Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP-PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. Osteoprotegerin (OPG), the product of this gene, is a key negative regulator of osteoclastogenesis and is secreted by osteoblasts/stromal cells. A previous study in Maltese postmenopausal women showed positive association of low bone mineral density (BMD) with a polymorphism found within the promoter region of this gene (C950T). In this study, direct DNA sequencing revealed 12 variants with polymorphisms C950T, G1181C and rs4876869 observed to be in strong linkage disequilibrium. The constructed haplotype T-G-T was found to increase the risk for a low BMD, while C-G-T and C-C-C have a protective role; thus, we investigated the functional role of both C950T and rs4876869 in vitro. The promoter region, including the C950T alleles, was amplified by PCR, cloned into pGL3 enhancer vector and transfected into HeLa, COS-7 and RAW264.7 cell lines. After incubation, luciferase activity was measured. The T/C (rs4876869) change was tested for its possible effect on pre-mRNA splicing, using an exon-trapping vector. A statistical significant difference in gene expression was observed between the alleles for T950C, with the T allele showing a lower luciferase expression in all cell lines (P!0 . 01). For rs4876869, exon skipping was observed for the C allele, while only one transcript harbouring the whole exon was observed for the T allele. Our findings suggest that the T-G-T haplotype might be increasing the risk for osteoporosis due to lower quantities of the full OPG transcript being expressed resulting in a higher bone resorption.

Deregulation of the Wnt pathway has been implicated in oncogenesis of numerous tissues including the pituitary gland. Immunohistochemical localization and quantification of β-catenin, Cyclin D1, c-MYC and Survivin expression in 47 pituitary adenomas (35 non-functioning, seven GH-secreting, three prolactinomas, two ACTH-secreting tumour) and six normal controls was undertaken in this study and correlation of protein expression to patient and tumour characteristics analysed. β-catenin was strictly membrane-bound with no difference observed between normal and tumour tissue. In contrast, Cyclin D1 and c-MYC localization was nuclear and significantly higher in tumour versus normal tissue (p < 0.05). c-MYC expression correlated negatively with age at diagnosis (p = 0.006, R = -0.395) while Cyclin D1 expression correlated positively with age (p = 0.036, R = 0.306) and was higher in males than in females (p = 0.036). c-MYC expression was significantly lower in patients with functional tumours requiring octreotide treatment and in patients with non-functioning tumours suffering from hypopituitarism. Survivin expression was extremely low in tumours and absent in normal controls. Involvement of the canonical Wnt pathway appears to be minimal, given the segregation of β-catenin to the membrane. Our data suggest that c-MYC may have an important role in early pituitary tumorigenesis while Cyclin D1 is likely to promote tumour growth at a later stage. We also report a novel gender difference in Cyclin D1 expression, the biological significance of which merits further analysis. The reported reduction of c-MYC in functional tumours subsequently treated with octreotide further supports a role of c-MYC in early tumorigenesis and not in recurrence. The decrease in c-MYC in patients with hypopituitarism provides the first in vivo evidence for hormonal regulation of c-MYC expression.

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Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G0/G1 phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands.

Interesting and novel observations on the differences in expression of tumour markers studied are reported. Correlation between Ki-67 expression, PTTG nuclear expression and recurrence/regrowth of PAs, emphasizes the role that Ki-67 and PTTG expression have as markers of increased proliferation. c-MYC and PTTG nuclear expression levels were correlated providing evidence that PTTG induces c-MYC expression in PAs and we propose that c-MYC might principally have a role in early pituitary tumorigenesis. Evidence is shown that the anti-proliferative effect of somatostatin analogue treatment in vivo occurs through regulation of the cell cycle.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor best known for its ability to mediate the effects of environmental toxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), polycyclic aromatic hydrocarbons (PAHs), benzene, and polychlorinated biphenyls (PCBs) through the initiation of transcription of a number of metabolically active enzymes. Therefore, the AHR has been studied mostly in the context of xenobiotic signaling. However, several studies have shown that the AHR is constitutively active and plays an important role in general cell physiology, independently of its activity as a xenobiotic receptor and in the absence of exogenous ligands. Within the pituitary, activation of the AHR by environmental toxins has been implicated in disruption of gonadal development and fertility. Studies carried out predominantly in mouse models have revealed the detrimental influence of several environmental toxins on specific cell lineages of the pituitary tissue mediated by activation of AHR and its downstream effectors. Activation of AHR during fetal development adversely affected pituitary development while adult models exposed to AHR ligands demonstrated varying degrees of pituitary dysfunction. Such dysfunction may arise as a result of direct effects on pituitary cells or indirect effects on the hypothalamic-pituitary-gonadal axis. This review offers in-depth analysis of all aspects of AHR biology, with a particular focus on its role and activity within the adenohypophysis and specifically in pituitary tumorigenesis. A novel mechanism by which the AHR may play a direct role in pituitary cell proliferation and tumor formation is postulated. This review therefore attempts to cover all aspects of the AHR's role in the pituitary tissue, from fetal development to adult physiology and the pathophysiology underlying endocrine disruption and pituitary tumorigenesis.

Mutations spanning the entire aryl hydrocarbon receptor-interacting protein (AIP) gene have been found in isolated familial cases of pituitary adenomas (PA). Missense mutations located in the N-terminus of the gene have been identified in several patients. However, the functional significance of these mutations remains a matter of controversy. In most studies, the N-terminus of AIP has been shown to regulate protein stability and subcellular localization of the AIP-AHR-HSP90 complex but not to be involved in protein-protein interactions. Other studies found that the N-terminal domain interacts directly with other proteins. The aim of this study was to analyze whether specific N-terminus AIP mutations identified in PA patients would be functionally different from wild-type (WT) AIP. In vitro analyses were used to assess the role of known N-terminus variants, a locally identified mutant, R9Q, and three other commonly genotyped N-terminus mutations R16H, V49M and K103R are found in PA patients. Given the functional effect of WT AIP on cAMP signalling alterations caused by N-terminus mutants on this pathway were also analyzed in GH3 cells. Results indicate that N-terminus mutations lead to de-regulation of the effect of WT AIP on cAMP signalling and increased cAMP thresholds in GH3 cells resulting in increased growth hormone (GH) secretion. Cycloheximide chase analysis identified a variation in protein degradation patterns between WT and N-terminus variants. Therefore, both functional and structural studies reveal that N-terminus mutations in the AIP gene alter protein behaviour significantly and hence can truly be pathogenic in nature.