Oncology
Oncology 2005;68:414-421 DOI: 10.1159/000086983
Received: May 25, 2004 Accepted after revision: December 12, 2004 Published online: July 12, 2005
Loss of Growth Hormone Secretagogue Receptor 1a and Overexpression of Type 1b Receptor Transcripts in Human Adrenocortical Tumors
Luisa Barzon Monia Pacenti Giulia Masi Anna-Lisa Stefani Karina Fincati Giorgio Palù
Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy
Key Words
Adrenal gland neoplasms . Ghrelin . Growth hormone secretagogue receptors · Real-time polymerase chain reaction . Adrenocortical carcinoma . Cushing’s syndrome
Abstract
Objective and Methods: Quantitative analysis of mRNA expression of ghrelin and its receptors GHS-R1a and -R1b in a large series of normal and neoplastic human adrenocortical tissues. Evaluation of the effects of ghre- lin on GHS-R expression and proliferation of human ad- renocortical carcinoma (ACC) cell lines. Results: Ghrelin and GHS-R transcripts are expressed in normal adrenal cortex, with GHS-R1b mRNA levels being 5- to 10-fold higher than GHS-R1a mRNA. A significant increase in ghrelin expression was observed in adrenocortical ade- nomas, but not in carcinomas. GHS-R1a was undetect- able in about 60% of both benign and malignant tumor samples, except for cortisol-producing adenomas, which showed increased receptor expression. At variance, GHS-R1b was overexpressed in both benign and malig- nant adrenocortical tumors. In vitro studies in human ACC cell lines demonstrated that GHS-R1a is downregu- lated and GHS-R1b mRNA expression is upregulated by ghrelin, while inhibiting cell proliferation. Conclusion:
Downregulation of GHS-R1a in adrenal tumors and the presence of high levels of GHS-R1b transcripts in adre- nocortical tissue suggest a role for these receptors in adrenal function and growth. In this regard, ghrelin in- hibits cell proliferation and modulates GHS-R expression in ACC cells in vitro.
Copyright @ 2005 S. Karger AG, Basel
Introduction
Tumors of the adrenal cortex have become a rather common finding in clinical practice, being identified in 0.5-2% of abdominal CT scans, often as an incidental discovery during the assessment of unrelated complaints [1]. The majority of adrenal tumors are nonfunctional, but some secrete hormones and are thus responsible for endocrine syndromes, e.g. hypersecretion of aldosterone (Conn’s syndrome), cortisol (Cushing’s syndrome), or an- drogens (virilization). Adrenocortical carcinoma (ACC) is a rare but highly malignant tumor carrying a poor prog- nosis, with a survival rate of 20% at 5 years [2, 3].
Although progress has been made in the elucidation of the molecular mechanism of adrenal tumorigenesis, and several genes have been reported to have diagnostic sig- nificance in adrenocortical neoplasms [4], the pathogen- esis of adrenal tumors remains poorly understood, and
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molecular markers still seem less accurate than pathology in predicting malignancy.
Insulin-like growth factor (IGF) 1 and, more potently IGF2, have been found to induce steroidogenesis and mi- togenesis in adrenocortical cells in vitro [5, 6] and to be overexpressed in adrenocortical tumors [4]. Expression of other components of the growth hormone (GH) axis, including GH receptor, has been demonstrated in normal and pathologic adrenocortical tissues [8, 9], thus suggest- ing that these growth factors may have a direct effect on the adrenal cortex.
Ghrelin is a recently identified 28-amino-acid peptide capable of stimulating pituitary growth hormone release in humans [10]. It was initially isolated from rodent stom- ach tissue based on its ability to bind with high affinity and specificity to the growth hormone secretagogue recep- tor (GHS-R) [11]. It was found to stimulate GH release from rat pituitary cells in culture, with specificity being comparable to growth-hormone-releasing hormone, but, more recently, it has been demonstrated to have a number of different central and peripheral effects on appetite, car- bohydrate metabolism, heart function, the gonadal axis, endocrine function, and cell proliferation [10].
