cells and that CRH acts on adrenal DHEA pro- duction. The data support the assertion of a direct action of CRH on human adrenocortical cells in addition to an intra-adrenal CRH receptor/adrenocorticotropin system. Enhanced CRH1R expression may be involved in adre- nocortical tumorigenesis.
Copyright @ 2005 S. Karger AG, Basel
Introduction
Corticotropin-releasing hormone (CRH) is not only the principal regulator of the hypothalamic-pituitary-ad- renal (HPA) axis and activator of the sympathoadrenal and systemic sympathetic systems, but also exerts pro- found actions on a number of peripheral tissues [1-4]. As an executive organ of the stress system the adrenal gland hosts the chromaffin and adrenocortical tissue, both of which show a remarkable interdependence [5, 6] and are controlled by CRH through activation of the HPA axis and the sympathoadrenal system [7].
However, there is evidence for a direct involvement of CRH in regulating adrenal function on the level of the adrenal gland as well. It was observed that, after ovine
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CRH infusion and insulin-induced hypoglycemia in healthy men, cortisol levels were higher than values that would correspond to corticotropin (ACTH) levels [8]. In addition, in functionally hypophysectomized calves, CRH increased adrenal steroidogenesis [9], and in hypophysec- tomized rats high doses of CRH reduced adrenocortical atrophy [10]. Besides, neutralizing antibodies to CRH sig- nificantly attenuated the ACTH-mediated rise in corticos- terone in rats [11] and ACTH failed to stimulate corticos- terone secretion in CRH type-1 receptor (CRH1R) knock- out mice despite upregulation of the ACTH receptor [12]. In vitro experiments showed that the CRH-mediated rise in cortisol production by adrenocortical cells in the pres- ence of chromaffin cells could be reversed completely by a-helical CRH (9-41) [13] or by co-incubation with the CRH1R antagonist antalarmin [14] .
Since 125I-labelled CRH binds to functional receptors in the adrenal medulla of rats [15] and monkeys [16], since adrenal chromaffin cells [17] and peripheral lym- phocytes [18] have the power to secrete ACTH and pro- opiomelanocortin (POMC)-derived peptides in response to CRH, and since rat adrenals and human adrenal cells in culture respond to CRH exposure with a significant rise in corticosterone secretion [14, 19, 20], a local CRH- ACTH-glucocorticoid system has been postulated [21, 22]. On the other hand, recent studies propose a rather direct action of CRH on fetal adrenocortical cells, dem- onstrating an increase in DHEA synthesis by adrenocor- tical cells after stimulation with CRH [23, 24].
However, little is known about the mechanisms of this complex proposed interaction, except for the interaction between adrenal medulla and cortex [6, 25, 26].
With the recent development of a commercially avail- able antibody directed to CRH1R and the combination of light microscopy-guided laser microdissection with the possibility of subsequent molecular studies, we have been put in the position to study the distribution of immuno- reactive CRH-receptor protein and mRNAs coding for type-1 and -2 CRH receptors. In this study we therefore addressed the questions as to which cells of the adrenal gland express CRH receptors, and whether the interfer- ence of CRH with the adrenal steroidogenesis on the lev- el of the adrenal gland could also be the consequence of a direct action on adrenocortical cells.
Materials and Methods
The procedure was approved by the Ethical Committee of the University of Düsseldorf, Germany. Formalin-fixed tissues from normal human adrenal glands (n = 4) after removal for renal carci-
noma, cortisol-producing adrenocortical (n = 4) and catechol- amine-producing adrenomedullary (n = 4) adenomas and adreno- cortical carcinomas (n = 4) were embedded in paraffin for immu- nohistological studies. In addition, we obtained tissue from normal human adrenal glands (n = 3) after removal for renal carcinoma, from benign adrenocortical adenomas with (n = 3) and without (n = 3) apparent glucocorticoid excess, and from catecholamine- secreting adrenal pheochromocytomas (n = 3) which were trans- ferred to dry ice and kept frozen for microdissection and down- stream molecular studies. Cells of one normal human adrenal gland were cultured for in vitro studies. In addition, the adrenocortical cancer cell line NCI-H295R was employed for in vitro studies, in- cluding DHEA secretion experiments.
