Molecular dissection of the GIP/GIPR axis in a stable cell model of GH-secreting pituitary adenomas
D. Regazzoa (Dr), E. Gallettaa (Dr), G. Perbellinia (Dr), M. Dalle Nogarea (Mr), F. Ceccatoa (Dr), K. Lefkimmmiatisb (Prof), C. Scaronia (Prof), G. Occhi*a (Prof)
a University of Padova, Padova, ITALY ; b University of Pavia, Pavia, ITALY
Background: Nearly one-third of GH-secreting pituitary adenomas (GH-PAs) aberrantly express the glucose-dependent insulinotropic polypeptide receptor (GIPR), a G-protein coupled receptor involved in the incretin response after nutrient stimulation. Acromegalic patients with GIPR positive (GIPR+) tumors show a paradoxical increase of GH after an oral glucose load, a milder tumoral phenotype including a better response to somatostatin analogs1.
Objectives: Looking for the molecular basis underlying GIP/GIPR signaling influence on GH-PAs phenotype, here we aimed at dissecting the signaling pathways associated with GIPR stimulation and investigated the comprehensive transcriptomic landscape of GIPR expressing GH3-cell line.
Methods: Stable GIPR-expressing clones (GH3GIPR) were generated using the Flp-In T-REx System and the GH3 cells as the host cell line. The GH3GIPR have been validated by luciferase-based assays, Western Blot, Calcium Imaging, FRET, and cell viability assay. We then performed RNA-seq profiling of GH3GIPR cells to investigate the signaling pathways linked to the GIP/GIPR axis.
Results: In GH3GIPR, the stimulation of GIPR induced cAMP elevation - with the consequent activation of the PKA cascade - and a significant fluctuation in Ca2+, both likely associated with increased GH and PRL promoter activity. Differential gene expression (DEG) analysis of RNA-seq data revealed that GIPR aberrant expression is a necessary and sufficient condition to modulate molecular pathways associated with GH synthesis/secretion as well as cell proliferation including cAMP, MAPK/ERK e JAK/STAT. Besides confirming the modulation of these pathways by dual-luciferase assays, we also demonstrated that GIPR overexpression, regardless of its stimulation, was associated with a milder proliferating phenotype, apparently recapitulating GIPR+ GH-PAs.
Conclusions: The GH3GIPR cells generated during this study mimic the GIPR+ GH-PAs, suggesting it may represent a valuable model to study the GIP/GIPR axis in this tumor type. In this preliminary work, we started clarifying some signaling pathways, possibly associated with the more benign phenotype of these tumors. Further studies, however, with specific provocative tests, will be mandatory to fully characterize these pathways and determine the magnitude of their involvement in these processes.
1 Scaroni, C. et al. J. Clin. Endocrinol. Metab. 104, 856-862 (2019)
The author has declared no conflict of interest.