Recently, ghrelin expression has been demonstrated in human adrenocortical cells [12, 13], but it has never been investigated in adrenocortical tumors to date. In addition to ghrelin, adrenocortical cells express high levels of its receptors, i.e. the functional GHS-R1a receptor and the apparently non-functional receptor variant GHS-R1b [12, 14], which have been localized mainly in the adrenal zona glomerulosa and outer zona fasciculata [13]. How- ever, functional studies demonstrated that ghrelin was unable to affect either basal or ACTH- and angiotensin- II-stimulated steroidogenesis in adrenocortical slices in vitro [13]. Similar observations were obtained from stud- ies on rat adrenocortical cells [15, 16].
In an attempt to identify new molecular mechanisms involved in the development of adrenocortical tumors and potential targets for therapeutic intervention, we an- alyzed the expression of ghrelin mRNA and its receptors in human normal adrenal cortex and in a large series of adrenocortical tumors. We also investigated the relation- ship between the ghrelin expression status and clinical and pathological parameters, in order to define their as- sociation with hormone activity or malignant behavior. Finally, in ACC cell lines, we assessed the ability of hu- man ghrelin to modulate the expression of genes encoding ghrelin and its receptors and its effect on cell prolifera- tion.
Patients and Methods
Patient Samples. Forty adrenal tumor tissues obtained from patients undergoing adrenalectomy for sporadic adrenal tumors were analyzed, including 12 non-functioning adrenocortical ade- nomas (NFA), 7 cortisol-producing adenomas (CPA, including 4 adenomas associated with overt Cushing’s syndrome and 3 associ- ated with subclinical Cushing’s syndrome), 13 aldosterone-pro- ducing adenomas (APA), 1 sporadic androgen-producing adeno- ma, 1 androgen-producing adenoma associated with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, and 6 ACCs (table 1). ACCs were histologically diagnosed based on the criteria of Weiss [17]. Normal adrenal cortex samples were macroscopi- cally dissected from adrenal glands, obtained from 14 donors un- dergoing kidney explantation, and employed as controls. Refer- ence RNA from human adrenal cortex (Human Adrenal Cortex Total RNA, Ambion, Austin, Tex., USA) was also employed as control. All patients gave informed consent in accordance with the University of Padova Institutional Review Board using Declara- tion of Helsinki guidelines. After surgical removal and dissection, tissue samples were immediately frozen in liquid nitrogen and stored at -80℃.
Adrenocortical Carcinoma Cell Lines
The human ACC cell lines NCI-H295 (American Type Culture Collection, ATCC, CRL-10296) and SW13 (ATCC, CCL-105) were used to investigate the effect of ghrelin on GHS-R mRNA expres- sion and cell proliferation. NCI-H295 cells were maintained in RPMI-1640 phenol-red-free medium supplemented with 10 mM glutamine, antibiotics (penicillin/streptomycin), insulin, transfer- rin, selenium, and 2% fetal bovine serum in a humidified atmo- sphere of 95% O2, 5% CO2 at 37°C. SW13 cells were maintained in Leibovitz medium supplemented with 10 mM glutamine, anti- biotics (penicillin/streptomycin), and 10% fetal bovine serum in a humidified atmosphere of 95% O2 at 37°C.