Immunohistology
A rabbit polyclonal antibody directed to the rat and cross-react- ing with the human ACTH receptor (MC2R; clone ab1579, abcam, Cambridge, UK) served to characterize adrenocortical cells in par- affin-embedded tissue sections and was used in dilutions of 1:100. A mouse monoclonal antibody to human chromogranin A (clone DAK-A3, DakoCytomation, Hamburg, Germany) was used to iden- tify chromaffin cells in the dilution of 1:500, as described previ- ously [27]. For the detection of CRH receptors we made use of a polyclonal goat antibody raised against the rat CRH1R (sc-1757, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) in a 1:200 dilution [4]. This antibody was reported to also recognize the hu- man CRH1R and CRH type-2 receptor (CRH2R), the latter with a much lower affinity. Exposure of tissue sections to the primary antibodies at room temperature lasted for 60 min. Detection of bound immunoglobulins was achieved by applying a biotinylated secondary pig anti-rabbit, pig anti-mouse, or pig anti-goat antibody (DakoCytomation) at room temperature for 30 min, followed by incubation with peroxidase-linked streptavidin-biotin complexes (DakoCytomation) at room temperature for 30 min or, in a second independent technique, through binding of a goat anti-rabbit (or anti-mouse) antibody- and peroxidase-linked polymer (En Vision, DakoCytomation) to bound primary antibodies, for 60 min at room temperature. For detection of digoxigenin-labelled probes we used the catalyzed signal amplification method as described by Bobrow et al. [28] and DakoCytomation (Cat No. 20157).
For visualization 0.05% DAB or 0.01% AEC and 0.0003% H2O2 in 0.5 M Tris-HCI buffer (pH 7.6) was used for 5-7 min. Washing steps were performed between all incubation steps except after pre- incubation with 2% normal swine serum (DakoCytomation) in 0.5 M Tris-HCl (pH 7.6) before applying the fi rst antibody and in- cluded rinsing in tap water for 10 s before being allowed to sit in 0.5 M Tris-HCl (pH 7.6) for 10 min. All sections were counter- stained with hematoxylin (Merck, Darmstadt, Germany) and mounted onto gelatin-coated glass slides. Control experiments were carried through at different levels, skipping the incubation step with the primary antibody, skipping the incubation step with the secondary antibody or the polymer, and for control of the CRH1R antibody, it was preabsorbed with the corresponding blocking pep- tide (sc-1757 P, Santa Cruz Biotechnology).
Laser Microdissection [29], RNA Isolation, and Reverse- Transcription Polymerase Chain-Reaction (RT-PCR)
Sections (8 pm) of all frozen adrenal tissues were fixed in 70% (v/v) ethanol, hematoxylin-stained and dehydrated in rising etha- nols and, eventually, xylol. Adrenocortical cells were separated
| Product | Primer | Sequences (5'-3') |
|---|---|---|
| CRH1R | Sense | CgC ATC CTC ATg ACC AAg CT |
| Antisense | TCA CAg CCT TCC TgT ACT gAA Tg | |
| TaqMan probe | Cgg gCA TCC ACC ACg TCT gAg A | |
| CRH2R | Sense | CCC ggg CCA TgT CCA T |
| Antisense | ACA gCg gCC gTC TgC TT | |
| TaqMan probe | CTA CAT CAC CCA CAC ggC TCA gCT TCC | |
| 18 S | Sense | Cgg CTA CAA CAT CCA Agg AA |
| Antisense | gCT ggA ATT ACC gCg gCT | |
| TaqMan probe | TgC Tgg CAC gAg ACT TgC CCT C |
from chromaffin cells using the AS LMD microscope (Leica Micro- systems, Wetzlar, Germany). Microdissected material was subject- ed to isolation of RNA using the RNeasy Micro Kit (Qiagen, Hilden, Germany), including a DNase I digestion step. The ob- tained RNA (1 µg) was reversely transcribed to cDNA using the Ready-To-Go random-primed first strand kit (Amersham Biosci- ences AB, Uppsala, Sweden), skipping reverse transcriptase for negative control reactions. For semiquantitative TaqMan-PCR amplification, we used primers and probes for CRH1R, CRH2R, GAP-DH and 18S for normalization as given in table 1, and ap- plied the conditions as follows: 40 thermal step cycles of denatur- ation at 95°℃ for 15 s and annealing/elongation at 62℃ for 45 s. All experiments were performed three times in triples for one cal- culation. Semiquantitative analysis was performed applying the comparative CT method in separate tubes, and the 18S primer set was used for calculations as an internal reference standard and for normalization. Results are expressed as a percentage of mRNA ex- pression in the tissue of interest compared to mRNA expression in the permanent cancer cell line NCI-H295R ± SEM.
In situ Hybridization
Single-stranded DNA antisense probes were amplified by poly- merase chain reaction (PCR) with 3’ primers for CRH1R. Control sense probes were designed in the same manner, with 5’ primers (table 1). Isolated and transcribed mRNA from normal human ad- renal glands served as the template. Probes were purified using Mi- croSpin S-300 HR columns (Amersham Biosciences) and labelled with digoxigenin (Roche, Mannheim, Germany). The labelled probes were hybridized to 4% paraformaldehyde-fixed cryostat sections of normal human adrenal cortex. Hybridization was carried out at 60°℃ for 24 h. After hybridization, slides were washed sequentially up to 0.4 × SSC at 60℃. Hybridization and post-hybridization treatments were concomitantly carried out with antisense probe on control sec- tions. Tissue-bound probe was stained with a monoclonal antibody to digoxigenin (Roche) and detected as described above. Due to the lack of normal adrenal medulla, we used sections of adrenal pheo- chromocytomas as a control. A sense probe was used to control the specificity of the probe and the hybridization method.
