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Gene Transcription of Receptors for Growth Hormone-Releasing Peptide and Somatostatin in Human Pituitary Adenomas¹

Medical Department M, Aarhus Kommunehospital, Aarhus University Hospital (S.N., S.M., J.W., J.O.L.J.); Laboratory for Molecular Pathology, Aarhus Kommunehospital, Aarhus University Hospital (L.M.R.); Research Laboratory for Biochemical Pathology, Aarhus Kommunehospital, Aarhus University Hospital (T.L.); Department of Neurosurgery, Aarhus Kommunehospital, Aarhus University Hospital (J.A.), DK-8000 Aarhus C, Denmark

Address correspondence and requests for reprints to: Steen Nielsen MD, Medical Department M (Diabetes and Endocrinology), Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark. E-mail: stn{at}afdm.aau.dk

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Growth hormone (GH)-releasing peptides (GHRP) or secretagogs (GHS) constitute a family of synthetic compounds with potent and specific GH releasing activity. The receptor (GHS-R) has recently been cloned even though the endogenous ligand remains to be identified. GHRPs act both at the hypothalamic and the pituitary level through mechanisms involving amplification of GH-releasing hormone activity and functional somatostatin antagonism. In the present study we examined the co-expression of messenger RNA (mRNA) for GHS-R and all 5 somatostatin receptor subtypes (sstr 1–5) in 28 human pituitary tumors by RT-PCR. GHS-R transcription was detected in 11 out of 12 somatotroph adenomas and in 2 out of 2 prolactinomas, whereas GHS-R expression was detected in only 2 out of 14 clinically nonfunctioning adenomas (NFPA), and no expression was seen in the only ACTH secreting adenoma. Almost all tumors expressed sstr 2 mRNA (n = 24), whereas only 1 tumor expressed sstr 4 mRNA. The expression of sstr 3 mRNA was inversely associated with GHS-R expression (P < 0.001), which could be attributed to a high prevalence of sstr 3 expression in NFPA.

This study suggests that GHS-R expression is predominantly observed in somatotroph adenomas and much less so in NFPA. Moreover, the presence of a distinct pattern of somatostatin receptor subtype co-expression is suggested, which may provide a molecular basis for the complex interaction between GHRPs and somatostatin.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GROWTH hormone (GH)-releasing peptides (GHRPs) constitute a heterogenous family of synthetic GH secretagogues originally described by Bowers et al. (1) one year before the characterization of GH releasing hormone (2, 3). Several lines of evidence suggested the existence of a specific receptor and, consequently, of an endogenous ligand (4). Recent achievements within this field include synthesis of nonpeptide GHRP agonists (peptidomimetics) and, more importantly, cloning of the pig and human GHRP receptor (GHS-R) (5). Localization studies in rat brain and pituitary gland have shown expression of the GHS-R in the anterior pituitary gland and in the hypothalamus, in addition to regions of the brain such as the hippocampus and substantia nigra.

The modes and sites of action of GHRPs are complex and not fully clarified. The pronounced increase in GH secretion is corroborated by direct effects on somatotrophs, amplification of GHRH activity and release, and functional somatostatin antagonism (4, 5). In addition to this, both in vitro and in vivo studies show that GHRPs stimulate the secretion of prolactin and ACTH from the normal pituitary.

The degree to which human pituitary adenomas are regulated by GHRPs has not been extensively investigated. Stimulation of GH secretion by GHRP-6 in cell cultures of human somatotroph adenoma has been published by Renner et al. (6), and recently, expression of GHS-R mRNA in human pitutitary adenomas secreting GH, prolactin, and ACTH has been reported (7).

In the present study we examined the expression of GHS-R messenger RNA (mRNA) in human pituitary tumors, including a large number of clinically nonfunctioning adenomas. To gain further insight into the complex interplay between GHRPs and somatostatin, we also measured mRNA expression for all five somatostatin receptor subtypes.

	Subjects and Methods

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Twenty-eight subjects with pituitary adenomas from a prospective series were included in the study. Tumor types were determined according to a preoperative standard endocrinological examination. For the purpose of RT-PCR analysis, tissue samples were collected at the time of transsphenoidal adenomectomy.

Material

The tissue samples were collected at the time of operation, immediately frozen in liquid nitrogen, and thereafter kept at -80 C until analyzed.