RNA Extraction and Real-Time RT-PCR Analysis
Total RNA was isolated from tissue samples following a single- step acid guanidium phenol-chloroform extraction procedure em- ploying OMNIzol™ (Euroclone, Wetherby, UK). Random primed cDNAs were generated from 3 µg of total RNA using MuLV reverse transcriptase (Applied Biosystems, Foster City, Calif., USA) in a total volume of 100 ul. Quantitative real-time RT-PCR of ghrelin and its receptors GHS-R1a and GHS-R1b was performed using oligonucleotide primers and probe sequences reported by Korbo- nits et al. [18]. PCRs were performed in triplicate using the ABI Prism 7700 Sequence Detector System (Applied Biosystems) in a total volume of 50 ul reaction mixture, containing 5 ul cDNA tem- plate, 25 ul TaqMan Universal PCR Master Mix (Applied Biosys- tems), 0.1 µM probe, and 0.1 p.M of each primer. Negative controls contained water instead of cDNA. PCR conditions were as follows: 50℃ for 2 min and denaturing at 95℃ for 10 min, followed by 45 cycles at 95℃ for 15 s and 60℃ for 60 s. Absolute quantification of transcripts was performed against standard curves obtained by amplification of serially diluted solutions (from 10 copies/ul to 106 copies/pl) of plasmid clones containing ghrelin, GHS-R1a, or GHS- R1b sequences as templates. In particular, plasmids for standard curves were generated by cloning PCR amplicons of target sequenc- es into the pCR2.1 vector (Invitrogen Life Technologies, Milan, Italy). Levels of mRNA expression were normalized to the endog-
| Type of tumor | n | F/M | Mean age, years | Mean diameter, cm |
|---|---|---|---|---|
| NFA | 12 | 6/6 | 44±6 | 3.1±1.0 |
| CPA | 7 | 3/4 | 52±7 | 2.9±0.7 |
| APA | 13 | 7/6 | 47±5 | 2.2±0.6 |
| Androgen-producing adenoma | 1 | 1/0 | 44 | 6.5 |
| Adrenal adenoma associated with CAH | 1 | 0/1 | 39 | 5.0 |
| ACC | ||||
| Functioning | 3 | 3/0 | 32±4 | 9.3±1.1 |
| Non-functioning | 3 | 1/2 | 34±3 | 8.5±1.2 |
| Normal adrenal cortex | 14 | 8/6 | 45±6 |
CAH = Congenital adrenal hyperplasia.
| Median copy numbers/µg total RNA (range) ghrelin GHS-R1a GHS-R1b | |||
|---|---|---|---|
| Normal | 324 (181-1,820) | 35 (4-112) | 320 (38-746) |
| CPA | 5,382 (901-130,515)* | 1,220 (219-6,252)* | 4,568 (814-952,538)* |
| APA | 1,089 (137-5,072)* | 0 (0-602) | 380 (45-9,526)* |
| NFA | 2,100 (0-4,8021)* | 0 (0-3,961)* | 2,234 (385-550,386)* |
| ACC | 549 (48-915) | 0 (0-323) | 1,633 (23-7,900)* |
* p < 0.05 vs. normal adrenal cortex by Mann-Whitney test.
enous control 18S ribosomal RNA, as quantified by real-time RT- PCR analysis using a TaqMan Ribosomal RNA Control Reagent Kit (Applied Biosystems) following the manufacturer’s protocol.
Ghrelin and GHS-Rs mRNA Analysis in ACC Cell Lines
In order to evaluate the effect of ghrelin peptide on the expres- sion of ghrelin and its receptors at mRNA level, NCI-H295R and SW-13 cells were seeded in triplicate at a density of 7.5 x 105 and 3.5 × 105 cells/well, respectively, in 6-well plates. After 24 h, cells were grown in the absence (control) or in the presence of human ghrelin (Bachem, Bubendorf, Switzerland) at concentrations of 10-10, 10-8, and 10-6 M. After 4 and 8 h, cells were collected for real-time RT-PCR analysis of ghrelin, GHS-R1a, and GHS-R1b mRNA levels.
Cell Survival Analysis
For cell proliferation studies, NCI-H295 and SW-13 cells were seeded at density of 5 × 103 cells/well in 96-well microtiter plates. After 24 h, cells were grown in the absence (control) or in the pres- ence of human ghrelin (Bachem) concentrations ranging from 10-10 to 10-6 M in 100 ul medium. Cell survival was quantified by the MTT assay (Sigma-Aldrich, Milano, Italy) after 24, 48, 72 and 96 h, and expressed as percentage relative to untreated control cells. Experiments were performed three times in sextuplicate and values were calculated as means and SD.
