Analysis of somatostatin receptors 2 and 5 polymorphisms in patients with acromegaly.

OBJECTIVE: The aim of the study was to investigate the possible correlation of single nucleotide polymorphisms in somatostatin receptor (SSTR)2 and SSTR5 genes with the responsiveness to somatostatin analogs in a cohort of acromegalic patients. STUDY DESIGN: Three single nucleotide polymorphisms (a-83 g, c-57 g, and t80c) of SSTR2 and three (t-461c, c325t, and c1004t) of SSTR5 were analyzed in 66 acromegalic patients with different responsiveness to somatostatin analogs and 66 healthy controls. RESULTS: Allele frequencies in patients and controls were similar. No association between SSTR2 genotypes and GH and IGF-I levels was found. When considering SSTR5 variants, patients homozygous or heterozygous for the substitution c1004 (P+) showed basal IGF-I levels significantly lower than patients homozygous for 1004t (P-). Moreover, serum GH levels were lower in patients with P+/T- haplotype (having c1004 allele and no t-461 allele) than in those with P-/T+. No correlation between SSTR2 and SSTR5 genotypes, responsiveness to somatostatin therapy, and mRNA expression in the removed adenomas (n = 10) was found. CONCLUSIONS: These data suggest a role for SSTR5 t-461c and c1004t alleles in influencing GH and IGF-I levels in patients with acromegaly, whereas SSTR2 and SSTR5 variants seem to have a minor role in determining the responsiveness to somatostatin analogs.

Assessing the presence of abnormal regulation of cortisol secretion by membrane hormone receptors: in vivo and in vitro studies in patients with functioning and non-functioning adrenal adenoma.

Regulation of cortisol secretion by aberrant hormone receptors may play a role in the pathogenesis of ACTH-independent Cushing's syndrome. In this study, the topic was evaluated by combining in vivo and in vitro approaches. Cortisol responses to various stimuli (standard meal, GnRH + TRH, cisapride, vasopressin, glucagon) were assessed in 6 patients with clinical or subclinical adrenal Cushing's syndrome, and non-functioning adrenal adenoma in two cases. Abnormal responses were observed in three patients with Cushing's syndrome; one patient showed a gastric inhibitory polypeptide (GIP)-dependent cortisol rise after meal, together with responses after GnRH and cisapride; the second patient showed an LH-dependent cortisol response to GnRH, and in the third cortisol rose after cisapride. The pattern of receptor expression performed by RT-PCR showed that while GIP-R was only expressed in tumor from the responsive patient, 5-hydroxytryptamine type 4 receptor and LH-R were also present in normal adrenal tissues and tissues from non-responsive patients. Interestingly, an activating mutation of Gsalpha gene was identified in one of these tumors. Therefore, cortisol responses to agents operating via Gs protein coupled receptors (in one case associated with Gsalpha mutation) were found in Cushing's patients, while these responses were absent in the others. The finding of receptor expression in normal and non-responsive tumors suggests that different mechanisms are probably involved in inducing in vivo cortisol responses.

Loss of heterozygosity at the SS receptor type 5 locus in human GH- and TSH-secreting pituitary adenomas.

SS receptor types 2 and 5 (sst2 and sst5) are involved in the control of secretion and proliferation of normal and tumoral somatotrophs and thyrotrophs. The mechanisms leading to reduced responsiveness to SS analogues in patients with pituitary tumors are poorly understood. The aim of the study was to verify the possible loss of heterozygosity (LOH) at the sst5 gene locus in somatotroph and thyrotroph adenomas by screening leukocyte and tumor DNA for two single nucleotide polymorphisms, i.e. C1004T leading to P335L change and T-461C in the 5'-upstream region. Among the 13 informative samples, 1 GH- and 1 TSH-secreting adenoma showed LOH at sst5 gene locus with the retention of Leu335 variant. By analyzing other polymorphic markers spanning from telomere to 16p13.3-13.2 boundaries, DNA deletion of at least 1 megabase was found in both tumors. LOH in thyrotroph adenoma was associated with unusual tumor aggressiveness that required a second surgery and resistance to SS analogs, while no obvious phenotype was identified in the case of the somatotroph adenoma. In conclusions, LOH at the sst5 gene locus is a rare phenomenon, occurring in about 10% of pituitary tumors, that seems to be associated with an aggressive phenotype, at least in thyrotroph adenomas. Further studies are required to confirm this association and to identify the genes, in addition to sst5, lost in these tumors.

