Somatotropinomas, But Not Nonfunctioning Pituitary Adenomas, Maintain a Functional Apoptotic RET/Pit1/ARF/p53 Pathway That Is Blocked by Excess GDNF

Abstract

Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF (Glial Cell Derived Neurotrophic Factor), RET is processed intracellularly to induce overexpression of Pit1 [Transcription factor (gene : POUF1) essential for transcription of Pituitary hormones GH, PRL and TSHb], which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 8 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p19Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p19Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 CCAAT/enhancer binding protein alpha (cEBPα) consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR (real time RT-PCR) for RET, Pit1, Arf, TP53, GDNF, steroidogenic factor 1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNF's survival function might constitute a new therapeutic route in acromegaly.

MiRNAs Mediate GDNF-Induced Proliferation and Migration of Glioma Cells

“…Diaz-Rodriguez et al . reported that GDNF and p53 levels were negatively correlated in somatotropinomas [68], which is consistent with our findings in U251 and U87 glioma cells (Fig. 6A, E).…”

Octreotide and pasireotide (dis)similarly inhibit pituitary tumor cells in vitro

“…Additionally, six normal pituitaries resected during surgical removal of a pituitary adenoma were also used. In all cases, tissue phenotype confirmation was supported by three separate methods as described previously (Luque et al 2013): detailed histological examination by an anatomopathologist, testing the hormonal phenotype using single-cell secretion on cells seeded onto PVDF membranes to evaluate the secretion of all pituitary hormones using specific antibodies, as previously reported (Vazquez-Martinez et al 2008, Luque et al 2013, Diaz-Rodriguez et al 2014; and molecular screening by qPCR, as shown in Fig. 1.…”

Pituitary Cell Turnover: From Adult Stem Cell Recruitment through Differentiation to Death

“…In somatotrophs, when GDNF is not present RET forms a complex with caspase 3 and PKC-delta (PKCd). Once this complex is formed, caspase 3 is activated to aCasp3, which processes RET and PKCd, and this phosphorylates transcription factors such as CREB and cEBPa, leading to uncontrolled Pit-1 transcription [92,94]. Excess Pit-1 in turn induces p19Arf transcription, p53 accumulation and apoptosis [95].…”

“…A recent report described the expression of RET, GDNF, ARF and p53 in somatotrophs but not in NFPA adenomas in order to ascertain whether the dependence death pathway is affected in those tumors [94]. The apoptotic pathway is conserved in acromegaly primary cultures when the cells are deprived of GDNF, so that RET gets processed and the intracellular fragment induces cEBP-alpha binding to the human Pit-1 promoter to accumulate Pit-1, leading to p14Arf expression, p53 stabilization and apoptosis.…”