Melanocortin 2 Receptor-Associated Protein (MRAP) and MRAP2 in Human Adrenocortical Tissues: Regulation of Expression and Association with ACTH Responsiveness
Johannes Hofland, Patric J. Delhanty, Jacobie Steenbergen, Leo J. Hofland, Peter M. van Koetsveld, Francien H. van Nederveen, Wouter W. de Herder, Richard A. Feelders, and Frank H. de Jong
Departments of Internal Medicine (J.H., P.J.D., J.S., L.J.H., P.M.v.K., W.W.d.H., R.A.F., F.H.d.J.) and Pathology (F.H.v.N.), Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands
Context: ACTH stimulates adrenocortical steroid production through the melanocortin 2 receptor (MC2R). MC2R trafficking and signaling are dependent on the MC2R accessory protein (MRAP). The MRAP homolog MRAP2 also transports the MC2R to the cell surface but might prevent activation.
Objective: The objective of the investigation was to study the regulatory pathways of MRAP and MRAP2 and their contributions to ACTH responsiveness in human adrenal tissues.
Design and Setting: MRAP, MRAP2, and MC2R expression levels were studied in 32 human adre- nocortical samples. Regulation of these mRNAs was investigated in 43 primary adrenal cultures, stimulated with ACTH, forskolin, angiotensin II (AngII), phorbol-12-myristate-13-acetate (PMA), or dexamethasone. The induction of cortisol, cAMP, and ACTH-responsive genes after treatment with ACTH was related to MRAP, MRAP2, and MC2R expression levels.
Results: MRAP and MRAP2 levels were lower in adrenocortical carcinomas (ACC) than in other adrenal tissues (P < 0.001). Patient ACTH and cortisol levels were associated with adrenal levels of MRAP and MC2R in adrenal hyperplasia samples (P < 0.05) but not in tumors. ACTH induced the expression of MRAP 11 ± 2.1-fold and MC2R 20 ± 3.8-fold in all adrenal tissue types (mean ± SEM, both P < 0.0001), whereas AnglI augmented these mRNAs 4.0 + 1.2-fold and 12.6 + 3.2-fold (P < 0.0001) in all but the ACC. MRAP2 expression was suppressed by forskolin (-24 + 15%, P = 0.013) and PMA (-22 + 7%, P = 0.0007). MRAP, MRAP2, or MC2R levels were not associated with the induction of cortisol, cAMP, or gene expression by ACTH in vitro.
Conclusion: MRAP and MC2R expression is induced by ACTH and AngII, which would facilitate cell surface receptor availability. Physiological expression levels of MRAP, MRAP2, and MC2R were not limiting for ACTH sensitivity. (J Clin Endocrinol Metab 97: E747-E754, 2012)
T he hypothalamic-pituitary-adrenal axis is essential for adaptation to internal and external stressors (1). Ad- renal glucocorticoid production is controlled by ACTH. Circulating ACTH binds to the Gas protein-coupled mela-
nocortin 2 receptor (MC2R) in the adrenal cortex, leading to the formation of cAMP and the activation of protein kinase A (PKA). This in turn induces rapid phosphoryla- tion of the steroid acute regulatory protein that facilitates
Abbreviations: ACC, Adrenocortical carcinoma; ADA, adrenocortical adenoma; AIMAH, ACTH-independent macronodular adrenal hyperplasia; AnglI, angiotensin II; AT1R, Angli type 1 receptor; CYP11B1, cytochrome P450 11ß-hydroxylase; CYP17A1, cytochrome P450 17-hydroxylase; CYP21A2, cytochrome P450 21-hydroxylase; DST, dexamethasone overnight screening test; FGD, familial glucocorticoid deficiency; FSK, forskolin; HPRT1, hypoxanthine phosphoribosyltransferase 1; INHA, inhibin @-subunit; MC2R, melanocortin 2 receptor; MRAP, MC2R-associated protein; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate.
transport of cholesterol into the mitochondria for conver- sion into active steroid hormones (2). In addition, ACTH induces the transcription of multiple steroidogenic en- zymes and thus ensures short- and long-term stimulation of steroidogenesis (3, 4).
The MC2R is the smallest G protein-coupled recep- tor known to date and belongs to a family of melano- cortin receptors (types 1-5) that bind to various deriv- atives of proopiomelanocortin, especially @-MSH (5). ACTH stimulates MC2R expression in the long term (6, 7) but also acutely decreases MC2R presence at the cell surface by causing its internalization (8). Mutations in MC2R lead to familial glucocorticoid deficiency (FGD), a potentially lethal syndrome characterized by undetect- able serum cortisol levels combined with highly elevated ACTH levels and ACTH unresponsiveness (9). Only about 25% of FGD is caused by mutations in the MC2R gene, suggesting that additional mechanisms are involved in ACTH signaling.
