Proliferative versus apoptotic signals in granulosa cells: β-arrestins as switch between life and death signals in vitro

Background: The immortalized human granulosa cell line hGL5 is not responsive to FSH and LH/hCG, which fail to activate the steroidogenic cAMP/PKA pathway, CREB phosphorylation and progesterone production. Conversely, the activation of adenylyl cyclase by forskolin results in intracellular cAMP increase and steroid production, cell rounding and apoptosis, suggesting a possible FSHR and LHCGR downregulation. Curiously, in hGL5 cells the expression of some receptors (e.g. the oxytocin receptor) is absent in serum starvation conditions and increases in a serum concentration-dependent manner. Aim of the study: To investigate the mechanism of FSHR expression regulation in hGL5 cells and to evaluate whether it is associated with life/death signals in vitro. Methods. We evaluated the FSHR expression in hGL5 cells maintained under different serum concentrations (between 0 and 15%) by real time PCR and Western blotting. The response to 50 nM FSH or 100 pM LH was evaluated by measuring cAMP and progesterone production by ELISA, as well as ERK1/2 and CREB phosphorylation by Western blotting. Cell viability was assessed by proliferation assay and confocal imaging. These endpoints were evaluated in the presence and in the absence of selective inhibitors or agonists (i.e. the PKA inhibitor H-89, the phorbol ester PMA as a PKC-ERK1/2 activator, and siRNA against β-arrestin1/2). Results. The expression of FSHR and LHCGR was serum-dependent at both mRNA and protein level, being absent under starvation and increasing progressively with serum concentrations (linear regression of expression-fold values plotted against serum concentration; p<0.05; n=3). However, FSH/LH stimulation was ineffective both on cAMP and progesterone production and CREB phosphorylation (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p≥0.05; n=3), suggesting uncoupling of the receptors to the Gs alpha protein. ERK1/2 phosphorylation was FSH/LH dose-dependent in the presence of serum (linear regression; p<0.05; n=3), resulting in a significant increase of cell proliferation over 4 days (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p<0.05; n=3). A similar increase of cell proliferation and ERK1/2 phosphorylation was provoked by PMA (Mann-Whitney’s U-test; p<0.05; n=3). β-arrestin1/2 siRNA transfection unlocked the cAMP/PKA pathway, leading to cAMP and progesterone accumulation and CREB phosphorylation, at high basal levels (Mann-Whitney’s U-test; p<0.05; n=3). Moreover, siRNA-treated cells underwent cell rounding, pro-caspase 3 cleavage and apoptosis. The pro-apoptotic effects of cAMP/PKA pathway activation were augmented by FSH- but not LH treatment, and inhibited by selective PKA blockade by H-89. Accordingly, transfected hGL5 cells permanently overexpressing the FSHR (but not LHCGR) for 4-8 weeks showed high basal cAMP levels, cell rounding and apoptosis (Mann-Whitney’s U-test; p<0.05; n=3), revealing the dual role of the FSHR in the activation of proliferative and apoptotic signals. Conclusions. Our results suggest that β-arrestins determine the FSHR-mediated (but not LH-mediated) signaling in vitro towards proliferative- or cell death-related pathways. Our results corroborate the relationship between cAMP/PKA pathway activation and cell death in granulosa cells.