The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

The impact of SST2 trafficking and signaling in the treatment of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (Pan-NETs), are heterogeneous neoplasms, whose incidence and prevalence are increasing worldwide. Pan-NETs are characterized by the expression of somatostatin receptors (SSTs). In particular, SST2 is the most widely distributed SST in NETs, thus representing the main molecular target for somatostatin analogs (SSAs). SSAs are currently approved for the treatment of well-differentiated NETs, and radionuclide-labeled SSAs are used for diagnostic and treatment purposes. SSAs, by binding to SSTs, have been shown to inhibit hormone secretion and thus provide control of hypersecretion symptoms, when present, and inhibit tumor proliferation. After SSA binding to SST2, the fate of the receptor is determined by trafficking mechanisms, crucial for the response to endogenous or pharmacological ligands. Although SST2 acts mostly through G protein-dependent mechanism, receptor-ligand complex endocytosis and receptor trafficking further regulate its function. SST2 mediates the decrease of hormone secretion via a G protein-dependent mechanism, culminating with the inhibition of adenylyl cyclase and calcium channels; it also inhibits cell proliferation and increases apoptosis through the modulation of protein tyrosine phosphatases. Moreover, SST2 inhibits angiogenesis and cell migration. In this respect, the cross-talk between SST2 and its interacting proteins, including Filamin A (FLNA) and aryl hydrocarbon receptor-interacting protein (AIP), plays a crucial role for SST2 signaling and responsiveness to SSAs. This review will focus on recent studies from our and other groups that have investigated the trafficking and signaling of SST2 in Pan-NETs, in order to provide insights into the mechanisms underlying tumor responsiveness to pharmacological treatments.

GPR101 Mutations are not a Frequent Cause of Congenital Isolated Growth Hormone Deficiency.

Patients with Xq26.3 microduplication present with X-linked acrogigantism (X-LAG) syndrome, an early-childhood form of gigantism due to marked growth hormone (GH) hypersecretion from mixed GH-PRL adenomas and hyperplasia. The microduplication includes GPR101, which is upregulated in patients' tumor tissue. The GPR101 gene codes for an orphan G protein coupled receptor that is normally highly expressed in the hypothalamus. Our aim was to determine whether GPR101 loss of function mutations or deletions could be involved in patients with congenital isolated GH deficiency (GHD). Taking advantage of the cohort of patients from the GENHYPOPIT network, we studied 41 patients with unexplained isolated GHD. All patients had Sanger sequencing of the GPR101 gene and array comparative genome hybridization (aCGH) to look for deletions. Functional studies (cell culture with GH secretion measurements, cAMP response) were performed. One novel GPR101 variant, c.589 G>T (p.V197L), was seen in the heterozygous state in a patient with isolated GHD. In silico analysis suggested that this variant could be deleterious. Functional studies did not show any significant difference in comparison with wild type for GH secretion and cAMP response. No truncating, frameshift, or small insertion-deletion (indel) GPR101 mutations were seen in the 41 patients. No deletion or other copy number variation at chromosome Xq26.3 was found on aCGH. We found a novel GPR101 variant of unknown significance, in a patient with isolated GH deficiency. Our study did not identify GPR101 abnormalities as a frequent cause of GH deficiency.

Chromium-induced diffuse dermatitis with lymph node involvement resulting from Langerhans cell histiocytosis after metal-on-metal hip resurfacing.

Total hip arthroplasty (THA) is a highly effective surgical treatment for severe joint involvement. However, due to the release of metal ions in the blood, the patients who undergo hip replacement with metal-on-metal (MOM) bearings may develop signs of allergic skin disease. We report a case of a 60-year-old man who had received MOM hip resurfacing 5 years earlier for osteoarthritis. He presented with a 3-year history of diffuse dermatitis that did not respond to antihistamines and corticosteroids and also had elevated serum levels of chromium and cobalt. A patch test revealed chromium-sulfate hypersensitivity. A skin biopsy showed nonspecific perivascular lymphocytic infiltrate associated with histiocytes. A biopsy of an inguinal lymph node demonstrated large aggregates of Langerhans cells, suggesting type IV delayed-type hypersensitivity. The prosthesis was replaced using ceramic-on-ceramic bearings and the dermatitis resolved after 3 months. The lymph nodes decreased in volume and the serum chromium levels normalized within 24 months of revision surgery. The high levels of serum ions associated with the metal debris from MOM-THAs may induce sensitization and type IV hypersensitivity reactions. Replacing the prosthesis using alternative coupling surfaces is the only approach that has the capacity to resolve these symptoms. Physicians who are not familiar with this issue may misdiagnose systemic symptoms and provide inadequate treatment.

MicroRNAs: Suggested role in pituitary adenoma pathogenesis.

MicroRNAs (miRNAs) are small non-coding RNA molecules that represent a major class of molecular regulators. miRNAs have been implicated in the pathogenesis of several human tumors, including pituitary adenomas. Altered expression of miRNAs has been described in pituitary adenomas, and specific miRNA signatures are related to clinical and therapeutic characteristics of the tumors. The data suggest that miRNAs influence various genes known to be associated with the pathogenesis of pituitary adenomas and in this review we summarize these currently available studies focusing on miRNAs in pituitary adenomas.

Familial isolated pituitary adenomas: an emerging clinical entity.

Familial pituitary tumors are increasingly recognized. While some of these cases are related to wellknown syndromic conditions such as multiple endocrine neoplasia type 1 (MEN1) or Carney complex, others belong to the familial isolated pituitary adenoma (FIPA) patient group. The discovery of heterozygous, loss-of-function germline mutations in the gene encoding the aryl hydrocarbon receptor interacting protein (AIP) in 2006 has subsequently enabled the identification of a mutation in this gene in 20% of FIPA families and 20% of childhood-onset simplex soma- totroph adenomas. The exact mechanism by which the lack of AIP leads to pituitary adenomas is not clear. AIP mutations cause a low penetrance autosomal dominant disease with often a distinct phenotype characterized by young-onset, aggressive, large GH, mixed GH and PRL or PRL-secreting adenomas. This review aims to summarize currently available clinical data on AIP mutation-positive and negative FIPA patients.