A family segregation study in FGD patients revealed that mutations in a gene termed melanocortin 2 receptor- associated protein (MRAP) could also cause abrogated ACTH signaling (10). MRAP was found to be an MC2R- trafficking protein crucial for the translocation of the re- ceptor from the endoplasmatic reticulum to the cell sur- face (10). Moreover, MRAP facilitated signaling of the MC2R (11-13). Loss of function of MRAP thus prevents membrane expression of MC2R and completely prevents ACTH signaling. Interestingly, MRAP forms a unique an- tiparallel homodimer in close proximity to the MC2R (14, 15). The accessory protein can also interact with other melanocortin receptors, particularly melanocor- tin receptor 5, but exerts negative effects on their sig- naling (16, 17). Expression of MRAP was recently shown to be predominantly present in the zona fascicu- lata in the rat adrenal gland (18), consistent with its facilitating role in glucocorticoid production. mRNA levels of MRAP were found to be up-regulated by ACTH and cAMP in murine Y1 adrenocortical cells (19) and normal human adrenal cells (4).
MRAP2, a protein with 39% amino acid homology to MRAP, was found to share the MC2R-trafficking func- tion (17). Because MRAP2 is not capable of rescuing ACTH signaling in FGD patients with MRAP mutations (10), MRAP2 does not appear to play a major supportive role in adrenocortical ACTH signaling. On the contrary, MRAP2 overexpression caused suppression of MC2R ac- tivation, and positive effects on signaling have been de- tected only at supraphysiological levels of ACTH (18, 19). However, it is unclear whether these effects observed in vitro might have functional consequences in vivo. Fur-
thermore, although it is now known that the expression of MRAP2 is restricted to the adrenal gland and brain tissue (17), the factors that regulate MRAP2 expression remain to be determined.
Adrenocortical tumors have an altered responsiveness to ACTH in vivo, which could partly be explained by modified expression levels of the MC2R (20-24). It is also possible that MRAP and MRAP2 modulate ACTH re- sponsiveness in the adrenal and that this control mecha- nism is dysregulated in adrenal tumors. For this reason, we have studied the expression of MC2R, MRAP, and MRAP2 in both normal and pathological human adrenal tissues and determined the ACTH responsiveness of pri- mary cells from these tissues in vitro. MRAP and MC2R levels were found to be potently stimulated by ACTH and angiotensin II (AngII), whereas fluctuations in expression levels of the (co)receptors were not related to ACTH sen- sitivity in these cells. Furthermore, we detected a dysregu- lation of MRAP in adrenal tumors.
Materials and Methods
Tissue collection
Samples of adrenal tissues were obtained from patients op- erated between 2007 and 2011. Normal adrenal samples were obtained at nephrectomy due to renal cell carcinoma or adre- nalectomy due to adrenal cyst. Samples of hyperplastic adrenal tissues were collected at biadrenalectomy because of incurable Cushing’s disease or ectopic ACTH secretion or because of ACTH- independent macronodular adrenal hyperplasia (AIMAH) (25). Tissue samples of adrenocortical adenomas and carcinomas were also obtained after adrenalectomy. Adrenocortical carcinomas were diagnosed as such if the van Slooten index exceeded 8 during the pathological evaluation (26). This study was approved by the Medical Ethics Committee of the Erasmus Medical Center, and all patients gave written informed consent. Fasting cortisol and ACTH levels, cortisol after 1 mg dexamethasone overnight screening test (DST), and 24-h cortisoluria were measured during routine clinical diagnostic evaluation by chemiluminescence-based immunoassays (Immulite 2000; Siemens, Deerfield, IL).
Cell culture
Adrenal or intratumoral samples were dissected shortly after resection. Parts of the samples were stored in Tissue Tek and kept at -80 C until the isolation of RNA. If sufficient material was available, the tissues were minced into small pieces and taken up in DMEM/F12 medium containing 5% fetal calf serum and pen- icillin/streptomycin (Invitrogen, Carlsbad, CA) for the develop- ment of primary cell cultures as previously described (27). In short, minced tissue was washed twice with culture medium be- fore incubation with 2.5 mg/ml type 1 collagenase (Sigma-Al- drich, St. Louis, MO) for 2 h at 37 C. After obtaining single-cell suspensions, cells were washed and separated from cellular de- bris by centrifugation through a Ficoll gradient. Thereafter, lipid-laden cells were counted and plated in 24-well plates at 100,000 cells/well.
A
B
1-
1000
*
0.1-
100
*
MRAP2
**
MRAP
10-
0.01-
1.
