The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. However, about one third of patients displays resistance to SSAs and, to date, the responsible molecular mechanisms are still under investigation. Recently, the cytoskeletal protein Filamin A (FLNA) and the scaffolding proteins β-arrestins have emerged as key modulators of the responsiveness of GH-secreting pituitary tumors to SSAs, by regulating SSTR2 signaling and desensitization, respectively. The aim of this study is to further explore FLNA involvement in SST2 intracellular trafficking in somatotroph cells, since alterations in this system might affect the amount of available receptor at the plasma membrane and lead to unresponsive tumor features. By biotinylation assay we first found that FLNA silencing strongly reduces octreotide-mediated SST2 internalization in rat GH-secreting pituitary tumor cell line, GH3 cells, (26.9±2.7% vs 4±1.7% SST2 internalization, control vs FLNA SiRNA cells, respectively, P<0.001) and in one human GH-secreting primary culture tested (93.2% vs 31.6% SST2 internalization, control vs FLNA SiRNA cells, respectively). Then, co-immunoprecipitation and immunofluorescence experiments performed in GH3 cells showed that FLNA knock down does not prevent the recruitment of β-arrestin2 to cell surface ligand-activated SST2, but rather impairs receptor localization into cytosolic vesicles positive for the early endocytic marker Rab5 (Pearsons coefficient mean 0.29±0.07 vs 0.16±0.03 of SST2-Rab5 colocalization in control vs FLNA SiRNA, P<0.01). Furthermore, after octreotide removal, SST2 recycling fate was affected by the loss of FLNA. Conversely to control cells, SST2 failed to accumulate into intracellular recycling vesicles linked to the early recycling marker Rab4, as resulted by Rab4-SST2 colocalizaition analysis (Pearsons coefficient mean 0.24±0.05 vs 0.16±0.04 of SST2-Rab4 colocalization in control vs FLNA SiRNA, P<0.05) and could not reach the plasma membrane in wash out experiments. Altogether these data unveil a crucial role of FLNA in the regulation of octreotide-induced SST2 trafficking in GH-secreting cells, suggesting the possibility of targeting FLNA-SST2 complexes for the treatment of pharmacologically resistant GH-secreting pituitary tumors.
18 - 21 May 2019
European Society of Endocrinology