RNA extraction

Total RNA was extracted from the tissue samples with Trizol (GIBCO BRL, Bethesda, MD). The amount and the quality of the RNA were assessed by measurement of the optical density at 260 and 280 nm.

RT-PCR

From each sample 1 µg of RNA was used for RT in 30 µL buffer with final concentrations of 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dCTP, 0.5 mM dGTP, 30 pmol random hexamers/30 µL, and 200 U MMLV RT. The samples were incubated for 75 min at 37 C, followed by incubation for 15 min at 95 C. A similar procedure was performed in a duplicate sample but without the addition of the MMLV-RT. This nonreverse transcribed material served as a negative control for the PCR procedures.

For the polymerase chain reaction 2 µL of the RT product was used in 20 µL of solution, which contained final concentrations of 20 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dCTP, 0.5 mM dGTP, 2.5 U Taq DNA polymerase/20 µL, and 500 pmol of each of the specific primers. The following primers were used: GHS-R (base 662-1011): 5'-TCTTCCTTCCTGTCTTCTGTC-3' and 5'-AAGTCTGAACACTGCCACC-3'; somatostatin receptor subtypes (sstr) 1: 5'-GGAACTCTATGGTCATCTAC-3' and 5'-GCTGAGCACAGTCAGACAGT-3' (8); sstr 2: 5'-TGACAGTCATGAGCATCGAC-3' and 5'-GCAAAGACAGATCATGGTGA-3' (8); sstr 3: 5'TCATCTGCCTCT-GCTACCTG-3' and 5'-GAGCCCAAAGAAGGCAGGCT-3' (8); sstr 4: 5'-ATCTTCGCAGACACCAGACC-3' and 5'-ATCAAGGCTGGTCACGACGA-3' (8); sstr 5: 5'-CGTCTTCATCATCTACACGG-3' and 5'-GGCCAGGTTGACGATGTTGA-3' (8); and GAPDH: 5'-GCCAAAAGGGTCATCATCTC-3' and 5'-GTAGAGGCAGGGATGATGTTC-3'. Ampli-fication of DNA was performed with two initial cycles at 95 C for 60 sec, followed by 60 sec at a temperature of annealing specific for each primer, and 60 sec at 73 C; this was followed by a number of cycles at 94 C for 45 sec, followed by 45 at the temperature of annealing and 45 sec at 73 C. In the final cyclus the samples were incubated for 2 min at 73 C. The temperature of annealing was 60 C in the case of sstr 1–4, 65 C in the case of sstr 5, and finally 50 C in the case of GAPDH and the GHS receptor. The amplification of DNA was carried out in 30 cycles in the case of GAPDH, 40 cycles for the GHS-R, and finally 38 cycles for the sstr 1–5. The PCR product was separated by gel electrophoresis in 2% agarose gel containing ethidium bromide. To ensure that an appropriate sensitivity level was used, the number of PCR cycles was chosen after optimization experiments using a pool of cDNA containing reverse transcribed RNA from several tumor types. This cDNA pool was subjected to different numbers of cycles between 30 and 40. The number of cycles that gave rise to a weak band was chosen for evaluation of all samples.

The PCR products were visualized in ultraviolet light using equipment from Bio-Rad (Hercules, CA). As an arbitrary cut-off level, a clearly visible band was considered a sign of gene transcription. The results of the gel electrophoresis were determined by an independent investigator who was unaware of the clinical characteristics of the patients.

All PCR-reactions gave rise to DNA with the expected size. The specificity of the GHS-R PCR-product was further verified by cleavage with the restriction enzyme DdeI. This procedure resulted in DNA fragments of 183, 95 and 71 base pairs, as expected.

Statistics

Proportions were compared by the chi-square test. A P-value less than 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The study comprised 28 patients, of which 16 were females and 12 were males. Eleven patients with acromegaly and 2 patients with prolactinomas were included in the study, while 14 patients harbored nonfunctioning pituitary adenomas (NFPA), i.e. pituitary adenomas without clinical signs of hormonal hypersecretion. Expression of mRNA encoding GAPDH was found in all cases, and no PCR product was seen in samples without the addition of the MMLV reverse transcriptase (data not shown). Expression of mRNA encoding the GHS-R was present in 14 out of 28 pituitary adenomas, and these 14 adenomas comprised 10 somatotroph adenomas, both prolactinomas, and 2 nonfunctioning adenomas, whereas it was absent in the only ACTH-producing adenoma. In total, 10 of 11 somatotroph adenomas expressed GHS-R mRNA. The distribution of mRNA for the GHS-R and somatostatin receptor subtypes in the different types of pituitary adenomas is seen in Table 1. A clear discrimination between adenomas GHS-R mRNA positive and negative is apparent in Fig. 1.