0.001
0.1
r=0.45
0.01
0.0001
p=0.015
0.001
0.001
0.01
0.1
1
10
100
NI
Hyp
p AIMAH ADA
ACC
MRAP
C
D
10-
10-
1-
1-
MRAP2
0.1-
MC2R
0.1
0.01-
0.001-
0.01
0.0001
NI
Hyp AIMAH ADA ACC
0.001
NI
Hyp AIMAH
ADA
ACC
After attachment for at least 24 h in medium containing 5% fetal calf serum, media were replaced with serum free medium. The next day, ACTH1-24 (10 ng/ml; Novartis, Basel, Switzer- land), the PKA stimulator forskolin (FSK; 10 µM), AngII (100 nM), the protein kinase C (PKC) stimulator phorbol-12-myris- tate-13-acetate (PMA; 5 nm), or dexamethasone (1 µM; all Sigma-Aldrich) were added in culture medium to quadruplicate wells. The supernatants were removed after an incubation period of 48 h, and the plates were snap frozen on dry ice and stored at -80 C until further processing. Supernatant cortisol and cAMP levels were measured by Immulite and RIA (Beckman Coulter, Woerden, The Netherlands), respectively.
mRNA measurements
Hematoxylin and eosin-stained slides of frozen tissue samples were checked for tissue composition and the presence of exten- sive necrosis or fibrosis. Representative viable tissue sections were cut by microtome and used for RNA isolation. Isolations from frozen tissue and plated cells were performed with TriPure reagent (Roche, Penzberg, Germany). After RNA quantification by spectrophotometry, cDNA was created from 1 µg of RNA by reverse transcription using Moloney murine leukemia virus re- verse transcriptase (Promega, Leiden, The Netherlands) as re- ported before (28). The equivalent of 20 ng of RNA was used in a quantitative PCR for the detection of hypoxanthine phospho- ribosyltransferase 1 (HPRT1), MC2R, MRAP, MRAP2, cyto- chrome P450 11ß-hydroxylase (CYP11B1), cytochrome P450 17-hydroxylase (CYP17A1), inhibin @-subunit (INHA), and cy- tochrome P450 21-hydroxylase (CYP21A2) mRNA in duplicate.
MRAP (Hs01588793_m1, which measures both known transcript splice variants), MRAP2 (Hs00536621_m1), and CYP21A2 (Hs00416901_g1) assays were purchased from Applied Biosystems (Nieuwerkerk aan den IJssel, The Netherlands). A FAM- TAMRA duolabeled probe was used for the detection of HPRT1, CYP11B1, CYP17A1 and INHA (methods and sequences in Refs. 28 and 29), whereas FastStart Universal SYBR green master mix (Roche) was used for the MC2R assay (forward primer: CCCA- GAAAGTTCCTGCTTCA, reverse: TCTT- CAGGATCTTTTCTTCCTTG). The ex- pression levels of the housekeeping gene HPRT1 were not affected by incubation with any of the secretagogues. Expression was cal- culated relative to that of HPRT1 using the 8 threshold cycle method.
Statistics
All statistical analyses were performed in GraphPad Prism (GraphPad Software, La Jolla, CA). mRNA expression levels were log converted before analysis. Analysis was per- formed by t test or one-way ANOVA, fol- lowed by Tukey’s multiple comparison test. Associations were analyzed by Pearson’s cor- relation coefficient and linear regression. All tests were calculated as two tailed and signif- icance was assumed at a P < 0.05.
Results
MRAP, MRAP2, and MC2R in patient samples
Basal levels of MRAP, MRAP2, and MC2R mRNAs were studied in frozen tissue samples of normal adrenal glands (n = 5), ACTH-dependent hyperplasia (n = 4), AIMAH (n = 7), adrenocortical adenomas (ADA; n = 8), and carcinomas (ACC; n = 8). Of the adenomas, four tumors secreted cortisol (one concomitantly with aldoste- rone), one aldosterone, and two sex steroids, whereas one adenoma was nonfunctional. The carcinomas produced cortisol in one patient, sex steroids in another, and a com- bination of both in four patients, whereas the other two ACC were nonfunctional.
Overall, MRAP expression levels (cycle threshold range 24-36) exceeded those of MRAP2 (range 31 to >40) 95 ± 24-fold (relative to HPRT1, mean ± SEM), although the ex- pression levels of these mRNAs were correlated within indi- vidual samples (r = 0.45, P = 0.015, Fig. 1A). MRAP mRNA expression levels in ACTH-dependent adrenal hyperplasia, AIMAH, and adrenal adenomas were not significantly dif- ferent from levels in normal adrenal tissues. The main finding was that MRAP and MRAP2 gene expression levels were uniformly suppressed to near undetectable levels in ACC
2500
r=0.79
200-
2500-
r=0.66
2000
Hyperplasia
p=0.0036
r=0.69
p=0.020
Cortisol after DST (nM)
r=0.85
200-
r=0.67
Fasting cortisol (nM)
Fasting cortisol (nM)
p=0.026
p=0.0037
p=0.025
ACTH (PM)
ACTH (PM)
ACTH-dependent
2000
150-
2000
1500
150-
1500
1500
100-
1000
100-
1000-
1000-
AIMAH
500-
50-
500
500-
50-
0
0
0
0
0
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
MRAP (A.U.)
MRAP (A.U.)
MC2R (A.U.)
MC2R (A.U.)
MC2R (A.U.)