Statistical Analysis
Data are presented as means + SD or medians and ranges. Comparisons between groups were performed by Mann-Whitney test, two-tailed unpaired Student’s t test, or analysis of variance, where appropriate. Correlation was analyzed by simple linear re- gression analysis, whereas discrete variables were compared by x2 test. p <0.05 was considered statistically significant.
Results
Ghrelin and GHS-R mRNA Expression in Adrenocortical Tissues
Quantitative real-time RT-PCR analysis confirmed the expression of mRNA of ghrelin and subtypes of its receptor in the normal adrenal cortex, although wide variability among samples was observed (table 2). In particular, the mean ghrelin mRNA copy number was 683 + 472/µg total RNA, the mean GHS-Rla mRNA copy number was 51 + 32/µg total RNA, and that of GHS-R1b mRNA was 345 ± 175/µg total RNA (means ± SD).
GHS-R1a (copies/ug total RNA)
140
120
100
80
60
40
20
0
0
1,000
2,000
3,000
4,000
5,000
6,000
Ghrelin (copies/ug total RNA)
GHS-R1b (copies/µg total RNA)
1,600
1,400
1,200
1,000
800
600
400
200
0
0
1,000
2,000
3,000
4,000
5,000
Ghrelin (copies/µg total RNA)
GHS-R 1b (copies/ug total RNA)
1,600
1,400
1,200
1000
800
600
400
200
0
0
25
50
75
100
125
GHS-R1a (copies/µg total RNA)
Expression of ghrelin mRNA in benign tumor sam- ples, including NFAs, CPAs, and APAs, was higher than in the normal adrenal cortex, whereas it was unchanged or slightly decreased in ACCs. GHS-R1a mRNA was un- detectable in 9/12 NFAs, 8/13 APAs, and 4/6 ACCs.
When expressed, GHS-R1 a mRNA levels ranged from 50 to 5,325 copy numbers/µg total RNA, without any appar- ent association between expression levels and clinical and pathological features of the tumor (i.e. endocrine activity, size or histological features). Interestingly, GHS-R1a mRNA levels were very high in CPAs, both associated with overt and subclinical Cushing’s syndrome (table 2). At variance with GHS-R1a, GHS-R1b mRNA was sig- nificantly increased in all adrenocortical tumor types as compared with the normal adrenal cortex (table 2).
Of note, the case of the androgen-secreting adenoma showed very high levels of both ghrelin mRNA (964% of control adrenal cortex) and its receptors GHS-R1a and GHS-R1b (67,466 and 543,513% of control, respective- ly). We also studied the case of an androgen-producing adrenal adenoma, which developed in a male patient with congenital adrenal hyperplasia due to 21-hydroxylase de- ficiency. Expression of ghrelin was within the normal range, but GHS-R1a was undetectable and GHS-R1b overexpressed (8,849% of control).
In the normal adrenal cortex, regression analysis showed a negative relationship between absolute amounts of ghrelin and GHS-Rla mRNA (r =- 0.53; p<0.05) and between GHS-Rla and GHS-R1b mRNAs (r = - 0.59; p < 0.05), but a significant positive correlation between ghrelin and GHS-R1b mRNA (r = 0.74; p<0.005; fig. 1). Although not statistically significant, this relationship was also observed in the different groups of adrenocorti- cal tumors.
Effect of Ghrelin on Ghrelin and GHS-R mRNA Expression in ACC Cell Lines
The functioning NCI-H295R cell line and the non- functioning SW13 cell line expressed both ghrelin mRNA (208 ± 27 and 36 ± 9 copy numbers/µg total RNA, re- spectively) and its receptors, with GHS-R1b mRNA lev- els (208,000 ± 6,400 and 90,000 ± 4,100 copy numbers/ µg total RNA, respectively) about 2-3 logs higher than GHS-Rla mRNA values (790 ± 21 and 2,200 ± 346 copy numbers/µg total RNA, respectively).