View larger version (53K): [in this window] [in a new window]   Figure 1. This figure shows the PCR produce of mRNA encoding the GHS-R. Patient numbers 22, 23, 24,12, 25, 13, and 14 are seen in lanes 1–7, respectively. A clearly visible band of the expected size of 349 base pairs is seen in lanes 1, 2, 3, and 5.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The recent cloning of the GH secretagogue receptor (GHS-R) by Howard et al. (5) represents the latest achievement within the research of GHRPs. It is a G-protein coupled receptor that activates phospholipase C, and the signal transduction pathway includes activation of Ca⁺⁺ channels, which increases free intracellular Ca⁺⁺ (9). Localization studies in rat brains have shown that GHS-R is expressed in the anterior pituitary gland and in several regions of the hypothalamus, as well as in extrahypothalamic areas.

Studies in humans have mainly included in vivo administration of GHRPs alone and in combination with GHRH and somatostatin in healthy subjects, as well as in patients with pituitary adenomas. Moreover, GHRP has been shown to stimulate GH secretion from human somatotroph adenoma cells in vitro (6). More recently, two preliminary reports have documented GHS-R mRNA expression in a small number of human pituitary adenomas and normal pituitaries (7, 10).

The present study aimed to further assess the prevalence of GHS-R mRNA expression in human pituitary adenomas, with special emphasis on GH-secreting tumors and clinically nonfunctioning pituitary adenomas. Moreover, we were interested in the distribution and co-expression of mRNA for all five somatostatin receptors (sstr 1–5) to gain further insight into the complex interplay between GHRPs and somatostatin (4). Our main finding is that GHS-R is expressed in GH and prolactin secreting adenomas, but not in NFPA. Moreover, there is evidence to suggest that the presence of GHS-R may be associated with a specific composition of somatostatin receptor subtypes.

In the present study GHS-R mRNA was demonstrated in 10 of 11 somatotroph adenomas and both prolactinomas. In accordance with this, it has previously been shown that GH secretion is stimulated by GHRPs in vitro (6), and hexarelin, a GHRP, stimulates the secretion of both GH and prolactin in acromegalic subjects in vivo (11, 12).

Expression of GHS-R mRNA was absent only in ACTH-secreting adenoma in this series, and furthermore, expression of GHS-R mRNA was found in only 2 of 14 clinically nonfunctioning adenomas. There were no immunohistochemical signs of GH or prolactin in tissue sections from these adenomas (data not shown), which supports the hypothesis that GHS-R mRNA expression is characteristic of lactosomatotroph adenoma and a rare finding in other pituitary adenomas.

The pathophysiological role of GHRPs in active acromegaly is presently unclear, but a possible interplay between GHRPs and somatostatin is suggested from the fact that GHRP-stimulated GH secretion is blunted by octreotide in some somatotroph adenomas (6). The presence of somatostatin receptors in pituitary adenomas has previously been shown by the demonstration of somatostatin binding sites, and the receptor subtypes have been documented by RT-PCR analysis in several papers (13, 14, 15, 16). In accordance with our results these studies generally report sstr 2 to be the most widely distributed receptor subtype in pituitary adenomas, whereas sstr 4 mRNA is absent in most series. The prevalence of sstr 3 mRNA expression in our population appeared to be high compared with previous studies (15). This is attributed to a large proportion of NFPA, in most of which sstr 3 mRNA was expressed. The significant negative association between GHS-R and sstr 3 mRNA expression in our series is also largely accounted for by the low prevalence of GHS-R mRNA in NFPA. Whether these associations imply causality cannot be deduced from our series, but it is tempting to speculate that the functional antagonism between GHRPs and somatostatin may rely on a specific pattern of co-expression of these receptors. Indeed, the frequent co-expression of sstr 2 mRNA and GHS-R mRNA and the negative association between GHS-R mRNA and sstr 3 mRNA could provide a molecular basis of the interplay between the GHRPs and somatostatin.