2500-
r =- 0.023
8-
r=0.029
2500
r =- 0.10
2000-
[ =- 0.19
8-
r =- 0.57
Tumors
Fasting cortisol (nM)
2000-
p=0.94
p=0.93
Fasting cortisol (nM)
2000
p=0.76
ACTH (pM)
Cortisol after DST (nM)
p=0.60
p=0.053
6-
1500
6-
Adenomas
Carcinomas
1500-
1500
ACTH (pM)
4-
1000
4-
1000-
1000
500-
·
2-
500
500-
2-
0
0
.
0
0-
.
.
·
0
..
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
MRAP (A.U.)
MRAP (A.U.)
MC2R (A.U.)
MC2R (A.U.)
MC2R (A.U.)
samples compared with normal and the other pathological samples (Fig. 1, B and C, P < 0.001), whereas mean MC2R expression was not significantly altered (Fig. 1D).
Patient serum steroid and plasma ACTH levels were not available for the normal adrenal samples. When an- alyzing the other adrenal samples, only MRAP expression was significantly correlated with plasma ACTH levels in all patients before operation (r = 0.42, P = 0.039, data not shown). There was no association between adrenal ex- pression levels of MRAP, MRAP2, or MC2R and fasting cortisol, cortisol after DST, or 24-h cortisoluria. How- ever, when analyzing the hyperplasia subgroups (ACTH dependent and independent) separately, there were clear associations between MRAP expression and fasting cor- tisol (r = 0.79, P = 0.0036) and ACTH levels (r = 0.69, P = 0.020, Fig. 2, upper panel). MC2R levels were also correlated with fasting cortisol (r = 0.66, P = 0.026), cortisol after DST (r = 0.85, P = 0.0037), and ACTH levels (r = 0.67, P = 0.025) but only in the combined hyperplasia group. There was no association between the levels of MRAP, MRAP2, or MC2R expression with se- rum cortisol or plasma ACTH levels in the adrenal ade- noma and carcinoma samples (Fig. 2, lower panel). Sim- ilarly, expression levels of MRAP, MRAP2, or MC2R did not relate to clinical steroid secretion of the adrenocortical tumors (aldosterone, cortisol, or sex steroids, data not shown).
Regulation of MRAP, MRAP2, and MC2R expression
We successfully developed primary cell cultures from 43 adrenocortical samples of varying pathological enti- ties. Because previous studies revealed that MRAP and MC2R expression in Y1 and human normal adrenal cells
were stimulated by ACTH (4, 19), we studied regulation of expression of MRAP, MRAP2, and MC2R in these cultures by ACTH as well as by AngII and dexamethasone. Furthermore, direct adenylyl cyclase and PKC stimulation was performed by the addition of FSK and PMA, respectively.
ACTH and FSK both significantly stimulated expres- sion of MRAP and MC2R in primary cultures (Fig. 3, P < 0.0001), whereas FSK suppressed MRAP2 expression to 0.76 ± 0.15-fold (mean ± SEM, P = 0.013). ACTH stim- ulated MRAP 11 + 2.1-fold without differences between the various tissue types. MC2R expression was increased 20 ± 3.8-fold after the addition of ACTH; this induction was larger in ACTH-dependent hyperplasia, compared with that in adrenocortical adenomas and carcinomas (both P < 0.05, Fig. 3). AngII also stimulated MRAP and MC2R expression to 4.0 ± 1.2-fold (P < 0.0001) and 12.6 ± 3.2-fold (P < 0.0001, Fig. 3), respectively. This inductive effect was apparent in all groups with the ex- ception of ACC. Addition of PMA to cultures of adreno- cortical cells stimulated MRAP expression to 2.0 ± 0.3- fold (P = 0.005) and concurrently decreased expression of MRAP2 0.78 + 0.07-fold (P = 0.0007, Fig. 3) but had no effect on MC2R transcription (data not shown, P > 0.05). Dexamethasone incubation in a subset of samples (n = 7) did not influence expression levels of MRAP, MRAP2, or MC2R (data not shown, P > 0.05).
MRAP, MRAP2, MC2R, and ACTH responsiveness
The AIMAH patients all underwent in vivo testing for hormonal stimuli according to Lacroix et al. (25). We found no association between the maximal increase in se- rum cortisol after 250 µg ACTH1-24 iv and adrenal levels
ACTH
FSK
Angli
100-
100
100;
MRAP
10
MRAP
10
MRAP
10
1
1
1
Poverall<0.0001
Poverall
<0.0001
Poverall
<0.0001
0.1
0.1
0.1
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
1000
100
100
100
MC2R
MC2R
10
MC2R
10
10
1
1
1
Poverall
<0.0001
Poverall
<0.0001
Poverall
<0.0001
0.1
0.1
0.1
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
PMA
PMA
FSK
10
10
10
MRAP
MRAP2
MRAP2
1
1
1
Poverall
=0.0048
Poverall
=0.0007
=0.013
0.1
0.1
0.1
Poverall
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
NI
Hyp AIMAH ADA ACC
of MRAP, MRAP2, or MC2R mRNA in these seven pa- tients (data not shown).
