Growth hormone receptor mRNA expression in non-functioning and somatotroph pituitary adenomas: implications for growth hormone substitution therapy?

The use of growth hormone (GH) in patients with GH deficiency induced by pituitary adenoma is widely accepted, but the safety of this mitogenic hormone, particularly in patients with residual tumor after neurosurgery, continues to be a concern. Since the mitogenic potency of GH is dependent upon the presence of the GH receptor (GH-R) and the subsequent IGF-1/IGF receptor (IGF-1-R) system we investigated the expression of the members of the growth hormone cascade in endocrine inactive and GH-producing pituitary adenomas. Tissue specimens of 18 clinically non-functioning pituitary adenomas and 6 GH-producing adenomas were collected following transsphenoidal surgery while normal cadaver pituitary glands served as controls. After RNA extraction, semi-quantitative RT-PCR amplification with specific primers for GH, GH-R, IGF-1 and IGF-1-R was performed. Applying this sensitive RT-PCR based approach, GH-R expression was demonstrated in all normal pituitaries, most inactive adenomas (94%), and the majority of GH-producing adenomas (66%). Both IGF-1 and IGF-1-R mRNA was detectable in the majority of inactive (72% and 77%, respectively) and somatotrophic adenomas (83% and 83%). While IGF-1-R mRNA was expressed in all normal pituitary specimen studied, IGF-1 was detectable in only 55% of them. In summary, expression of members of the GH-IGF-1 cascade could be demonstrated in a substantial subset of patients with non-functioning and GH-producing pituitary adenomas. These factors might serve as a substrate for the transduction of mitogenic effects of GH on remnant pituitary tumors during GH replacement therapy. Therefore, GH therapy should be carefully considered and patients on GH therapy kept under close observation.     

Prevalence of Gs alpha mutations in Korean patients with pituitary adenomas

The reported frequencies of Gs alpha mutations (gsp mutations) in growth hormone (GH)-secreting pituitary adenomas are variable (ranging from 4.4 to 43%), and the presence of these mutations in the other pituitary adenomas is still a matter of controversy. Previous clinical and biochemical analyses of patients with GH-secreting pituitary adenomas and gsp mutations produced conflicting results and did not demonstrate obvious characteristics. Therefore, we investigated the prevalence of gsp mutations in Korean patients with pituitary adenomas and elucidated the characteristics of these patients. Forty-four GH-secreting adenomas, 7 prolactin (PRL)-secreting adenomas and 32 clinically non-functioning adenomas were examined for the presence of point mutations in codon 201 and 227 of the Gs alpha gene using a nested PCR and direct sequencing of DNA extracted from fresh tissue or paraffin-embedded pituitary adenoma samples. Seven of the 44 GH-secreting pituitary adenomas had point mutations at codon 201 or 227; of these, five mutations were in codon 201 and two were in codon 227. In patients with gsp mutations, mean tumor size was significantly smaller than in patients without gsp mutations (15.9+/-8.7 mm vs. 24.9+/-14.9 mm, P<0.05). Age, sex, basal GH levels, GH response to oral glucose loading, GH response to octreotide and surgical outcome were not different in the two groups. One of the 32 clinically non-functioning pituitary adenomas had a point mutation at codon 201; none of the seven prolactinomas had these mutations. These results show that gsp mutations are not rare in Korean acromegalic patients and mean tumor size is significantly smaller in acromegalic patients with gsp mutations. Our results also confirm the low frequency of gsp mutations in clinically non-functioning pituitary adenomas and the absence of gsp mutations in prolactinoma.     

Differential expression of genes related to drug responsiveness between sparsely and densely granulated somatotroph adenomas