Treatment of NCI-H295R and SW13 cells with hu- man ghrelin for 4 and 8 h led to a significant increase in ghrelin and GHS-R1b mRNA levels, but GHS-R1a mRNA values were decreased compared to untreated control cells (fig. 2).
Ghrelin and GHS-R mRNA values were measured by real-time RT-PCR in both ACC cell lines, either in basal condition or during exposure to ghrelin peptide in adre- nal tissue samples, and GHS-R1a mRNA demonstrated a significant negative correlation with ghrelin and GHS-
700
4- SW13 4 h
GHS-R1a mRNA (copies/µg total RNA)
600
SW13 8 h
500
NCI-H295R 4 h
NCI-H295R 8 h
400
*
300
I
200
*
*
100
*
*
5
*
I
*
*
*
0
0
10-10
10-8
10-6
Ghrelin (M)
106
*
GHS-R1b mRNA (copies/µg total RNA)
*
*
*
105
*
*
*
*
104
103
0
10-10
10-8
10-6
Ghrelin (M)
200
180
Ghrelin mRNA (copies/µg total RNA)
160
*
*
140
120
*
*
100
80
I
60
40
20
I
0
0
10-10
10-8
10-6
Ghrelin (M)
NCI-H295R
120
Cell survival (% of control)
T
T
100
天
+
+
*
*
80
1
Z
*
*
*
F
*
*
1
*
I
*
60
1
*
I
40
24 h
48 h
20
72 h
96 h
0
0
0.0001
0.001
0.01
0.1
1
Ghrelin (p.M)
SW13
120
Cell survival (% of control)
100
I
£
56
*
80
1
2
I
*
*
L
* *
I
60
40
24 h
48 h
20
72 h
96 h
0
0
0.0001
0.001
0.01
0.1
1
Ghrelin (p.M)
Barzon/Pacenti/Masi/Stefani/Fincati/Palù
R1b mRNAs, and GHS-R1b showed a positive correla- tion with ghrelin.
Effect of Ghrelin on the Proliferation of ACC Cell Lines
Treatment with human ghrelin significantly reduced survival of NCI-H295R and SW13 cells in a dose-depen- dent manner. This antiproliferative effect was more evi- dent after 72-96 h of exposure to the hormone (fig. 3).
Discussion
This study represents the first report on quantitative analysis of ghrelin and GHS-R expression in a large series of human adrenocortical tumors.
Evidence accumulates indicating that the ghrelin/ GHS-R axis could play a role in tumorigenesis, possibly via autocrine/paracrine mechanisms. Ghrelin and its re- ceptors are coexpressed in normal and adenomatous pi- tuitary cells [18-20], as well as in neuroendocrine tumors [18, 21-23], breast cancer [24], thyroid carcinomas [25, 26], testicular tumors [27], prostate cancer [28], and pan- creatic adenocarcinoma cell lines [29]. The effects of ghre- lin on cell proliferation and tumor growth have been in- vestigated in vitro in primary cell cultures and in tumor cell lines, demonstrating opposite activity in different cell types. Ghrelin significantly increases proliferation of prostate [28], hepatocellular [30] and pancreatic [29] car- cinoma cell lines, for example, but it induces a dose-de- pendent inhibition of growth in thyroid carcinoma cell lines [26].
Our results show that ghrelin and GHS-R mRNAs are coexpressed in the human normal adrenal cortex, con- firming previous observations which reported higher lev- els of GHS-R1b than GHS-R1a transcripts[13] .
