PGE2 Acts via PTGER2 to Induced FGF2 and Epiregulin Expression in Bovine Granulosa Cells: Involvement of PKA and EPAC Signaling

Introduction: PGE2, epiregulin (EREG) and FGF2 are elevated in the early corpus luteum (CL) immediately after ovulation. Although LH is known to stimulate these factors, their expression is also interdependent. For instance, PGE2 induces EREG and other EGF-like-peptides. Classically PGE2 acts via cAMP but its detailed mechanism of action is still unclear.

Aim: Examine whether PGE2 can induce FGF2 along with EREG and elucidate the signaling pathways involved in their expression in granulosa cells (GCs).

Methods: GCs collected from large healthy bovine follicles. Drug treatments: PGE2; PD173074 (FGF-receptor1 inhibitor); PGE-receptors’ agonists (PTGER1-4); H-89-dihydrochloride (PKA-inhibitor); 8-(4-Chlorophenylthio)-2’-O-Me-cAMP-AM (exchange-protein directly activated by cAMP (EPAC)-activator); ESI09 (EPAC1/2-inhibitor) and ESI05 (EPAC2-specific-inhibitor). Transfection: scrambled or PTGS2-siRNA.

Results: PGE2 maximally induced FGF2 and EREG at 3hr decreasing gradually until 24h. Similarly, knocking-down PGE2 levels in PTGS2-silenced GCs, exhibited low FGF2 and EREG. PGE2 induced FGF2 protein and increased viable GCs’ numbers. PD173074 significantly abolished this last effect, thus verifying PGE2 affects GCs’ survival via FGF2. Amongst four PTGER agonists, only PTGER2-agonist (butaprost) mimicked PGE2 effect on FGF2 and EREG. To check the involvement of PKA, a canonical downstream target of cAMP, H89 was used. With this inhibitor EREG stimulation was abolished while FGF2 remains unchanged. FGF2 stimulation was EPAC-dependent: EPAC activator upregulated FGF2 but also EREG. FGF2 was inhibited by ESI09 while EREG was partially reduced by ESI05.

Conclusions: This study unravels the mechanism of PGE2 actions; both FGF2 and EREG were induced via PTGER2 subtype elevating cAMP in GCs. Interestingly, while FGF2 expression was EPAC dependent, EREG was mediated by both PKA and EPAC. These results demonstrate the diverse roles and mode of PGE2 action in CL development.