There are two main subtypes of GH-producing pituitary adenoma: densely granulated (DG-type) and sparsely granulated (SG-type). Despite the difference in drug responsiveness between the two subtypes, their molecular mechanisms remain unknown. The aim of this study is to evaluate the differential expression of genes related to drug responsiveness between the two subtypes of somatotroph adenoma, and their relationship to the clinical characteristics. Eighty-two acromegaly patients (44 DG-type, 38 SG-type) were studied retrospectively. Clinical characteristics were compared between the two subtypes. Among them, 36 tumor tissue specimens (19 DG-type, 17 SG-type) were available for investigation of the expression of SSTR2, SSTR5 and D2R that are reported to be involved in drug responsiveness by realtime RT-PCR. Protein level was evaluated by immunohistochemical study. Patients with SG-type adenomas were younger in age and showed greater GH suppression by octreotide, but not by bromocriptin, and bigger in size and more invasiveness than DG-type adenomas. The mRNA expression of SSTR2 in DG-type adenomas were greater than those in SG-type adenomas and showed significantly positive correlation with GH suppression by octreotide. There was positive correlation between mRNA and protein levels of SSTR2. These data suggested that the differences of responsiveness to octreotide between DG- and SG-type adenomas are based on the expression levels of SSTR2.     

Genetic changes in human pituitary adenomas

In the last few years molecular biology technologies have provided important insights into mechanisms possibly involved in pituitary tumor formation. Several evidences indicate that the majority of pituitary adenomas is monoclonal, thus implying that they derive from the replication of a single mutant cell. In about 30-40% of GH-secreting adenomas mutations at codon 201 and 227 of GNAS1 gene that codes for the Gs alpha subunit have been identified. These mutations, named gsp for Gs protein, cause the constitutive, hormone-independent, activation of adenylyl cyclase and consequent overproduction of cAMP, that is mitogenic in somatotropes. Screening studies carried out on large series of acromegalic patients carrying tumors with or without gsp oncogene failed to detect clinical differences between the two groups. The existence of mechanisms induced by gsp mutations and able to counteract the mitogenic potential of this oncogene has been hypothesized. In particular, several events including the low expression of mutant Gs, the induction of phosphodiesterases, that are involved in cAMP degradation, and of the inducible cAMP early repressor (ICER) that represses cAMP induced gene expression, together with a high sensitivity to somatostatin have been characterized in gsp positive tumors. As far as the loss of oncosuppressors is concerned, no mutations of these genes have been so far reported, while they are frequently expressed at low levels in pituitary tumors. However, the nature of initiating and promoting events involved in tumor formation remains to be clarified in the majority of pituitary tumors.     

The clinical response to somatostatin analogues in acromegaly correlates to the somatostatin receptor subtype 2a protein expression of the adenoma

Objective Reduced expression of the somatostatin receptor subtype 2 (SSTR2) has been suggested as an explanation for the poor response to octreotide in acromegaly, but studies correlating levels of SSTR2 mRNA to octreotide efficacy have been contradictory. Some studies have found better responses to somatostatin analogues in G‐protein α subunit (Gsα) mutation (gsp oncogene)‐positive adenomas. The aim of this study was to determine adenoma SSTR2a protein expression and gsp status in a large group of patients with acromegaly, and relate this to the clinical effect of octreotide. Patients Seventy‐one patients were included. All underwent transsphenoidal surgery, 23 patients after preoperative octreotide treatment. Measurements The adenoma SSTR2a expression was examined by immunohistochemistry and Western blot analysis, and gsp status determined. An acute octreotide test was performed, and the change in IGF‐1 level after 6 months preoperative octreotide treatment was recorded. Results The acute octreotide response in non‐pretreated patients and the preoperative long‐term octreotide response were significantly better in patients with adenomas containing a large proportion of cells that stained positively for SSTR2a by immunohistochemistry. However, the SSTR2a protein level assessed by Western blot did not correlate with the octreotide response. The preoperatively treated group had lower SSTR2a protein levels and fewer adenomas with a large percentage of positively stained cells. The gsp oncogene was detected in 43% of the adenomas but did not correlate to the octreotide response. Conclusion The clinical effect of octreotide correlates with the proportion of cells positive for SSTR2a in immunohistochemical staining, rather than the adenoma SSTR2a protein level. There may be a down‐regulation of SSTR2a during octreotide treatment.     

Expression of somatostatin receptors on human pituitary adenomas in vivo and ex vivo.