Expression of ghrelin in benign adrenocortical tumors was higher than in the normal adrenal cortex, but not in ACCs, which, at variance, showed a slight decrease in transcript levels. About 60% of benign and malignant ad- renocortical tumors did not express GHS-R1a, with the exception of CPAs associated with overt or subclinical Cushing’s syndrome, which showed very high levels of GHS-R1a mRNA. This observation does not agree with the reported inhibitory effect of glucorticoids on GHS-R expression through a glucocorticoid-responsive element [31, 32]. GHS-R1b had a different expression profile than GHS-R1a, with higher transcript levels in tumor tissue than in the normal adrenal cortex. These results, together with the observation of a differential relationship be-
tween transcript levels of ghrelin and the two types of GHS-R, suggest that expression of the two receptors is regulated by different mechanisms in the adrenal gland and that ghrelin could be involved in the regulation of its own receptor, by downregulating GHS-R1a and upregu- lating GHS-R1b. Indeed, we demonstrated that exposure of human ACC cell lines to ghrelin significantly decreased GHS-R1a mRNA levels, but increased GHS-R1b and, interestingly, ghrelin mRNA values. Dose-dependent in- hibition of GHS-R mRNA expression by ghrelin was also demonstrated in rat adrenocortical cell cultures in vitro [15] . In a recent study, binding of ghrelin to its type 1a receptor was followed by internalization of the ghrelin/ GHS-R1a complex and accumulation in the perinuclear region. Afterwards, the ligand appears to be degraded in lysosomes, whereas the receptor slowly recycles to the plasma membrane [33]. Down- and upregulation of re- ceptor expression by modulation of mRNA and protein synthesis resulting from continuous exposure of cells to agonists could represent another mechanism of GHS-R regulation [34].
Coexpression of ghrelin and GHS-R receptors has been demonstrated in rat [15, 16] and human adrenal cortex [13], in particular in the zona glomerulosa and outer zona fasciculata, but did not affect their endocrine activity [13, 16]. Despite this zonal distribution of GHS- Rs in the adrenal cortex, the levels of GHS-Rs in tumors arising from zona glomerulosa cells (i.e. APAs) were not increased compared to other tumor types.
Our in vitro experiments demonstrated that ghrelin significantly inhibited the proliferation of human ACC cell lines. These results are in agreement with the pro- apoptotic activity of ghrelin on human ACC cells [35] and concordant with the lack of GHS-R1a expression in most benign and malignant adrenocortical tumors. It is thus conceivable that type 1a receptor has a role in the control of adrenocortical cell growth. On the other hand, high levels of type 1b receptor in the normal adrenal cortex and its overexpression in tumor samples, together with ghrelin overexpression, suggest that GHS-R1b could be functional in the adrenal gland and involved in adrenal tumorigenesis.
In conclusion, our study demonstrates that ghrelin and GHS-R1a and -R1b transcripts are expressed in normal adrenal cortex. Loss of GHS-R1a expression and GHS- R1b mRNA overexpression is frequently observed in both benign and malignant adrenocortical tumors, re- gardless of their endocrine function, with the exception of cortisol-producing adenomas, which reveal upregula- tion of both receptors. Although our results should be
confi rmed by protein data, loss of GHS-R1a in adreno- cortical tumors and the presence of high levels of GHS- R1b in normal and tumor tissues suggest a role for these receptors in adrenal function and/or growth. This is also indicated by in vitro studies in human ACC cell lines, which demonstrated that ghrelin downregulates GHS- R1a and upregulates ghrelin and GHS-R1b mRNA ex- pression, while inhibiting cell proliferation. Further stud-
ies are needed to investigate the role of the ghrelin/ GHS-R axis in the adrenal gland and its disorders.
Acknowledgments
This work was supported in part by grants from the ‘Azienda Ospedaliera’ of Padova and the University of Padova, Italy.
References
1 Barzon L, Sonino N, Fallo F, Palù G, Boscaro M: Prevalence and natural history of adrenal incidentalomas. Eur J Endocrinol 2003;149: 273-285.
2 Luton JP, Cerdas S, Billaud L, Thomas G, Guilhaume B, Bertagna X, Laudat MH, Lou- vel A, Chapuis Y, Blondeau P, Bonnin A, Bri- caire H: Clinical features of adrenocortical car- cinoma, prognostic factors, and the effect of mitotane therapy. N Engl J Med 1990;322: 1195-1201.