In vitro Studies
After mechanical dissection of the adrenal cortex of one human adrenal gland, small pieces (ca. 1 mm3) were digested enzymati- cally using DNA se I (No. 9003-98-9, Sigma-Aldrich, Germany) and
collagenase from Clostridium histolyticum (Sigma-Aldrich). After dispersal primary adrenocortical cells were cultured in medium. NCI-H295R and primary adrenocortical cells were cultured in DMEM/F12 supplemented with insulin (66 nM), hydrocortisone (10 nM), 17-estradiol (10 nM), transferrin (10 µg/ml), selenite (30 nM), penicillin (100 U/ml), streptomycin (100 µg/ml), ascorbic acid (10 µg/ml), and 2 or 10% fetal bovine serum, respectively. Cells were initially grown in 75-cm2 flasks (Becton Dickinson, Heidel- berg, Germany) at 37℃ in a humidified atmosphere of 5% CO2/95% air. The medium was changed 3 times a week, and cells were sub- cultured every 7-10 days using Accutase (PAA Laboratories, Cölbe, Germany) for cell detachment. For experimental settings cells were subcultured from 70% confluent stock cultures in quadruplicates into 24-well culture plates (Becton Dickinson Falcon) at a density of 100,000 cells/cm2 for 48 h. The cells were then washed and incu- bated with 100 nM human CRH (Ferring, Kiel, Germany) in serum- free medium for 24 h before DHEA and cortisol concentrations were measured by radioimmunoassay (both DPC Biermann, Bad Nauheim, Germany) [4, 14]. For comparison with other studies we chose an incubation period of 24 h [14, 24, 30]. Exposure of the cells to 10 u.M forskolin (Sigma-Aldrich GmbH, Seelze, Germany), 100 nM ACTH or CRH vehicle served as positive and negative con- trols, respectively. Results are presented as a percentage of basal/ve- hicle secretion + SD. Intra- and interassay coefficients were less than 5%. Statistical analysis was performed using the ANOVA test with Bonferroni post hoc but done with the data obtained from the in vitro experiments with primary human adrenal cells (n = 1).
Results
Immunohistochemistry
Human adrenocortical cells could be identified using the antibody directed against the human MC-2 receptor (fig. 1A, C). In serial sections, the same cell type stained with the antibody to the CRH1R (fig. 1B, D). Adrenocor- tical cells of all zones could be labeled using this antibody, with a predominant staining in the zona reticularis (zR) and the zona glomerulosa (zG) and a weaker staining in the zona fasciculata (zF). Besides, benign adrenocortical
M
M
C
C
C
C
C
C
A
B
ZF
ZF
ZR
ZR
ZG
ZG
C
D
C
C
C
C
C
C
M
E
F
A
B
C
D
E
F
Willenberg/Haase/Papewalis/Schott/ Scherbaum/Bornstein
✽
CRH1R
✽
6,200
CRH2R
**
6,012
225
mRNA expression, %
Basal DHEA secretion, %
200
1,400
1,200
175
1,000
150
800
600
125
400
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0
75
Normal adrenals
Incidentalomas
Cortisol- producing adenomas
Pheochromo- cytomas
Basal
Forskolin 10 μ.Μ
ACTH 100 nM
CRH 100 nM
CRH 10 nM
A
B
C
D
tumors also stained with antibodies against ACTH and CRH receptors (fig. 2A, B, respectively) as well as 3 of 4 adrenocortical carcinomas (fig. 2C), while the normal ad- renal chromaffin cells and adrenal pheochromocytoma cells did not (fig. 1A-D, fig. 2D). However, chromaffin cells in normal adrenal medulla and in pheochromocy- toma did stain with the antibody to chromogranin A.
mRNA Expression Studies in Microdissected Tissue The adrenocortical tissue could be successfully dissect- ed from the adrenomedullary tissue (fig. 3C, D). Thus, we received medullary-free preparations from normal hu- man adrenal cortex, adrenal pheochromocytoma cells free from remaining cortical tissue and chromaffin cell- free preparations of adrenocortical tumors. There was al-
most no detectable expression of mRNA coding CRH1R or CRH2R in normal adrenal medulla and in pheochro- mocytomas (fig. 3A). There was a constant expression of CRH1R or CRH2R mRNA in those preparations which were rich in adrenocortical cells. If the expression in NCI-H295R cells is assumed to be 100%, the expres- sion in normal adrenal cortices was 101.5%. There was a 6-fold higher expression of messages for the CRH re- ceptors in adrenocortical adenomas without and a 10- to 60-fold higher expression in adrenocortical adeno- mas with autonomous secretion of glucocorticoids (fig. 3A) in comparison to the expression in NCI-H295R cells.