Effects of hypothalamic neuropeptides on extracellular signal-regulated kinase (ERK1 and ERK2) cascade in human tumoral pituitary cells.

The G protein-coupled receptor (GPCR) activation has been demonstrated to affect the ERK1/2 cascade in different cell lines. We investigated the effects of hypothalamic neuropeptides acting via GPCR on this pathway in GH-secreting (GH-oma) and nonsecreting (NFPA) pituitary adenomas. GHRH increased ERK1/2 activity (236 +/- 80%) in both gsp- and gsp+ GH-omas, this effect being almost completely abolished by protein kinase C (PKC) blockade. Both GnRH and pituitary adenylate-activating peptide caused a similar PKC-dependent activation of ERK1/2 in most NFPA. Increasing cAMP by forskolin caused a protein kinase A-dependent increase of ERK activity (287 +/- 37%) in GH-omas and had no effect in NFPA. ERK cascade blockade in GH-omas did not affect basal and GHRH-stimulated GH release, whereas it totally prevented the 3-fold increase in cyclin D1 protein expression induced by GHRH. In conclusion, this study demonstrated that in pituitary adenomas the activation of GPCR by neurohormones caused a PKC-dependent activation of ERK1/2 cascade that, at least in GH-omas, resulted to be involved in cyclin D1 induction by GHRH. Moreover, a stimulatory effect of the protein kinase A-dependent pathway on ERK1/2 cascade occurred selectively in GH-omas, probably contributing to the mitogenic potential of the cAMP pathway in this cell type.

Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells.

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.

Mutation of somatostatin receptor type 5 in an acromegalic patient resistant to somatostatin analog treatment.

Introduction of somatostatin analogs has greatly contributed to improving the prognosis of acromegaly. Although the majority of patients are effectively treated by these agents, resistance occurs in a subset of patients. So far, resistance to somatostatin has never been associated with mutations of the somatostatin receptor subtypes (sst2 and sst5) that inhibit GH secretion. Molecular analysis of genomic DNA from pituitary tumor and peripheral blood obtained from an acromegalic resistant to octreotide showed a somatic activating mutation of Gsalpha (Arg201Cys), no mutation in sst2, and one polymorphism (Pro109Ser) and one germ line mutation (Arg240Trp) in sst5. Wild-type (WT) and mutant sst5 PCR products were cloned and transfected into Chinese hamster ovary K1 cells. In Chinese hamster ovary K1 cells stably expressing mutant sst5, somatostatin-28 was less potent in inhibiting cyclic AMP levels than in WT cells. Proliferation of mutant cells exceeded that of WT by 50%. Moreover, somatostatin reduced cell growth and MAPK activity in WT but not in mutant cells in which the peptide even increased MAPK activity. We suggest that this mutation that abrogates the antiproliferative action of somatostatin and activates mitogenic pathways may be involved in the resistance to somatostatin treatment.

Expression of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein and inducible-cAMP early repressor genes in growth hormone-secreting pituitary adenomas with or without mutations of the Gsalpha gene.

