Endocrine Abstracts

Background: The immortalised human granulosa cell line (hGL5) is not responding to gonadotropins, which fail to activate the cAMP/PKA/CREB pathway, progesterone production, cell rounding and apoptosis, suggesting that FSH- and LH/hCG-receptors (FSHR; LHCGR) are downregulated.

Aim: To investigate whether the mechanism of FSHR/LHCGR downregulation is associated with life/death signals in hGL5 cells.

Methods: We evaluated the FSHR/LHCGR expression in cultured hGL5 cells (0–15% serum) 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 by proliferation assay and confocal imaging, in the presence or absence of selective inhibitors/agonists (i.e. the PKA inhibitor H-89, PMA as a PKC-ERK1/2 activator, siRNA against β-arrestin1/2).

Results: Despite the positive expression of FSHR/LHCGR in the presence of serum at both mRNA and protein level (linear regression; P<0.05; n=3), FSH/LH stimulation was ineffective on cAMP/pCREB activation and progesterone production (Mann-Whitney’s U-test; P≥0.05; n=3), suggesting uncoupling of the receptors to the Gs-α protein. Conversely, ERK1/2 phosphorylation was FSH/LH- and dose-dependent in the presence of serum, increasing cell proliferation within 3 days, an effect similar to that obtained 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/CREB activation and progesterone accumulation at high levels (Mann-Whitney’s U-test; P<0.05; n=3), cell rounding, pro-caspase three cleavage and apoptosis. The pro-apoptotic effects of cAMP/PKA basal activation were augmented by FSH -but not LH- treatment and inhibited by H-89 selective PKA blockade. Accordingly, long-term (4–8 weeks) overexpression of FSHR resulted in high basal cAMP levels, cell rounding and apoptosis (Mann-Whitney’s U-test; P<0.05; n=3), revealing the peculiar, dual role of FSHR in the activation of proliferative/apoptotic signals.

Conclusions: β-arrestins determine the FSHR-, rather than LHCGR-mediated signalling towards proliferative/apoptotic pathways, acting as selective switch between ERK or cAMP/PKA pathway activation.

Disclosure: This work was supported by a grant of the Italian Ministry of Education, University and Research (PRIN 2010C8ERKX).