Reduced protein expression of the phosphodiesterases PDE4A4 and PDE4A8 in AIP mutation positive somatotroph adenomas.

Type 4 phosphodiesterases (PDE4s) of the large PDE enzyme superfamily have unique specificity for cAMP and may, therefore, be relevant for somatotroph tumorigenesis. Somatotroph adenomas typically overexpress PDEs probably as part of a compensatory mechanism to reduce cAMP levels. The rat PDE4A5 isoform (human homolog PDE4A4) interacts with the AIP protein, coded by a tumour suppressor gene mutated in a subgroup of familial isolated pituitary adenomas (FIPAs). PDE4A8 is the closest related isoform of PDE4A4. We aimed to evaluate the expression of both PDE4A4 and PDE4A8 in GH cells of AIP-mutated adenomas and compare their expression with that in GH cells from sporadic AIP-mutation negative GH-secreting adenomas, where we had shown previously that both PDE4A4 and PDE4A8 isoforms had been over-expressed. Confocal immunofluorescence analysis showed that both PDE4A8 and PDE4A4 had lower expression in AIP-mutated somatotropinoma samples compared to sporadic GH-secreting tumours (P < 0.0001 for both). Based on the association of low PDE4A4 and PDE4A8 expression with germline AIP-mutations positive samples we suggest that lack of AIP hinders the upregulation of PDE4A8 and PDE4A4 protein seen in sporadic somatotrophinomas. These data point to a unique disturbance of the cAMP-PDE pathway in AIP-mutation positive adenomas, which may help to explain their well-described poor response to somatostatin analogues.

Effects of AMPK activation and combined treatment with AMPK activators and somatostatin on hormone secretion and cell growth in cultured GH-secreting pituitary tumor cells.

We investigated the effects of the AMPK activator AICAR as compared to somatostatin-14 on cell viability and GH secretion in human GH-secreting pituitary adenomas in vitro and in rat GH3 cells. Overnight treatment with AICAR increased phospho-(threonine-172) AMPK levels (activated AMPK) in cultured human adenomas. As to the effects on cell viability, four adenomas out of 15 were responsive to AICAR (0.4mM) and five adenomas were responsive to SS-14 (100 nM). One adenoma was responsive to both somatostatin and AICAR. The effects of cotreatment with SS-14 and AICAR were investigated in eight adenomas. In two adenomas, the effects of AICAR+SS-14 did not exceed the effect of AICAR. In two adenomas which were not responsive to either AICAR or SS-14, the cotreatment was able to reduce cell viability versus control. Two adenomas were not responsive to any treatment. As to the effects on GH secretion, nine adenomas out of 15 were responsive to AICAR. Twelve adenomas were responsive to SS-14. Eight adenomas were responsive to both AICAR and SS-14. Cotreatment exceeded the effect of single treatments in 4 out of 10 adenomas. In GH3 cells, AICAR reduced the activity of p70S6 kinase, which plays an important role in cell growth. SS-14 did not affect significantly AMPK phosphorylation and p70S6K activity but it was able to enhance the inhibitory effect of AICAR on phospho-S6 levels. Moreover, AICAR and SS-14 reduced ERK phosphorylation with a different time course. The combined treatment reduced phospho-ERK levels at any time point.

Loss of 15-hydroxyprostaglandin dehydrogenase indicates a tumor suppressor role in pituitary adenomas.

15-hydroxyprostaglandin dehydrogenase (15-PGDH) may function as a tumor suppressor that antagonizes the action of the cyclooxygenase-2 (COX-2) oncogene in several types of tumors. However, it is unknown if it has a role in the pituitary. Recently, our group found that 15-PGDH expression was low in prolactin (PRL) secreting adenomas (prolactinomas) and growth hormone (GH) secreting adenomas (GHomas) using fiber-optic BeadArray technology. In this study, we examined the relative expression of 15-PGDH and COX-2 mRNA in clinical specimens and examined the effects of 15-PGDH on GH3 rat pituitary tumor cell proliferation, apoptosis and hormone secretion. 15-PGDH expression was lower and COX-2 expression was higher in prolactinomas and GHomas compared with normal controls. Overexpressed 15-PGDH inhibited tumor cell proliferation and induced apoptosis. It had a significant suppressive effect on mRNA levels and on the secretion of PRL and GH in GH3 cells. The inhibition of cell proliferation was accompanied by the decreased expression of cox-2, matrix metalloproteinase-9 (MMP-9) and B cell leukemia/lymphoma-2 (Bcl-2). These data are suggestive of a previously unrecognized pathway in pituitary tumorigenesis, and this novel observation may shed light on therapeutic strategies for pituitary tumors.

G-protein and signalling in pituitary tumours.

The genesis of pituitary tumours is still under debate. Although these neoplasias are monoclonal in origin, mutations of GNAS1, the gene encoding the alpha subunit of the stimulatory G-protein, Gs, are the only mutational changes unequivocally associated with growth hormone (GH)-secreting adenomas. However, despite the growth advantage that this oncogene has been demonstrated to confer in vitro, patients carrying this mutation have a similar clinical and biochemical phenotype to those who do not carry it. This discrepancy is due to the occurrence of events able to counteract the biological effect of the mutation. Consistent with a potential role of the cyclic adenosine monophosphate pathway in the proliferation of somatotrophs, germline mutations of the gene encoding the type 1alpha regulatory subunit of protein kinase A (PRKAR1A) have been found in patients with the Carney complex, a syndrome including GH-secreting adenomas, whereas alterations in the expression levels of this subunit are frequently observed in sporadic adenomas.

Evidence that PKA activity is constitutively activated in human GH-secreting adenoma cells in a patient with Carney complex harbouring a PRKAR1A mutation.

CONTEXT: The GHRH-protein kinase A (PKA) signalling pathway is essential for cell proliferation and GH synthesis/secretion in somatotrophs. An inactivating mutation of PRKAR1A is one of the causes of somatotrophinoma in Carney complex (CNC). The basal PKA activity of somatotroph adenoma cells from CNC has not been evaluated because of a limited amount of available tissue. OBJECTIVE: This study examined how the PRKAR1A mutation affects the PKA signalling pathway in a human somatotrophinoma with a PRKAR1A mutation. DESIGN AND SETTING: Somatotrophinoma cells from a 40-year-old male patient with CNC were used. The patient had a novel somatic heterozygous germline frameshift mutation (227delT) in PRKAR1A leading to a premature stop codon. The tumour showed loss of heterozygosity (LOH) at 17q23-24. Primary cultured adenoma cells were subjected to electrophysiological experiments to evaluate PKA signalling in individual cells. RESULTS: GHRH did not increase the nonselective cation current or the voltage-gated calcium current in these adenoma cells, in contrast to nonadenomatous somatotroph cells in which these currents increase through the PKA pathway. Application of a PKA inhibitor inhibited the basal currents in these adenoma cells, results that were not observed in nonadenomatous somatotrophs. These data indicate that the basal currents are already increased and cannot be further increased by GHRH. CONCLUSIONS: The results demonstrate that PKA is activated at the basal state in these adenoma cells. The data also show that both the nonselective cation current and the voltage-gated calcium current, vital regulators of GH secretion downstream of PKA, are maximally increased in these cells. These maximally increased currents probably account for the excessive GH secretion.