The distribution and biologic activity of somatostatin receptor subtypes (SSTR) in pituitary adenomas is not clarified, especially regarding clinically non-functioning adenomas (NFPA). We therefore characterized SSTR in human pituitary adenomas by combining molecular biology and in vivo scintigraphy. Co-expression of gonadotropin-releasing hormone receptor (GnRH-R) mRNA was also assessed to see whether this feature was associated with adenoma subtype and SSTR status. Pituitary tumor biopsies were obtained during transsphenoidal adenomectomy from 21 patients (11 NFPA, 7 acromegalics, 2 prolactinomas, 1 Cushing's disease). Expression of mRNA encoding the 5 known SSTR subtypes and the GnRH-R was determined by RT-PCR. Twelve patients also underwent a pre-operative somatostatin receptor scintigraphy. Most adenomas (no.=18) expressed mRNA for more than one SSTR. SSTR2 mRNA was expressed in 18 cases, whereas SSTR4 was absent in all but one. SSTR3 was frequently expressed in NFPAs. Somatostatin receptor scintigraphy was positive in most cases, and with a significantly higher uptake index in GH-producing adenomas all of which expressed SSTR2 mRNA. The uptake index appeared to be related to receptor density rather than tumor volume. Expression of GnRH-R mRNA was found in both NFPAs and GH-producing adenomas and was not significantly associated with a particular SSTR subtype population. In conclusion: 1) the distribution of SSTR is not significantly different between NFPA and GH-producing adenomas; and 2) somatostatin receptor scintigraphy reveals a higher uptake in GH-producing adenomas which is not significantly related to either SSTR distribution or tumor volume.     

垂体转录活化物-1基因在人垂体腺瘤中的表达

目的　用RT PCR方法 ,定量研究垂体转录活化物 (Pit) 1mRNA在不同类型的垂体腺瘤中的表达。方法　 3 5例垂体腺瘤患者根据血清激素水平和临床表现确定腺瘤类型 ,根据影像学和术中所见对肿瘤分级和分期。用RT PCR方法检测垂体腺瘤组织中Pit 1mRNA的表达。结果　 3 5例垂体腺瘤患者有PRL腺瘤 13例 ,GH腺瘤 6例 ,GH/PRL腺瘤 2例 ,无功能瘤 11例以及ACTH腺瘤 3例。Pit 1mRNA在所有PRL瘤、GH/PRL瘤、GH瘤和 81.8% (9/ 11)无功能腺瘤中有表达。在ACTH腺瘤组中无表达。Pit 1mRNA在PRL、GH和GH/PRL 3组腺瘤中的表达量差异无显著性 ,均显著高于无功能瘤组(均P <0 .0 5)。Pit 1表达量与肿瘤分级分期无明显相关性。PRL瘤术前血清PRL值与腺瘤组织Pit 1表达量呈显著的正相关 (r=0 .92 ,P <0 .0 1) ,GH腺瘤术前血清GH值与腺瘤组织Pit 1表达量呈显著的正相关 (r =0 .98,P <0 .0 1)。结论　Pit 1mRNA在PRL、GH、GH/PRL瘤以及大部分无功能腺瘤中有表达 ,其中在PRL、GH以及GH/PRL瘤的表达量较高 ,Pit 1对垂体GH和PRL腺瘤的细胞特异分化以及分泌功能是否具有作用 ,尚待进一步研究     

Heterozygous gsp mutation renders ion channels of human somatotroph adenoma cells unresponsive to growth hormone-releasing hormone

Ionic mechanisms play an important role in the regulation of hormone secretion. The GHRH-induced GH release by human GH-secreting cells is transmitted through protein kinase A (PKA), which activates nonselective cation current (NSCC) and induces membrane depolarization, intracellular Ca2+ increase, and GH secretion. To evaluate whether ionic mechanisms have pathophysiological significance in GH oversecretion of GH-secreting pituitary adenomas, we examined four adenomas with constitutively active Gs alpha mutation (gsp mutation) and compared with three gsp-negative adenomas. In primary-cultured cells of gsp-positive adenomas, GHRH did not increase the NSCC under voltage-clamp experiments. Detailed examination showed that NSCC was maximally activated at the basal level and application of GHRH did not increase the current in these adenomas. Furthermore, by using single-cell RT-PCR method, we demonstrated for the first time at the single cell level that gsp mutation is heterozygous in GH-secreting pituitary adenomas. These indicate that heterozygous gsp mutation fully activates NSCC at the basal level, which may account for the GH oversecretion in gsp-positive GH-secreting pituitary adenomas.     