In situ Hybridization
Bound CRH1R anti-sense probe could only be de- tected in adrenocortical and not in adrenal medullary tissue (fig. 1E, F). Control sense probes could not be detected in either adrenocortical or in adrenomedullary tissue.
In vitro Studies
NCI-H295R responded to a 100-nM concentration of CRH after 24-hour exposure with a 137.5 ± 15.1% (p < 0.05) increase in DHEA secretion (fig. 3B). As expected, the response to forskolin was significant with an increase in DHEA secretion of 202.1 ± 16.0% (p<0.01). Cortisol secretion by NCI-H295R cells in response to 100 nM CRH however was not significant (106.9 ± 8.8%). Pri- mary human adrenocortical cells also responded to expo- sure to 100 nM ACTH, 100 nM CRH or 10 nM CRH with a 1.46-, 1.37- or 1.07-fold increase in DHEA secre- tion, respectively (fig. 3B) and a 2.63-, 1.07- or 1.13-fold increase in cortisol secretion, respectively.
Discussion
Since there are different assertions concerning the di- rect action of CRH on adrenal tissue, the aim of this study was to look at the distribution of the CRH receptor in normal adrenal glands and to examine whether the ob- served direct effects of CRH on adrenal steroidogenesis are mediated via chromaffin cells or due to direct target- ing of adrenocortical cells by CRH.
We were able to characterize adrenocortical cells using the anti-MC2R antibody, which was also shown to recog- nize viable adrenocortical cells in vitro. Then we showed that the antibody raised against the CRH1R also marked the cells which could be stained using the anti-MC2R an-
tibody, providing evidence that these cells were adreno- cortical cells. Consistent with the results, we found that expression of mRNA coding for CRH1R and CRH2R was much higher in preparations rich in adrenocortical cells and that only small amounts of CRH receptor mes- sage could be detected in chromaffin cells of pheochro- mocytomas and normal adrenal medulla. This is support- ed by our data obtained from our in situ hybridization experiments that documented the presence of high amounts of CRH1R mRNA in adrenocortical cells only. Therefore, we believe that the CRH1R and CRH2R are predominantly expressed in the adrenal cortex rather than in the adrenal medulla.
The link of the CRH receptor expression to steroido- genesis is reflected by a significant rise in DHEA secretion by adrenocortical cells after stimulation with CRH. In- terestingly, in a previous study we found a better response of adrenocortical cells to CRH in the presence of chro- maffin cells, measuring cortisol when determining this effect [14]. Due to the absence of human chromaffin cells in the model employed here, this hypothesis could not be tested. However, there are data which suppose a differ- ential regulation of cortisol and DHEA secretion by ad- renal cells and which support our results of a direct action of CRH on adrenocortical cells [23, 24]. Recent studies propose a differential regulation of steroidogenic en- zymes, e.g. adrenal 17x-hydroxylase, by CRH [30].
Immunoreactive CRH has been found in the adrenal medulla and shown to be identical to hypothalamic CRH [17, 31]. Its secretion by adrenal cells follows physiologi- cal and pathological stimuli, including splanchnic nerve activation [32], K+-induced membrane depolarization, nicotine [33], hemorrhage [34], and various neuropep- tides and cytokines [6, 30, 34], and it is suppressed in a classical endocrine feedback loop [22, 35].
With the exception of semiquantitative RT-PCR of microdissected tissue, the methods we employed in our study were rather specific than highly sensitive with refer- ence to the antibody titer and probe concentrations in our non-radioactive in situ hybridization. This may explain why we did not localize CRH receptors to the adrenal medulla. On the other hand, it suggests that the occur- rence of CRH1R and CRH2R on adrenocortical cells is more relevant to adrenocortical function than the existing intra-adrenal CRH-ACTH system that includes CRH re- ceptors on nerve cell endings, chromaffin cells, and/or lymphocytes and their ability to secrete ACTH. However, both direct and indirect actions on adrenocortical cells may be necessary in the fine-tuning of adrenal steroido- genesis.
We conclude that CRH receptors are also expressed in adrenocortical cells. There are different physiological and pathological conditions that lead to the activation of such peripheral CRH receptors and different downstream
pathways, and there are conditions that seem to be asso- ciated with upregulation of peripheral CRH receptors, such as adrenal tumorigenesis.
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