In vitro cortisol levels after the 48-h incubation period were detectable in 21 of 43 primary cultures (49%). Over- all, ACTH stimulated supernatant cortisol levels 5.4 + 0.64-fold (P = 0.0081). The induction of cortisol after the addition of ACTH was not significantly associated with basal expression levels of MRAP, MRAP2, or MC2R (P > 0.05, data not shown). Supernatant cAMP levels were also measured in a subset of cultures. These levels were unde- tectable after 48 h in 67% (10 of 15) of cultures in un- stimulated conditions and 33% (five of 15) of cultures after ACTH treatment (data not shown). There was no relation between the expression levels of MRAP, MRAP2, or MC2R and the ACTH-induced cAMP levels in the su-
pernatant of the 10 adrenal cell cultures with detectable CAMP (P > 0.05, data not shown).
CYP11B1, CYP17A1, CYP21A2, INHA, and MRAP are the five most differentially ACTH-regulated genes in adult adrenocortical cells (4). The proteins of the first three genes are key steroidogenic enzymes of cortisol produc- tion, whereas the inhibin «-subunit is presumably in- volved in adrenocortical cell proliferation and can serve as a tumor marker for ACC (30, 31). We measured the in- duction of the above-mentioned genes by ACTH in a va- riety of adrenocortical primary cultures as an indicator of ACTH responsiveness. Average induction of CYP11B1, CYP17A1, INHA, CYP21A2 and MRAP by ACTH after 48 h was 43 + 26-fold (mean ± SEM), 10±2.1-fold, 25 ± 8.6-fold, 14 ± 4.2-fold, and 11 ± 2.0-fold, respectively.
Regression analysis uncovered no associations between the unstimulated levels of MRAP, MRAP2, or MC2R and the induction of any of these five genes or the combination thereof (P > 0.05, data not shown).
Nine primary cell cultures (one normal, three hyper- plasia, one AIMAH, two adenomas, and two carcinomas) were separately incubated with both ACTH and FSK. We calculated the ACTH-induced stimulation of gene expres- sion relative to that by FSK as a measure of MC2R-related signaling potential. When comparing these ratios with the expression levels of the MC2R-MRAP complex, we found a negative correlation between MRAP2 and the ACTH to FSK induction ratio of CYP21A2 (r = - 0.70, P = 0.036), but this failed to reach significance after Bonferroni cor- rection for the five genes tested (0.01). The average ratio of all five genes studied in these nine samples was not associated with MRAP2 expression (r = - 0.46, P > 0.05). MRAP and MC2R expression levels were also not associated with the ACTH to FSK induction ratio.
Discussion
ACTH is the principal regulator of adrenal cortisol production and signals through the MC2R in a cAMP/ PKA-dependent pathway. The discovery of the MC2R ac- cessory proteins has uncovered new insights into G pro- tein-coupled signaling. Adequate MRAP expression is obligatory for cell surface localization and activation of the MC2R (10, 11), whereas MRAP2 appears to inhibit ACTH signaling (19). Most studies on this subject have used overexpression systems in models devoid of endog- enous MC2R or MRAP expression or mouse Y1 cells (10- 17, 32). The role and effects of these accessory proteins in human primary adrenal disease have not been explored thus far, partly because of the lack of a suitable antibody to the coreceptors. Furthermore, regulation of endoge- nous levels of these proteins remains largely unknown. We now show that MRAP, concurrent with MC2R, is posi- tively regulated by ACTH and AngII in human adrenal tissue and that adrenal MRAP and MC2R levels are cor- related with high ACTH and cortisol production states in patients with ACTH-dependent and ACTH-independent adrenal hyperplasia. No clear relationship was found be- tween physiological levels of MRAP, MRAP2, or MC2R mRNAs and ACTH responsiveness in adrenal cells.
ACTH binding to the MC2R induces a rapid confor- mational change in the MC2R-MRAP complex and leads to the activation of adenylyl cyclase (14). Within minutes after the binding of ACTH, the MC2R is internalized to endocytic vesicles through a clathrin-dependent pathway (8). In that manner, ACTH decreases cell surface expres-
sion of its receptor and thus ACTH responsiveness (8). By increasing the transcription of MC2R and its accessory protein MRAP, ACTH increases expression of the MC2R- MRAP complex at the plasma membrane and would be expected to improve signaling in its target tissue, the ad- renal cortex (7). Moreover, the absence of concomitant stimulation of MRAP2 by ACTH, or even suppression of MRAP2 expression as seen after direct adenylyl cyclase stimulation by FSK, would prevent the additional forma- tion of MRAP2-MC2R complexes that signal poorly in response to ACTH (18, 19). Furthermore, MRAP mRNA expression in the adrenal cortex markedly exceeds that of MRAP2. Although mRNA expression levels are not uni- formly representative of protein levels, this excess would predispose to the formation of functional MC2R-MRAP complexes.
