Endocrine Abstracts

Despite the increasing evidence on the relevance of the actin binding protein filamin A (FLNA) in determining an efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors, there is a gap in the knowledge on the mechanisms regulating FLNA itself. FLNA is phosphorylated by PKA on Ser²¹⁵² located in a FLNA region crucial for SST2 or partner proteins binding. Although cAMP/PKA pathway plays a crucial role in GH-secreting tumors pathogenesis, the biological relevance of FLNA phosphorylation is unknown. Aim of this study is to investigate in rat and human GH-secreting pituitary tumor cells (N=3) the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function. We found a PKA-mediated increase (about 2-fold) and SST2 agonist-induced decrease (−50%) of FLNA Ser²¹⁵² phosphorylation (P-FLNA) in GH3, GH4C1 and primary somatotroph tumor cells. By transfecting phosphomimetic (S2152D) and phosphodeficient (S2152A) FLNA mutants in GH3 cells, we found that S2152D FLNA abolished the antiproliferative effects exerted by BIM23120 in wild type or S2152A FLNA transfected cells (−32±13% and −21±3%, respectively, P<0.05 vs basal). Moreover, BIM23120 increased caspase activity in wild-type and S2152A FLNA transfected cells (+39±13%+28±9%, respectively, P<0.05 vs basal), but not in S2152D FLNA transfected cells, suggesting a negative effect of FLNA phosphorylation on SST2 signal transduction. Co-immunoprecipitation and immunofluorescence analysis revealed that S2152D FLNA is able to bind SST2 on the plasma membrane both in basal conditions and after SST2 activation, whereas wild-type and S2152A FLNA are recruited to SST2 after 5 min of agonist stimulation. We can hypothesize that FLNA phosphorylation impair the ability of FLNA to bind proteins involved in SST2 signaling without affecting, or even increasing, its ability to bind SST2. In conclusion, our data suggest that cAMP pathway activation abolishes the ability of FLNA to function as scaffold for SST2 signal transduction by increasing FLNA phosphorylation, whereas SST2 activation induces FLNA dephosphorylation in a positive auto-regulatory loop. Since SST2 functions are regulated by FLNA, modulation of P-FLNA might suggest new pharmacological strategies for SSA resistant pituitary tumors as well as a new biomarker for tumor responsiveness to SSA.