Effects of corticotrophin releasing hormone (CRH) on cell viability and differentiation in the human BeWo choriocarcinoma cell line: a potential syncytialisation inducer distinct from cyclic adenosine monophosphate (cAMP)

Abstract

BackgroundPlacental production of corticotrophin releasing hormone (CRH) rises exponentially as pregnancy progresses, and has been linked with the onset of normal and preterm labour. CRH is produced in syncytiotrophoblast cells and production is increased by glucocorticoids and cAMP. It remains unclear whether cAMP acts by inducing differentiation of cytotrophoblasts and/or through induction of syncytialisation. As CRH can stimulate cAMP pathways we have tested whether a feed-forward system may exist in placental cells during syncytialisation.MethodsThe choriocarcinoma BeWo cell line was treated with cAMP, CRH or vehicle. Cell viability was determined by MTT assay, while apoptosis was analysed by DAPI staining and by FACS. Differentiation was measured by assaying message for hCG and ERVW-1 (syncytin1) by qRT-PCR, as well as the respective protein by ELISA. Fusion of BeWo cells was assessed by co-staining cell membrane and nuclei with CellMask and Hoechst 33342. CRHR1 and CRHR2 mRNA levels were measured by qRT-PCR.ResultsWe show that cAMP has an inductive effect on syncytialisation, as evidenced by induction of hCG secretion, by ERVW-1 mRNA expression and by formation of multinuclear cells. CRH mRNA expression was found to increase prior to the changes in the other syncytialisation markers. cAMP had an inhibitory effect on BeWo cell viability, but exogenous CRH did not. However, CRH did mimic the differentiation inducing effect of cAMP, suggesting a link between CRH and cAMP signalling in syncytialisation. We also found that treatment of BeWo cells with exogenous CRH resulted in elevated cellular CRHR1 levels.ConclusionsThis study suggests a positive feed-forward role exists for CRH in trophoblast cell differentiation, which may underlie the exponential rise in CRH observed as gestation advances.

Methylation of the Corticotropin Releasing Hormone Gene Promoter in BeWo Cells: Relationship to Gene Activity

“…8-Br-cAMP increased CRH mRNA abundance relative to vehicle at all time points (24 h, p = 0.0042; 48 h, p = 0.0066; 72 h, p = 0.0004; Figure 1 ), which confirmed previous findings of the stimulatory effects of 8-Br-cAMP on CRH gene expression in BeWo cells [ 28 ]. 5-AZA-dC treatment had no effect at 24 h and 48 h and reduced CRH mRNA abundance relative to vehicle at 72 h, effectively blocking the increase seen between 48 h and 72 h in vehicle-treated cells ( p = 0.0021, Figure 1 ).…”

Activation and regulation of endogenous retroviral genes in the human pituitary gland and related endocrine tumours

“…CRHR, POMC and the ACTHR have also been detected in the placenta [76,77], where we recently demonstrated a sixfold Syncytin-1 induction of expression in human primary trophoblasts after 48 h of CRH treatment [50]. It was also shown that CRH resulted in the induction of CRHR1 and syncytialization, similar to cAMP alone [78]. Our immunofluorescence and IHC experiments taken together with our primary PA cell culture further corroborates a possible and positive connection between ERV gene expression and cAMP signalling.…”

Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down

“…Raw CT values were processed using the DCT method relative to the geometric mean of two reference genes, ACTB and GAPDH. These reference genes have been widely utilized in other studies of choriocarcinoma cell line mRNA expression levels (Lee et al, 2004;Chiu et al, 2007;Li et al, 2008;Masumoto et al, 2011;Mparmpakas et al, 2012;Chen et al, 2013). DCT values generated relative to ACTB and GAPDH were highly correlated in all assays included in this study (Supplementary data, Fig.…”