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. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0±2.7% vs 4±4.3% SST2 internalization, control vs FLNA siRNA cells, respectively, P<0.001) and human GH-secreting primary cultured cells (70.3±21.1% vs 24±19.2% SST2 internalization, control vs FLNA siRNA cells, respectively, P<0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Co-immunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely-dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and Rab4, respectively (33.7±8.9% down to 25.9±6.9%, p<0.05, and 28.4±7.4% down to 17.6±5.7%, p<0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control vs FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and Rab4 sorting endosomes.

Cytoskeleton protein Filamin A is required for efficient Somatostatin receptor type 2 internalization and recycling through Rab5 and Rab4 sorting endosomes in tumor somatotroph cells / Treppiedi, Donatella; Mangili, Federica; Giardino, Elena; Catalano, Rosa; Locatelli, Marco; Lania, Andrea; Spada, Anna; Arosio, Maura; Calebiro, Davide; Mantovani, Giovanna; Peverelli, Erika. - In: NEUROENDOCRINOLOGY. - ISSN 0028-3835. - (2019). [10.1159/000503791]

Cytoskeleton protein Filamin A is required for efficient Somatostatin receptor type 2 internalization and recycling through Rab5 and Rab4 sorting endosomes in tumor somatotroph cells

Catalano, Rosa;
2019

Abstract

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. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0±2.7% vs 4±4.3% SST2 internalization, control vs FLNA siRNA cells, respectively, P<0.001) and human GH-secreting primary cultured cells (70.3±21.1% vs 24±19.2% SST2 internalization, control vs FLNA siRNA cells, respectively, P<0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Co-immunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely-dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and Rab4, respectively (33.7±8.9% down to 25.9±6.9%, p<0.05, and 28.4±7.4% down to 17.6±5.7%, p<0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control vs FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and Rab4 sorting endosomes.
2019
SST2, FLNA, intracellular trafficking
01 Pubblicazione su rivista::01a Articolo in rivista
Cytoskeleton protein Filamin A is required for efficient Somatostatin receptor type 2 internalization and recycling through Rab5 and Rab4 sorting endosomes in tumor somatotroph cells / Treppiedi, Donatella; Mangili, Federica; Giardino, Elena; Catalano, Rosa; Locatelli, Marco; Lania, Andrea; Spada, Anna; Arosio, Maura; Calebiro, Davide; Mantovani, Giovanna; Peverelli, Erika. - In: NEUROENDOCRINOLOGY. - ISSN 0028-3835. - (2019). [10.1159/000503791]
File allegati a questo prodotto
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1346913
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 9
  • Scopus 12
  • ???jsp.display-item.citation.isi??? 12
social impact