Somatostatin receptor subtypes mRNA in TSH-secreting pituitary adenomas: a case showing a dramatic reduction in tumor size during short octreotide treatment

TSH-secreting adenoma is a rare pituitary adenoma, and the expression levels of the specific subtypes of somatostatin receptors (sstr) mRNAs have remained obscure. To determine the quantitative expression of the sstr1-5 mRNAs in TSH-secreting adenomas that may be related to the efficacy of treatment with a somatostatin analogue, expression of the sstr1-5 mRNAs was examined and compared in TSH-secreting adenomas and other pituitary adenomas. The pituitary adenomas were obtained at transsphenoidal surgery from 4 cases of TSH-secreting adenoma, including 1 patient showing a significant shrinkage of the tumor size after only 10 days of octreotide treatment, 2 patients without tumor size reduction and 1 patient without treatment, and 5 GH-secreting adenomas, 6 prolactinomas, 5 nonfunctioning adenomas, 4 ACTH-secreting adenomas and normal pituitaries at autopsy from 4 normal subjects. In comparison to the normal pituitary, sstr2A>sstr1>sstr5>sstr3 mRNAs were expressed in the TSH-secreting adenomas examined. No expression of sstr2B or sstr4 mRNA was observed. The expression level of sstr2 mRNA was significantly higher than those in normal pituitary, prolactinomas, ACTH-secreting and nonfunctioning pituitary adenomas. The patient with marked shrinkage of the tumor showed the highest expression of both sstr2 and sstr5 mRNAs among all the cases of pituitary adenoma. A TSH-secreting tumor without shrinkage showed a similar expression level of sstr2 mRNA. These findings demonstrated that TSH-secreting adenomas express sstr1, 2A, 3 and 5 mRNAs, predominantly sstr2A, and in addition to the expression of sstr2 mRNA, the expression level of sstr5 mRNA may be a factor affecting the tumor shrinkage by somatostatin analogues against TSH-secreting adenomas.     

Quantitative in-situ hybridization histochemistry of anterior pituitary hormone mRNA species in human pituitary adenomas

We have examined the anterior pituitary hormone messenger (m) RNA species contained in biopsies of 41 pituitary tumours obtained at hypophysectomy using in-situ hybridization histochemistry. The adenoma were grouped clinically into 12 prolactinomas, 8 somatotrope adenomas, 16 non-functioning, 4 Nelson's syndrome, and 1 thyrotrope adenoma. Of these, 10 contained no detectable anterior pituitary hormone mRNA species and 11 appeared to be expressing the gene responsible for the patients' clinical state in isolation. In a number of cases the accumulation of specific mRNA species was not accompanied by an increase in the circulating levels of the corresponding hormone or subunit. Evidence of activation of more than 1 anterior pituitary hormone gene was present in 16 adenomas of which only 7 showed a pattern of activation or amplification of gene expression which would suggest deregulation of either the inositol phospholipid or cAMP second messenger pathway. It was therefore not possible from these data to postulate that isolated deregulation of a single second messenger transduction pathway is a common etiological factor in pituitary tumour formation.     

Molecular diagnosis of pituitary adenoma predisposition caused by aryl hydrocarbon receptor-interacting protein gene mutations

Pituitary adenomas are common neoplasms of the anterior pituitary gland. Germ-line mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause pituitary adenoma predisposition (PAP), a recent discovery based on genetic studies in Northern Finland. In this population, a founder mutation explained a significant proportion of all acromegaly cases. Typically, PAP patients were of a young age at diagnosis but did not display a strong family history of pituitary adenomas. To evaluate the role of AIP in pituitary adenoma susceptibility in other populations and to gain insight into patient selection for molecular screening of the condition, we investigated the possible contribution of AIP mutations in pituitary tumorigenesis in patients from Europe and the United States. A total of 460 patients were investigated by AIP sequencing: young acromegaly patients, unselected acromegaly patients, unselected pituitary adenoma patients, and endocrine neoplasia-predisposition patients who were negative for MEN1 mutations. Nine AIP mutations were identified. Because many of the patients displayed no family history of pituitary adenomas, detection of the condition appears challenging. Feasibility of AIP immunohistochemistry (IHC) as a prescreening tool was tested in 50 adenomas: 12 AIP mutation-positive versus 38 mutation-negative pituitary tumors. AIP IHC staining levels proved to be a useful predictor of AIP status, with 75% sensitivity and 95% specificity for germ-line mutations. AIP contributes to PAP in all studied populations. AIP IHC, followed by genetic counseling and possible AIP mutation analysis in IHC-negative cases, a procedure similar to the diagnostics of the Lynch syndrome, appears feasible in identification of PAP.     