In summary, we report the presence of GHS-R mRNA and somatostatin receptor mRNA in a prospective series of human pituitary adenomas. Our main findings are that GHS-R mRNA is expressed predominantly in somatotroph adenomas, and that a specific pattern of co-expression of GHS-R and the somatostatin receptor subtypes appears to exist. The physiological and clinical implications of these observations need to be further scrutinized.

	Acknowledgments

 
The technical assistance of Mrs. Merete Dixen is gratefully acknowledged.

	Footnotes

 
¹ The study was supported by Aarhus University - Novo Nordisk Centre for Growth and Regeneration.

Received March 18, 1998.

Revised May 5, 1998.

Accepted May 12, 1998.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Momany FA, Bowers CY, Reynolds GA, Chang D, Hong A, Newlander K. 1981 Design, synthesis, and biological activity of peptides which release growth hormone in vitro. Endocrinology. 108:31–39.[Abstract]
2. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB. 1982 Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science. 218:585–587.[Abstract/Free Full Text]
3. Rivier J, Spiess J, Thorner M, Vale W. 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumor. Nature. 300:276–278.[CrossRef][Medline]
4. Smith RG, Lex HTvdP, Howard AD, et al. 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev. 18:621–645.[Abstract/Free Full Text]
5. Howard AD, Feighner SD, Cully DF, et al. 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release [see comments]. Science. 273:974–977.[Abstract]
6. Renner U, Brockmeier S, Strasburger CJ, et al. 1994 Growth hormone (GH)-releasing peptide stimulation of GH release from human somatotroph adenoma cells: interaction with GH-releasing hormone, thyrotropin-releasing hormone, and octreotide. J Clin Endocrinol Metab. 78:1090–1096.[Abstract]
7. Keyzer Yd, Lenne F, Bertagna X. 1997 Widespread transcription of the growth hormone-releasing peptide receptor gene in neuroendocrine human tumors. Eur J Endocrinol. 137:715–718.[Abstract]
8. Kubota A, Yamada Y, Kagimoto S, et al. 1994 Identification of somatostatin receptor subtypes and an implication for the efficacy of somatostatin analogue SMS 201–995 in treatment of human endocrine tumors. J Clin Invest. 93:1321–1325.
9. Herrington J, Hille B. 1994 Growth hormone-releasing hexapeptide elevates intracellular calcium in rat somatotropes by two mechanisms. Endocrinology. 135:1100–1108.[Abstract]
10. Korbonits M, Aylwin SJB, Trainer PJ, Burrin JM, Besser GM, Grossman AB. Growth hormone secretagogue-receptor mRNA is expressed in human pituitary adenomas. Proceedings of the 79th Annual Meeting of The Endocrine Society, Minneapolis, MN, 1997 p. 128. (Abstract)
11. Alster DK, Bowers CY, Jaffe CA, Ho PJ, Barkan AL. 1993 The growth hormone (GH) response to GH-releasing peptide (His-DTrp-Ala-Trp-DPhe-Lys-NH2), GH-releasing hormone, and thyrotropin-releasing hormone in acromegaly. J Clin Endocrinol Metab. 77:842–845.[Abstract]
12. Ciccarelli E, Grottoli S, Razzore P, et al. 1996 Hexarelin, a synthetic growth hormone releasing peptide, stimulates prolactin secretion in acromegalic but not in hyperprolactinaemic patients. Clin Endocrinol (Oxf). 44:67–71.[CrossRef][Medline]
13. Greenman Y, Melmed S. 1994 Heterogeneous expression of two somatostatin receptor subtypes in pituitary tumors. J Clin Endocrinol Metab. 78:398–403.[Abstract]
14. Greenman Y, Melmed S. 1994 Expression of three somatostatin receptor subtypes in pituitary adenomas: evidence for preferential SSTR5 expression in the mammosomatotroph lineage. J Clin Endocrinol Metab. 79:724–729.[Abstract]
15. Miller GM, Alexander JM, Bikkal HA, Katznelson L, Zervas NT, Klibanski A. 1995 Somatostatin receptor subtype gene expression in pituitary adenomas. J Clin Endocrinol Metab. 80:1386–1392.[Abstract]
16. Panetta R, Patel YC. 1995 Expression of mRNA for all five human somatostatin receptors (hSSTR1–5) in pituitary tumors. Life Sci. 56:333–342.[CrossRef][Medline]

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