In about 30-40% of GH-secreting adenomas, gain-of-function mutations of the Gsalpha gene, which convert this gene into an oncogene termed gsp, occur. Gsalpha mutations have been related to pituitary tumorigenesis. We focused on 2 nuclear transcription factors that are final targets of the cAMP-dependent pathway and are positively regulated by cAMP signaling, i.e. the cAMP-responsive element binding protein (CREB) and the inducible cAMP early repressor (ICER), that derives from alternative splicing of cAMP-responsive element modulator gene. We examined 21 GH-secreting adenomas, 8 with (gsp(+)) and 13 without (gsp(-)) a mutated Gsalpha. Analysis of CREB and ICER I/II messenger RNA revealed that the levels of both transcripts were higher in gsp(+) than in gsp(-) tumors (CREB/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mean optical density +/- SE, 2.34 +/- 0.36 in gsp(+) vs. 0.99 +/- 0.22 in gsp(-), P = 0.003; ICER I/GAPDH, 0.53 +/- 0.15 in gsp(+) vs. 0.14 +/- 0.07 in gsp(-), P = 0.01; ICER II/GAPDH, 1.5 +/- 0.21 in gsp(+) vs. 0.83 +/- 0.13 in gsp(-), P = 0.01), although a few cases in both groups did not display this pattern of expression. Moreover, no positive correlation between the levels of CREB and ICER transcripts was observed, suggesting the possible presence of alterations in the mechanisms by which cAMP signaling directs the expression of CREB and/or ICER genes. Our results indicate a complex pattern of expression of nuclear transcription factors that mediate cAMP action in both gsp(+) and gsp(-) tumors, suggesting that, beside Gsalpha gene mutations, different and partially unknown molecular events may contribute to the pathogenesis of these tumors.

Somatostatin receptor subtype 2 and 5 in human GH-secreting pituitary adenomas: analysis of gene sequence and mRNA expression.

The role of somatostatin receptor subtypes 2 and 5 (SSTR2 and SSTR5) in determining the secretory and proliferative phenotype as well as the sensitivity to somatostatin analogue treatment is not clearly established. We quantified the expression of SSTR2 and SSTR5 mRNA using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 19 human growth hormone (GH) -secreting adenomas. Tumour characteristics and in vivo sensitivity to somatostatin analogues were assessed; tumours were screened for Gsalpha gene mutations. PCR products of SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were directly sequenced. All tumours expressed both SSTR2 and SSTR5 mRNA at variable levels. No significant correlation between SSTR2 and SSTR5 expression and the presence of Gsalpha mutation, GH levels, or tumour size and invasiveness was observed. A negative correlation between SSTR2 and SSTR5 mRNA levels was observed (r = 0.5; P < 0.05). No significant correlation between the levels of SSTR2 and SSTR5 expression and the in vivo responsiveness to somatostatin analogues was observed, although a tendency to a low SSTR2 expression in resistant tumours was found. No mutations in the coding or bordering regions of either SSTR2 or SSTR5 adenomatous DNA from patients totally or partially resistant to somatostatin analogues were found. The study shows that the different expression of SSTR2 and SSTR5 in GH-secreting adenomas is not significantly correlated with the secretory and proliferative phenotype, although the large, hypersecretory tumours and those with a poor sensitivity to somatostatin analogues seem to express low levels of SSTR2 mRNA. Moreover, both SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were found to possess intact coding sequences.

Activating mutations of the Gs alpha gene are associated with low levels of Gs alpha protein in growth hormone-secreting tumors.

Evidence suggests the existence of a direct relationship between cellular Gs alpha content and activation of the adenylyl cyclase system. Data on Gs alpha levels in endocrine tumors that depend on cAMP for growth, particularly pituitary adenomas, are still limited. The levels of Gs alpha protein were evaluated in 11 GH-secreting adenomas with Gs alpha mutations (gsp+) and 15 without (gsp). Complementary DNAs from gsp+ tumors contained very low amounts of wild-type Gs alpha sequences, indicating a preponderance of the mutant Gs alpha transcripts in these tumors. Immunoblotting of Gs alpha protein showed that the two isoforms were present at high levels in all gsp-, but were undetectable or barely detectable in gsp+. The low Gs alpha content in gsp+ tumors was not due to a reduction in ribonucleic acid synthesis or stability, as Gs alpha messenger ribonucleic acid levels were similar in wild-type and mutant tissues. Treatment of gsp- cells with cholera toxin caused a marked reduction of Gs alpha levels. As in other cell systems cholera toxin increases Gs alpha degradation, our data are consistent with an accelerated removal of mutant Gs alpha. This may represent an additional mechanism of feedback response to the constitutive activation of cAMP signaling in pituitary tumors with mutations in the Gs alpha gene.