The gsp oncogene disrupts Ras/ERK-dependent prolactin gene regulation in gsp inducible somatotroph cell line

The MAPK ERK1/2 cascade regulates all the critical cellular functions, and in many pathological situations, these regulatory processes are perturbed. It has been clearly established that this cascade is an integrative point in the control of the pituitary functions exerted by various extracellular signals. In particular, ERK1/2 cross talk with the cAMP pathway is determinant in the control of somatolactotroph hormonal secretion exerted via neuropeptide receptors. GH-secreting adenomas are characterized by frequent cAMP pathway alterations, such as constitutive activation of the α-subunit of the heterotrimeric Gs protein (the gsp oncogene), overexpression of Gsα, and changes in the protein kinase A regulatory subunits. However, it has not yet been established exactly how these alterations result in GH-secreting adenomas or how the ERK1/2 cascade contributes to the process of GH-secreting adenoma tumorigenesis. In this study on the conditional gsp-oncogene-expressing GH4C1 cell line, expression of the gsp oncogene, which was observed in up to 40% of GH-secreting adenomas, was found to induce sustained ERK1/2 activation, which required activation of the protein kinase A and the GTPases Ras and Rap1. All these signaling components contribute to the chronic activation of the human prolactin promoter. The data obtained here show that Ras plays a crucial role in these processes: in a physiopathological context, i.e. in the presence of the gsp oncogene, it switched from being a repressor of the cAMP/ protein kinase A ERK-sensitive prolactin gene control exerted by neuropeptides to an activator of the prolactin promoter.     

The release of leptin and its effect on hormone release from human pituitary adenomas

BACKGROUND: Leptin is the protein product of the obese gene, known to play an important role in body energy balance. The leptin receptor exists in numerous isoforms, the long isoform being the major form involved in signal transduction. Leptin expression has recently been demonstrated in the human pituitary, both in normal tissue and in pituitary adenomas. The long isoform of the leptin receptor has also been shown to be present in pituitary adenomas; however, contrasting results have been obtained regarding its expression in the normal human pituitary. AIM: The aim of this study was (i) to investigate the presence and pattern of distribution of leptin mRNA and the long isoform of its receptor mRNA in the normal pituitary and in different types of pituitary adenomas with RT-PCR; (ii) to study leptin secretion from human pituitary tumours in culture and (iii) to assess in vitro pituitary hormone release following stimulation with human leptin. RESULTS: Leptin receptor long isoform expression was detected in 2/4 GH-secreting adenomas, 12/17 non-functioning adenomas, 5/9 ACTH-secreting adenomas, 1/2 prolactinomas, 2/2 FSH-secreting adenomas and 5/5 normal pituitaries. The receptor long isoform did not segregate with any particular tumour type, and varying levels of expression were detected between the tissues studied. Leptin mRNA was detected at a low level of expression in 2/7 GH-secreting adenomas, 9/14 non-functioning adenomas, 2/3 ACTH-secreting adenomas, 1/3 prolactinomas and 1/3 FSH-secreting adenomas. We were unable to detect leptin mRNA in any of the five normal pituitaries removed at autopsy; however, immunostaining of a non-tumorous pituitary adjacent to an adenoma removed at transsphenoidal surgery showed scattered leptin positive cells. Culture of pituitary adenomas showed that 16/47 released leptin into the incubation media. Leptin release did not correlate with tumour type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumours caused stimulation of FSH and alpha-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. CONCLUSION: We conclude that not only is leptin stored within the pituitary, but it may also be released from pituitary cells and modulate other pituitary hormone secretion. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.