Consistent with their reduced responsiveness to ACTH, adrenocortical carcinomas showed lower levels of MRAP mRNA compared with all other types of adrenal tissue. The ACTH-dependent hyperplasia samples, which had been chronically stimulated by ACTH in vivo, showed the highest expression levels of MRAP. Interestingly, the ACTH respon- siveness, as measured by the induction of MC2R by ACTH, was higher in adrenal hyperplasia, compared with adeno- mas and carcinomas. Adrenocortical carcinomas have an impairment in their cAMP/PKA pathway due to decreased expression of cAMP response element-binding protein and inducible cAMP early repressor isoforms (33, 34), which could contribute to a reduced stimulation of MRAP after ACTH. The lower MRAP levels in ACC could be expected to decrease ACTH responsiveness, but because we found no relation between mRNA expression of MRAP and ACTH responsiveness, this remains specula- tive. Surprisingly, MC2R expression in ACCs was com- parable with that in other adrenal tissues, whereas a pre- vious study, with a larger sample size, detected lower MC2R mRNA levels in ACC by Northern blot (20). Our findings suggest that ACCs show a divergent regulatory control of MRAP and MC2R expression. In situ hybrid- ization and immunohistochemistry studies could provide further insight into MRAP, MRAP2, and MC2R presence and function in adrenal tumors.
Correlation analysis between clinical data and adrenal MRAP and MC2R levels revealed associations between ACTH and cortisol production with MRAP and MC2R levels but only in patients with adrenal hyperplasia. The increased cortisol secretion is a result of the elevated ACTH levels and in AIMAH of other hormonal factors that stimulate G protein-coupled receptors and cAMP for- mation (25). The relationship between cortisol and ACTH levels and adrenal mRNA levels indicate that ACTH/PKA is also a major regulator of MRAP and MC2R transcrip-
tion in adrenal hyperplasia in vivo. This was recently also observed in patients with Cushing’s disease, who showed decreased sensitivity to exogenous ACTH in the first week after successful surgical resection of ACTH-producing pi- tuitary adenomas (35). In adrenocortical tumors, how- ever, there appears to be an uncoupling between control of steroidogenesis and MRAP-MC2R levels.
The main stimulator of aldosterone production, AngII, also increased the expression of MC2R and MRAP. This confirms that AngII increases ACTH responsiveness in AngII type 1 receptor (AT1R)-positive cells (7). Because ACTH is responsible for approximately 10% of aldoste- rone production, this could be an important physiological link between AngII- and ACTH-controlled mineralocor- ticoid production. The ACCs showed no response to An- gII, consistent with the absent or minimal AT1R levels present in these tumors (36). AngII-induced MRAP ex- pression could be PKC dependent because the addition of PMA showed a similar effect. PMA did not increase MC2R expression, possibly linking the induction of MC2R by AngII to the Ca2+-dependent pathway of AT1R signaling. This regulatory mechanism could add to the differential expression of the receptor and its accessory protein, as stated above.
On the other hand, adrenal MRAP2 expression was not found to be affected by AngII. PMA did reduce MRAP2 expression, implying that other AT1R pathways such as the Ca2+-dependent pathway simultaneously inhibit MRAP2 transcription after AngII signaling. MRAP2 lev- els were also decreased in ACC and correlated with the levels of MRAP. The decreased MRAP2 expression could be speculated to result from the tumor formation itself or factors overexpressed in ACC, such as IGF-II (37).
The effects of ACTH, i.e. increased steroidogenesis, would be expected to be dependent on the expression lev- els of components of the MC2R complex. However, we were unable to find a direct relationship between expres- sion levels of MRAP, MRAP2, or MC2R with in vivo or in vitro induction of cortisol or ACTH-responsive gene expression after the administration of ACTH. Physiolog- ical mRNA levels of MRAP and MC2R were thus not limiting for the ACTH effect. For MRAP this was previ- ously also found in Y1 cells, in which overexpression of MRAP did not increase the ACTH-induced cAMP pro- duction over that of endogenous levels of MRAP (19).
When the ACTH response was corrected for maximum possible cAMP (FSK) response of the cells, an inverse as- sociation between ACTH responsiveness of CYP21A2 ex- pression and MRAP2 was uncovered in adrenal cells in vitro, but this failed to reach statistical significance after Bonferroni correction. Combined with the absence of as- sociations of MRAP2 levels with the ACTH-induced stim-
ulation of cortisol, cAMP, or the other gene expression levels studied, MRAP2 levels within the physiological range also do not appear to inhibit ACTH responsiveness in vitro. Although it has been shown that overexpression of MRAP2 suppressed ACTH signaling via the MC2R in one study (19), this was not confirmed in two other reports (17, 18), and the low levels of MRAP2 currently encoun- tered in various human adrenal tissues were found not to suppress ACTH sensitivity.
In conclusion, we found that MRAP and MC2R are positively regulated by ACTH and AngII in human adre- nocortical tissues. In vivo cortisol and ACTH levels were associated with adrenal levels of MRAP and MC2R, con- sistent with their regulation in vitro. We found no asso- ciation between the ACTH-induced stimulation of corti- sol, cAMP, or ACTH-responsive genes and expression levels of MRAP, MRAP2, or MC2R, suggesting that phys- iological levels of the ACTH (co)receptors are not limiting for ACTH responsiveness in adrenocortical cells.