- 3 Barzon L, Fallo F, Sonino N, Daniele O, Bos- caro M: Adrenocortical carcinoma: Experience in 45 patients. Oncology 1997;54:490-496.
4 Koch CA, Pacak K, Chrousos GP: The mo- lecular pathogenesis of hereditary and sporad- ic adrenocortical and adrenomedullary tu- mors. J Clin Endocrinol Metab 2002;87: 5367-5384.
5 Mesiano S, Mellon SH, Jaffe RB: Mitogenic action, regulation and localization of insulin- like growth factors in the human fetal adrenal gland. J Clin Endocrinol Metab 1993;76:968- 976.
-6 Fottner C, Engelhardt D, Weber MM: Regula- tion of steroidogenesis by insulin-like growth factors (IGFs) in adult human adrenocortical cells: IGF-I and, more potently, IGF-II prefer- entially enhance androgen biosynthesis through interaction with the IGF-I receptor and IGF-binding proteins. J Endocrinol 1998; 158:409-417.
7 Weber MM, Fottner C, Wolf E: The role of the insulin-like growth factor system in adrenocor- tical tumorigenesis. Eur J Clin Invest 2000; 30(suppl 3):69-75.
8 Mercado M, DaVila N, McLeod JF, Baumann G: Distribution of growth hormone receptor messenger ribonucleic acid containing and lacking exon 3 in human tissues. J Clin Endo- crinol Metab 1994;78:731-735.
9 Lin CJ, Mendonca BB, Lucon AM, Guazzelli IC, Nicolau W, Villares SM: Growth hormone receptor messenger ribonucleic acid in normal and pathologic human adrenocortical tissues - An analysis by quantitative polymerase chain reaction technique. J Clin Endocrinol Metab 1997;82:2671-2676.
10 Van der Lely AJ, Tschop M, Heiman ML, Ghigo E: Biological, physiological, pathophys- iological, and pharmacological aspects of ghre- lin. Endocr Rev 2004;25:426-457.
11 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth- hormone-releasing acylated peptide from stomach. Nature 1999;402:656-660.
12 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Car- penter R, Grossman AB, Korbonits M: The tis- sue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002;87:2988-2991.
13 Tortorella C, Macchi C, Spinazzi R, Malendo- wicz LK, Trejter M, Nussdorfer GG: Ghrelin, an endogenous ligand for the growth hormone- secretagogue receptor, is expressed in the hu- man adrenal cortex. Int J Mol Med 2003;12: 213-217.
14 Carraro G, Albertin G, Abudukerimu A, Ara- gona F, Nussdorfer GG: Growth hormone se- cretagogue receptor subtypes 1a and 1b are ex- pressed in the human adrenal cortex. Int J Mol Med 2004; 13:295-298.
15 Barreiro ML, Pinilla L, Aguilar E, Tena-Sem- pere M: Expression and homologous regula- tion of GH secretagogue receptor mRNA in rat adrenal gland. Eur J Endocrinol 2002; 147: 677-688.
16 Andreis PG, Malendowicz LK, Trejter M, Neri G, Spinazzi R, Rossi GP, Nussdorfer GG: Ghrelin and growth hormone secretagogue re- ceptor are expressed in the rat adrenal cortex: Evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells. FEBS Lett 2003;536:173-179.
17 Weiss LM: Comparative histologic study of 43 metastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163-169.
18 Korbonits M, Bustin SA, Kojima M, Jordan S, Adams EF, Lowe DG, Kangawa K, Grossman AB: The expression of the growth hormone se- cretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neu- roendocrine tumors. J Clin Endocrinol Metab 2001;86:881-887.