G protein abnormalities in pituitary adenomas.

It has been demonstrated that the majority of secreting and nonsecreting adenomas is monoclonal in origin suggesting that these neoplasia arise from the replication of a single mutated cell, in which growth advantage results from either activation of protooncogenes or inactivation of antioncogenes. Although a large number of genes has been screened for mutations, only few genetic abnormalities have been found in pituitary tumors such as allelic deletion of chromosome 11q13 where the MEN-1 gene has been localised, and mutations in the gene encoding the alpha subunit of the stimulatory Gs and Gi2 protein. These mutations constitutively activate the alpha subunit of the Gs and Gi2 protein by inhibiting their intrinsic GTPase activity. Both Gs alpha and Gi2alpha can be considered products of protooncogenes (gsp and gip2, respectively) since gain of function mutations that activate mitogenic signals have been recognized in human tumors. Gsp oncogene is found in 30-40% of GH-secreting adenomas, in a low percentage of nonfunctioning and ACTH-secreting pituitary adenomas, in toxic thyroid adenomas and differentiated thyroid carcinomas. The same mutations, occurred early in embriogenesis, have been also identified in tissues from patients affected with the McCune Albright syndrome. These mutations result in an increased cAMP production and in the subsequent overactivation of specific pathways involved in both cell growth and specific programmes of cell differentiation. By consequence, the endocrine tumors expressing gsp oncogene retain differentiated functions. The gip2 oncogene has been identified in about 10% of nonfunctioning pituitary adenomas, in tumors of the ovary and the adrenal cortex. However, it remains to be established whether Gi proteins activate mitogenic signals in pituitary cells. Since Gi proteins are involved in mediating the effect of inhibitory neurohormones on intracellular effectors, it has been proposed that in pituitary tumors the low expression of these proteins, particularly Gi1-3alpha, may contribute to uncontrolled pituitary cells growth by preventing the transduction of inhibitory signals. While by in vitro mutagenesis it has been demonstrated that activated mutant of Gq alpha, G12alpha, G13alpha and Gz alpha are fully oncogenic, it remains to be proved whether or not these abnormalities might naturally occur in human tumors and, in particular, in pituitary adenomas.

Growth hormone-releasing hexapeptide (GHRP-6) increases intracellular calcium concentrations in cultured cells from human pituitary adenomas of different types.

OBJECTIVE: The GH-releasing peptide GHRP-6, has been found to interact with specific receptors in somatotrophs, causing cytosolic Ca2+ ([Ca2+]i) rise and GH release. Moreover, this peptide has been demonstrated to stimulate the secretion of pituitary hormones other than GH, i.e. ACTH and prolactin, this effect being generally attributed to a central action. In this study we evaluated whether the pituitary action of this peptide is restricted to cell type of somatotroph lineage. METHODS: The effect opf GHRP-6 on [Ca2+]i was tested in cell preparations obtained from a series of human pituitary adenomas (9 GH-secreting adenomas, 7 nonfunctioning adenomas, 3 ACTH-secreting adenomas, 2 TSH-secreting adenomas and 1 prolactinoma) loaded with the Ca2+ indicator fura-2. RESULTS: GHRP-6, at concentrations higher than 1 nmol/l, significantly increased [Ca2+]i in all tumours, with the exception of the 3 ACTH-secreting adenomas in which the peptide was ineffective at any concentration tested (from 1 nmol/l to 1 micromol/l). By contrast, in all ACTH-secreting adenomas, both corticotrophin-releasing hormone and pituitary adenylate cyclase activating peptide caused a marked [Ca2+]i increase. In tumours responsive to GHRP-6, the peptide caused a typical biphasic [Ca2+]i rise due to Ca2+ mobilization from the intracellular stores and Ca2+ influx through voltage-dependent Ca2+ channels. CONCLUSIONS: These data indicate that almost all tumoral pituitary cell types are targets of GHRP-6 action, the only exception being corticotrophs.