Acknowledgments
Address all correspondence and requests for reprints to: J. Hofland, Department of Internal Medicine, Erasmus Medical Center, Room Ee-532, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: j.hofland@erasmusmc.nl.
Disclosure Summary: The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
1. Chrousos GP 2009 Stress and disorders of the stress system. Nat Rev Endocrinol 5:374-381
2. Stocco DM 2001 StAR protein and the regulation of steroid hor- mone biosynthesis. Annu Rev Physiol 63:193-213
3. Le Roy C, Li JY, Stocco DM, Langlois D, Saez JM 2000 Regulation by adrenocorticotropin (ACTH), angiotensin II, transforming growth factor-ß, and insulin-like growth factor I of bovine adrenal cell steroidogenic capacity and expression of ACTH receptor, ste- roidogenic acute regulatory protein, cytochrome P450c17, and 3ß- hydroxysteroid dehydrogenase. Endocrinology 141:1599-1607
4. Xing Y, Parker CR, Edwards M, Rainey WE 2010 ACTH is a potent regulator of gene expression in human adrenal cells. J Mol Endo- crinol 45:59-68
5. Mountjoy KG, Robbins LS, Mortrud MT, Cone RD 1992 The clon- ing of a family of genes that encode the melanocortin receptors. Science 257:1248-1251
6. Mountjoy KG, Bird IM, Rainey WE, Cone RD 1994 ACTH induces up-regulation of ACTH receptor mRNA in mouse and human ad- renocortical cell lines. Mol Cell Endocrinol 99:R17-R20
7. Lebrethon MC, Naville D, Begeot M, Saez JM 1994 Regulation of corticotropin receptor number and messenger RNA in cultured hu- man adrenocortical cells by corticotropin and angiotensin II. J Clin Invest 93:1828-1833
8. Kilianova Z, Basora N, Kilian P, Payet MD, Gallo-Payet N 2006 Human melanocortin receptor 2 expression and functionality: ef-
fects of protein kinase A and protein kinase C on desensitization and internalization. Endocrinology 147:2325-2337
9. Clark AJ, Weber A 1998 Adrenocorticotropin insensitivity syn- dromes. Endocr Rev 19:828-843
10. Metherell LA, Chapple JP, Cooray S, David A, Becker C, Rüschen- dorf F, Naville D, Begeot M, Khoo B, Nürnberg P, Huebner A, Cheetham ME, Clark AJ 2005 Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocor- ticoid deficiency type 2. Nat Genet 37:166-170
11. Webb TR, Chan L, Cooray SN, Cheetham ME, Chapple JP, Clark AJ 2009 Distinct melanocortin 2 receptor accessory protein do- mains are required for melanocortin 2 receptor interaction and pro- motion of receptor trafficking. Endocrinology 150:720-726
12. Roy S, Rached M, Gallo-Payet N 2007 Differential regulation of the human adrenocorticotropin receptor [melanocortin-2 receptor (MC2R)] by human MC2R accessory protein isoforms æ and ß in isogenic human embryonic kidney 293 cells. Mol Endocrinol 21: 1656-1669
13. Cooray SN, Almiro Do Vale I, Leung KY, Webb TR, Chapple JP, Egertov á M, Cheetham ME, Elphick MR, Clark AJ 2008 The mela- nocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse y1 cell line. Endocrinology 149:1935-1941
14. Cooray SN, Chung TT, Mazhar K, Szidonya L, Clark AJ 2011 Bioluminescence resonance energy transfer reveals the adrenocor- ticotropin (ACTH)-induced conformational change of the activated ACTH receptor complex in living cells. Endocrinology 152:495- 502
15. Sebag JA, Hinkle PM 2007 Melanocortin-2 receptor accessory pro- tein MRAP forms antiparallel homodimers. Proc Natl Acad Sci USA 104:20244-20249
16. Sebag JA, Hinkle PM 2009 Opposite effects of the melanocortin-2 (MC2) receptor accessory protein MRAP on MC2 and MC5 recep- tor dimerization and trafficking. J Biol Chem 284:22641-22648
17. Chan LF, Webb TR, Chung TT, Meimaridou E, Cooray SN, Guasti L, Chapple JP, Egertov á M, Elphick MR, Cheetham ME, Metherell LA, Clark AJ 2009 MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family. Proc Natl Acad Sci USA 106: 6146-6151
18. Gorrigan RJ, Guasti L, King P, Clark AJ, Chan LF 2011 Localisation of the melanocortin-2-receptor and its accessory proteins in the de- veloping and adult adrenal gland. J Mol Endocrinol 46:227-232
19. Sebag JA, Hinkle PM 2010 Regulation of G protein-coupled recep- tor signaling: specific dominant-negative effects of melanocortin 2 receptor accessory protein 2. Sci Signal 3:ra28
