CRH is known to activate the cAMP/PKA (protein kinase A) pathway via its receptors, CRHR1 and CRHR2, leading to phosphorylation of the cAMP response element binding protein (CREB1) . As a transcription factor, CREB1 binds to a cAMP response element (CRE) sequence in the promoter of target genes, regulating the transcription of those genes [26, 27]. CRH has been studied as one of those target genes containing a CRE in the promoter, and the CRE has been shown to be crucial for CRH gene expression [28–30]. Furthermore, CRH and its receptors (CRHR1/R2) are expressed in placental tissues [9, 31] and CRH can bind to syncytiotrophoblast membranes . So, the induction of CRH by cAMP hints that CRH may play a feedback role in cAMP mediated effects in placental trophoblasts.
Using BeWo choriocarcinoma cells as a well-established cellular model of placental trophoblast syncytialisation we showed that, in the presence of serum, 8-Br-cAMP significantly induces cell apoptosis and promotes differentiation. Our previous data also revealed that 8-Br-cAMP had less effect on BeWo cell viability and syncytialisation if cells were cultured in 10% charcoal stripped media , since charcoal stripping would remove other factors such as steroids, leading us to imply that CRH signal pathways may play a role in cAMP-mediated cell apoptosis and differentiation. The ability of CRH to regulate cell apoptosis in several cells of different origin has been reported [33, 34], and CRH has been shown to induce FASLG (Fas ligand) production and apoptosis in the rat pheochromocytoma cell line PC12 via activation of MAPK (p38 mitogen-activated protein kinase) . This prompted us to further propose that although CRH is produced by differentiated trophoblasts it may also play a role in regulating trophoblast viability, thereby helping to keep the syncytiotrophoblast pool in renewal. However, our experiments reveal that although CRH induces cell apoptosis in some cell lines it has no significant effect on BeWo cell viability, according to DAPI stain and FACS analysis. This also suggests that any effect that 8-Br-cAMP has on BeWo cell viability is mediated through a signal pathway other than a CRH pathway. Indeed, these results indicate that while CRH may activate GNAS pathways, cAMP may activate additional compartments of cAMP mediated signalling, or CRH may activate additional pathways that antagonise apoptosis effects mediated by cAMP. Nevertheless, the existence of CRH receptors in BeWo cells suggests that CRH could be involved in another bioactivity, such as modulation of differentiation or syncytialisation of trophoblast cells. Consistent with this idea, we demonstrated that CRH can induce cell differentiation, as evidenced by the induction of ERVW-1 and by cell fusion. For trophoblast cells, the widely accepted marker of differentiation is a combination of a biochemical index (induction of hCG and ERVW-1 proteins) and a morphological index (formation of cell fusion), with the latter being more convincing. Recently, the reliability of hCG as a trophoblast differentiation marker has been called into question, due to dissociation of the two differentiation indexes. We show here additional evidence that biochemical and morphological differentiation can be dissociated, since CRH facilitates cell fusion without any induction of hCG secretion. Also, in primary human trophoblasts, CRH stimulation induced ERVW-1 expression, without any induction of hCG secretion . Similar dissociations have been reported in villous cytotrophoblast cells cultured in the absence of serum  and in JEG3 cells stimulated by forskolin, where an induction of hCG occurred without any evidence of cell fusion . Recently, it was reported that the PKA inhibitor, H89, can reverse forskolin-induced BeWo cell fusion without altering forskolin-induced hCG secretion . Therefore, hCG protein expression may not necessarily be linked to syncytial fusion. This study adds to the growing evidence that differentiation and fusion are related but distinct events . However, the secretion of hCG can be enhanced by 8-Br-cAMP but not by CRH, suggesting that CRH and 8-Br-cAMP may regulate cell fusion through different signal pathways. Indeed, others have shown that charcoal scavenged hormones, such as estradiol, glucocorticoids and hCG, play roles in trophoblast differentiation at different stages of pregnancy .
We recently published evidence of a coherent relationship between the induction by cAMP of apoptosis and syncytial differentiation in BeWo cells. In cells cultured in media containing serum, a large increase in FASLG/FAS ratio and the occurrence of cell apoptosis coincided with a greater induction of ERVW-1 and hCG and the formation of syncytia. In contrast, in cells cultured in media lacking serum, the lower increase in FASLG/FAS ratio with no apparent cell apoptosis is in agreement with the lower induction of ERVW-1 and hCG with no sign of cell fusion . This relationship has prompted us to propose that the cytotrophoblastic syncytialisation process involves an apoptotic event. Clearly, the data in this paper shows that 8-Br-cAMP induces both cell apoptosis as well as cell fusion whereas CRH has no effect on apoptosis but can still promote cell fusion, indicating that syncytialisation is a process that can occur either with or without cell apoptosis. 8-Br-cAMP and CRH both elevate intracellular cAMP, with 8-BR-cAMP resulting in a sustained intracellular cAMP elevation resistant to phosphodiesterases, whereas elevations via cAMP are transient and compartmentalized and, as in other studies, this could determine whether elevating cAMP is pro- or anti- apoptotic . Thus, whether the syncytialisation process correlates with an apoptosis program or not is far more complex than previously realised, and may depend on either, or both, the inducer and the cell model used. Furthermore, we found that the expression of CRHR1 and CRHR2 can be up regulated by 8-Br-cAMP and also by CRH itself. This latter finding allows the possibility that CRH and the CRH-receptors are involved in a positive feed-forward system that may underlie the exponential rise in CRH observed across pregnancy that leads to delivery. This requires further investigation but, if correct, suggests the timing of birth in humans is an event determined by a placental maturation program.