RUOLO DELLA FILAMINA A NELLA REGOLAZIONE DELL'ESPRESSIONE E DELLA TRADUZIONE DEL SEGNALE DEL RECETTORE DELLA SOMATOSTATINA SST2 IN CELLULE TUMORALI GH-SECERNENTI

Increased and unregulated growth hormone (GH) production, usually caused by a GH-secreting pituitary tumor (somatotroph tumor), characterizes acromegaly. The pharmacological treatment involves the use of somatostatin analogs (SS), which binding to specific receptor subtypes (SST2, SST5) coupled to inhibitory G proteins, reducing tumor growth and hormonal secretion. SST- mediated signaling involves the inhibition of adenylate cyclase and the activation of K+ channels and closing of channels to the voltage-dependent Ca++, with a consequent inhibition of GH secretion. The antiproliferative effects of SS are mediated by multiple pathways, including MAPK pathway inhibition or stimulation of protein phosphatases activity by SST5 and SST2 receptor subtypes, respectively, moreover SST2 and SST3 subtypes induce apoptosis, through the tireosin protein phosphatase. However, about 30% of acromegalic patients is resistant to medical therapy and the molecular events involved in resistance to SS are not fully understood. One of the hypotheses focuses on the search for alterations in the downstream mechanisms of SSTs activation and proteins associated with them. Several studies identified specific protein-protein interactions as determinant in the regulation of receptor anchoring and signaling, in fact, it has been demonstrated the crucial role of a cytoskeleton ubiquitous protein, Filamin A (FLNA), in the regulation of expression, localization, and signaling of many GPCRs. A pituitary level, the FLNA is crucial for dopamine receptor D2R signal transduction and expression on the plasma membrane. Recently, has been demonstrated interaction with the receptor SST2 and FLNA. From these evidences, the aim of my study was to investigate the role of FLNA in the modulation of SST2 expression, intracellular localization and signal transduction in GH-secreting adenomas and in somatotroph rat GH3 cells. We have shown by co-immunoprecipitation the interaction between FLNA and SST2 in GH-secreting adenomas and GH3 cells. In addition, by immunohistochemisty and Western blot analysis, we found a variable pattern of FLNA expression between different GH- secreting adenoma samples, with no obvious correlation with SST2 expression. As demonstrated by gene silencing techniques in primary cultures of GH-secreting adenomas, FLNA do not seem to be necessary for the stability of SST2 expression and for its correct targeting to the plasma membrane. While, as shown by the expression of cyclin D1 level, FLNA is required to mediate the inhibition of cell proliferation.To further study the molecular mechanisms underlying these effects , we used GH3 cells , which express endogenously both FLNA that SST2 , and we transfected them with truncated forms of FLNA that act as dominant negative; in particular FLNA 19-20, containing the binding region to SST2 , abolishes the ability of FLNA to bind SST2 and FLNA 21-24 , containing the region mostly involved in the binding of signal transduction molecules, affects the function of FLNA scaffold. Our data show that the SST2 interaction with FLNA is crucial for the stability of SST2 expression after prolonged stimulation with the selective agonist BIM23120. In fact, agonist treatment does not influence the total amount of expressed receptor in control cells, while is observed a strong reduction of SST2 expression after incubation with BIM23120 (55 ± 5 % vs. basal) in cells transfected with FLNA 19-20. To investigate the role of FLNA in SST2 signal transduction, we evaluated the SST2 ability to induce apoptosis by measuring caspase activity after stimulation with BIM23120. We observed a significant increase of apoptosis in control cells (77 ± 54 % vs basal), while this effect was completely abolished in cells transfected with FLNA 19-20 and 21-24. The result obtained with FLNA 19-20 shows that SST2 signal transduction require association with FLNA , while the effect of FLNA 21-24 , which competes with the endogenous FLNA for the binding of molecules necessary for the signal transduction suggests that FLNA also has a functional role probably by acting as a scaffold protein. Further investigate the role of FLNA in SST2 regulation in this cellular model could be crucial to identify possible molecular mechanisms involved in drug resistance to SS analogs among a percentage of patients with acromegaly.