20. Reincke M, Beuschlein F, Latronico AC, Arlt W, Chrousos GP, Allolio B 1997 Expression of adrenocorticotrophic hormone recep- tor mRNA in human adrenocortical neoplasms: correlation with P450scc expression. Clin Endocrinol (Oxf) 46:619-626
21. Reincke M, Mora P, Beuschlein F, Arlt W, Chrousos GP, Allolio B 1997 Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigenesis. J Clin En- docrinol Metab 82:3054-3058
22. Imai T, Sarkar D, Shibata A, Funahashi H, Morita-Matsuyama T, Kikumori T, Ohmori S, Seo H 2001 Expression of adrenocortico- tropin receptor gene in adrenocortical adenomas from patients with Cushing syndrome: possible contribution for the autonomous pro- duction of cortisol. Ann Surg 234:85-91
23. Bertagna C, Orth DN 1981 Clinical and laboratory findings and
results of therapy in 58 patients with adrenocortical tumors admit- ted to a single medical center (1951 to 1978). Am J Med 71:855-875
24. Schubert B, Fassnacht M, Beuschlein F, Zenkert S, Allolio B, Re- incke M 2001 Angiotensin II type 1 receptor and ACTH receptor expression in human adrenocortical neoplasms. Clin Endocrinol (Oxf) 54:627-632
25. Lacroix A, Ndiaye N, Tremblay J, Hamet P 2001 Ectopic and ab- normal hormone receptors in adrenal Cushing’s syndrome. Endocr Rev 22:75-110
26. van’t Sant HP, Bouvy ND, Kazemier G, Bonjer HJ, Hop WC, Feelders RA, de Herder WW, de Krijger RR 2007 The prognostic value of two different histopathological scoring systems for adre- nocortical carcinomas. Histopathology 51:239-245
27. Lamberts SW, Bons EG, Bruining HA, de Jong FH 1987 Differential effects of the imidazole derivatives etomidate, ketoconazole and mi- conazole and of metyrapone on the secretion of cortisol and its precursors by human adrenocortical cells. J Pharmacol Exp Ther 240:259-264
28. Chai W, Hofland J, Jansen PM, Garrelds IM, de Vries R, van den Bogaerdt AJ, Feelders RA, de Jong FH, Danser AH 2010 Steroid- ogenesis vs. steroid uptake in the heart: do corticosteroids mediate effects via cardiac mineralocorticoid receptors? J Hypertens 28: 1044-1053
29. Hofland J, Timmerman MA, de Herder WW, van Schaik RH, de Krijger RR, de Jong FH 2006 Expression of activin and inhibin subunits, receptors and binding proteins in human adrenocortical neoplasms. Clin Endocrinol (Oxf) 65:792-799
30. Hofland J, Feelders R, van der Wal R, Kerstens MN, Haak HR, de Herder W, de Jong FH 2012 Serum inhibin pro-&C is a tumor marker for adrenocortical carcinomas. Eur J Endocrinol 166:281- 289
31. Hofland J, de Jong FH 22 June 2011 Inhibins and activins: Their roles in the adrenal gland and the development of adrenocortical tumors. Mol Cell Endocrinol 10.1016/j.mce.2011.06.005
32. Sebag JA, Hinkle PM 2009 Regions of melanocortin 2 (MC2) re- ceptor accessory protein necessary for dual topology and MC2 re- ceptor trafficking and signaling. J Biol Chem 284:610-618
33. Peri A, Luciani P, Conforti B, Baglioni-Peri S, Cioppi F, Crescioli C, Ferruzzi P, Gelmini S, Arnaldi G, Nesi G, Serio M, Mantero F, Mannelli M 2001 Variable expression of the transcription factors cAMP response element-binding protein and inducible cAMP early repressor in the normal adrenal cortex and in adrenocortical ade- nomas and carcinomas. J Clin Endocrinol Metab 86:5443-5449
34. Rosenberg D, Groussin L, Jullian E, Perlemoine K, Medjane S, Lou- vel A, Bertagna X, Bertherat J 2003 Transcription factor 3’,5’-cyclic adenosine 5’-monophosphate-responsive element-binding protein (CREB) is decreased during human adrenal cortex tumorigenesis and fetal development. J Clin Endocrinol Metab 88:3958-3965
35. Alwani RA, de Herder WW, de Jong FH, Lamberts SW, van der Lely AJ, Feelders RA 2011 Rapid decrease in adrenal responsiveness to ACTH stimulation after successful pituitary surgery in patients with Cushing’s disease. Clin Endocrinol (Oxf) 75:602-607
36. Opocher G, Rocco S, Cimolato M, Vianello B, Arnaldi G, Mantero F 1997 Angiotensin II receptors in cortical and medullary adrenal tumors. J Clin Endocrinol Metab 82:865-869
37. Almeida MQ, Fragoso MC, Lotfi CF, Santos MG, Nishi MY, Costa MH, Lerario AM, Maciel CC, Mattos GE, Jorge AA, Mendonca BB, Latronico AC 2008 Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors. J Clin Endocrinol Metab 93:3524-3531