The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)-promoter activity in GH3 cells.

Somatic mutations in the GNAS1 gene, encoding the α-subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)-secreting pituitary tumours. By altering the adenylate cyclase-cAMP-protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second-messenger cascade. Among them, the glucose-dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP-dependent cortisol secretion in patients affected by food-dependent Cushing's syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH-secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation-negative somatotropinomas. GIP stimulated adenylyl cyclase and GH-promoter activity in GIPR-transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

Adrenal lesions in acromegaly: do metabolic aspects and aryl hydrocarbon receptor interacting protein gene have a role? Evaluation at baseline and after long-term follow-up.

BACKGROUND: Adrenal lesions are discovered in acromegaly more frequently than in general population, without relationship with primary disease. Some patients, carriers of aryl hydrocarbon receptor interacting protein (AIP) gene mutations, developed an adrenal neoplasm. AIM: To evaluate the role of metabolic and genetic aspects and the follow-up of adrenal nodules in acromegaly. MATERIAL AND METHODS: We studied 69 acromegalic patients (30 male and 39 female, 56 ± 15 yr) who had been referred to the Endocrinology Unit of Padua. In all patients we determined body mass index (BMI) and waist-to-hip ratio (WHR); we performed an oral glucose tolerance test (OGTT) whenever possible. If adrenal computed tomography revealed a lesion, the patient underwent an endocrine and genetic study. RESULTS: Adrenal lesions were identified in 14 patients and were not related to gender, duration of disease, GH or IGF-I concentrations, basal and after-OGTT glucose and insulin levels, log(HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI) values, whereas BMI and WHR were higher in patients with adrenal lesions. Baseline endocrine and radiological study revealed benign lesions; during mean 4-yr follow-up none of the patients showed hormone excess, even though some lesions increased in size. We did not find any mutation in AIP gene, except heterozygous silent alteration (T48T). CONCLUSIONS: The frequency of non-functioning adrenal lesions in acromegaly is not associated with the considered aspects, except BMI and WHR. The prolonged follow-up showed that these lesions have a tendency to increase in size independently of the control of acromegaly, so a morphological follow- up is recommended.

Prevalence of AIP mutations in a large series of sporadic Italian acromegalic patients and evaluation of CDKN1B status in acromegalic patients with multiple endocrine neoplasia.

BACKGROUND: Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene and the p27(KIP1) encoding gene CDKN1B have been associated with two well-defined hereditary conditions, familial isolated pituitary adenoma (FIPA) and multiple endocrine neoplasia type 4 (MEN4). Somatotropinomas are present in most AIP mutated FIPA kindreds, as well as in two-thirds of MEN4 patients who carry pituitary tumors. METHODS: Germline DNA samples of 131 Italian sporadic acromegalic patients including 38 individuals with multiple tumors, and of six FIPA families (four homogeneous for prolactinomas and two heterogeneous with prolactin/nonfunctioning pituitary adenomas) were collected in a multicentric collaborative study. The prevalence of AIP and CDKN1B gene point mutations and copy number variations were evaluated. RESULTS: Two novel (IVS3+1G>A and c.871G>A) and one previously described (c.911G>A) AIP mutations were detected in four apparently sporadic cases (3.1%) with relatively high age at diagnosis (49+/-18, range 30-67). No mutations/rearrangements were detected in FIPA families. The highly conserved c.871G>A substitution was detected in a patient who also carried a MEN1 mutation suggesting that she is a double heterozygote. The possible pathogenic effect on AIP splicing of the silent substitution c.144G>A found in another patient was ruled out using a minigene-based approach. CDKN1B mutations/rearrangements were neither identified in patients with multiple neoplasia nor in FIPA families. CONCLUSION: AIP is mutated in about 3% of apparently sporadic acromegalic patients. The relatively high age at diagnosis, as well as its sporadic presentation, suggests that these patients are carriers of mutations with reduced pathogenicity. p27(KIP1) is unlikely to represent the common unifying nonendocrine etiology for acromegaly and cancer.

The R304X mutation of the aryl hydrocarbon receptor interacting protein gene in familial isolated pituitary adenomas: Mutational hot-spot or founder effect?

BACKGROUND: Mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene have been described in about 15% of kindreds with familial isolated pituitary adenomas and in a minority of early onset sporadic pituitary adenomas (PA). Among the AIP mutations reported so far, the R304X (AIPR304X) represents, together with the "Finnish mutation" Q14X, the most common one. METHODS: Three AIPR304X Italian families, including a newly reported kindred, have been genotyped for 12 genetic markers surrounding the AIP gene in order to look for a potential founder effect in Italy. Disease penetrance and genotype-phenotype correlations were also addressed. RESULTS: Analysis of chromosome 11' genetic markers revealed a common haplotype in 2 AIPR304X kindreds originating from central Italy. Overall, 17 mutations carriers were identified, including 7 patients and 10 unaffected subjects, respectively, arguing in this case for a disease penetrance of 41%. Mean age at diagnosis was 19.1±6.7 yr old, with females tending to be older than males. Though most PA were somatotropinomas (6/7), a great variability in disease severity was observed, even between subjects sharing the same at-risk haplotype. CONCLUSION: These data provide strong evidence for a new founder effect of the AIPR304X mutation in central Italy and the observed variations in disease severity point out the role of additional genetic or environmental factors in such kindreds.