Constitutively active Gs alpha is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas.

Because phosphodiesterase (PDE) expression and activity are controlled by cAMP, we investigated whether activating mutations of Gs alpha gene that occur in human GH-secreting adenomas are associated with increased PDE activity. We studied 10 adenomas with wild-type Gs alpha (gsp-) and 8 with mutant Gs alpha (gsp+). Although, in the absence of PDE inhibitors, intracellular cAMP levels were similar in gsp+ e gsp- adenomas, the PDE blockade with 3-isobutyl-1-methylxanthine induced a marked increase in cAMP in all but one gsp+ adenoma (% increase: from 77 to 2900) and a slight rise in only 2 gsp-. Similar results were obtained with the PDE4 selective inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl)]-2-pyrrolidinone. In vitro GH release was significantly higher in gsp+ than in gsp- adenomas (315 +/- 158 vs. 82 +/- 53 micrograms/well; P < 0.01), and PDE blockade caused a further increase in 3 of 5 gsp+ adenomas but not in gsp- tumors. By direct measurement, PDE activity was about 7-fold higher in gsp+ than in gsp- adenomas (320 +/- 213 vs. 48 +/- 23 pmol/min.mg protein; P < 0.05) and was largely 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone sensitive. This study first demonstrates that activating mutations of the Gs alpha gene that naturally occur in pituitary adenomas is associated with an increased PDE activity that might, at least partially, counteract the constitutive activation of the cAMP-dependent pathway.

Immunodetection of G proteins in human pituitary adenomas: evidence for a low expression of proteins of the Gi subfamily.

G proteins mediate signal transduction in a variety of cell systems. As the expression of these proteins has not yet been investigated in detail in human pituitary tumors, we evaluated the presence of G proteins in a series of tumors including six non-functioning adenomas, five GH-secreting adenomas, three prolactinomas and one TSH-secreting adenoma, using immunoblotting and immunohistochemistry. By immunoblotting, Gi1/2alpha was undetectable in six and barely detectable in nine tumors. A similar pattern of expression was observed by probing with the antibody to Gi3alpha, which detected a very weak band in 11 tumors and no protein in four. In contrast, using large amounts of membrane proteins (40 microg), both Gi1/2alpha and Gi3alpha were detected, although at very low levels, in the negative tumors. The low expression of these proteins appeared to be specific to tumoral tissues, as both Gi1/2alpha and Gi3alpha were abundant in normal human and rat pituitary. In all tumors, Go alpha, the two Gs alpha forms, Gq/11 and G beta were present in significant amounts. Semiquantitative analysis indicated that Gs alpha was clearly detected when 2.5 microg loaded proteins were used, whereas Gi1/2alpha and Gi3alpha were barely detected with 5 microg. By immunofluorescence, all tumors studied were markedly positive for Gs alpha that was immunolocalized at the cell periphery, whereas they showed a weak positivity for Gi1/2alpha and Gi3alpha. The study is the first to provide evidence for a low expression of Gi proteins, which are involved in the transduction of inhibitory signals, in pituitary adenomas.

Cyclic AMP and calcium in the transduction of hypothalamic neurohormone action in human pituitary tumors.

Although hypothalamic neurohormones were originally identified on the basis of their ability to influence hormone release from the target cells, it is at present clear that these agents are involved in the control of several biological processes that include cell proliferation and differentiation as well as hyperplasia and neoplastic transformation. Hypothalamic neurohormones bind specific receptors belonging to the G-protein-coupled receptor superfamily to generate intracellular effectors, in particular cAMP and calcium. Several in vivo and in vitro studies suggest the presence of functional and/or structural alterations in the receptor/effector molecules involved in the transduction of hypothalamic neurohormone action in the pituitary. The present study reports the frequent presence in pituitary tumors of cellular alterations that result in amplification of stimulatory signals, particularly activation of the cAMP-dependent pathway, and reduction of inhibitory inputs, both events having possible implications in tumor formation and/or progression.