19 Barlier A, Zamora AJ, Grino M, Gunz G, Pel- legrini-Bouiller I, Morange-Ramos I, Figarella- Branger D, Dufour H, Jaquet P, Enjalbert A: Expression of functional growth hormone se- cretagogue receptors in human pituitary ade- nomas: Polymerase chain reaction, triple in- situ hybridization and cell culture studies. J Neuroendocrinol 1999;11:491-502.
20 Kim K, Arai K, Sanno N, Osamura RY, Tera- moto A, Shibasaki T: Ghrelin and growth hor- mone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adeno- mas. Clin Endocrinol (Oxf) 2001;54:759- 768.
21 Papotti M, Cassoni P, Volante M, Deghenghi R, Muccioli G, Ghigo E: Ghrelin-producing endocrine tumors of the stomach and intestine. J Clin Endocrinol Metab 2001; 86:5052- 5059.
22 Arnaldi G, Mancini T, Kola B, Appolloni G, Freddi S, Concettoni C, Bearzi I, Masini A, Boscaro M, Mantero F: Cyclical Cushing’s syn- drome in a patient with a bronchial neuroen- docrine tumor (typical carcinoid) expressing ghrelin and growth hormone secretagogue re- ceptors. J Clin Endocrinol Metab 2003;88: 5834-5840.
23 Volante M, Allia E, Gugliotta P, Funaro A, Broglio F, Deghenghi R, Muccioli G, Ghigo E, Papotti M: Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocri- nol Metab 2002;87:1300-1308.
24 Cassoni P, Papotti M, Ghe C, Catapano F, Sa- pino A, Graziani A, Deghenghi R, Reissmann T, Ghigo E, Muccioli G: Identification, charac- terization, and biological activity of specific re- ceptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. J Clin Endocrinol Metab 2001;86:1738-1745.
25 Cassoni P, Papotti M, Catapano F, Ghe C, De- ghenghi R, Ghigo E, Muccioli G: Specific bind- ing sites for synthetic growth hormone secreta- gogues in non-tumoral and neoplastic human thyroid tissue. J Endocrinol 2000; 165:139- 146.
26 Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, Papotti M: Ghrelin in fetal thyroid and follicular tumors and cell lines: Expression and effects on tumor growth. Am J Pathol 2003; 162:645-654.
27 Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, Pinilla L, Pa- niagua R, Nistal M, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M: Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in nor- mal human testis and testicular tumors. J Clin Endocrinol Metab 2004;89:400-409.
28 Jeffery PL, Herington AC, Chopin LK: Expres- sion and action of the growth hormone releas- ing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocrinol 2002; 172:R7- R11.
29 Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, Whang EE: Ghre- lin promotes pancreatic adenocarcinoma cel- lular proliferation and invasiveness. Biochem Biophys Res Commun 2003; 309:464-468.
30 Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chi- hara K: Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem 2002;277:5667-7564.
31 Tamura H, Kamegai J, Sugihara H, Kineman RD, Frohman LA, Wakabayashi I: Glucocor- ticoids regulate pituitary growth hormone se- cretagogue receptor gene expression. J Neuro- endocrinol 2000; 12:481-485.
32 Petersenn S, Rasch AC, Penshorn M, Beil FU, Schulte HM: Genomic structure and transcrip- tional regulation of the human growth hor- mone secretagogue receptor. Endocrinology 2001;142:2649-2659.
33 Camina JP, Carreira MC, El Messari S, Llo- rens-Cortes C, Smith RG, Casanueva FF: De- sensitization and endocytosis mechanisms of ghrelin-activated growth hormone secreta- gogue receptor 1a. Endocrinology 2004; 145: 930-940.
34 Bohm SK, Grady EF, Bunnett NW: Regula- tory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J 1997; 322:1-18.
35 Belloni AS, Macchi C, Rebuffat P, Conconi MT, Malendowicz LK, Parnigotto PP, Nuss- dorfer GG: Effect of ghrelin on the apoptotic deletion rate of different types of cells cultured in vitro. Int J Mol Med 2004; 14:165-167.