ABSTRACT
BACKGROUND: Placental 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: This 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.
Subject(s)
Cell Differentiation/drug effects , Corticotropin-Releasing Hormone/pharmacology , Cyclic AMP/pharmacology , Trophoblasts/drug effects , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Cell Differentiation/genetics , Cell Fusion , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Choriocarcinoma/genetics , Choriocarcinoma/metabolism , Choriocarcinoma/pathology , Chorionic Gonadotropin/genetics , Chorionic Gonadotropin/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression/drug effects , Gene Products, env/genetics , Gene Products, env/metabolism , Giant Cells/drug effects , Giant Cells/metabolism , Humans , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Corticotropin-Releasing Hormone/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trophoblasts/metabolism , Uterine Neoplasms/genetics , Uterine Neoplasms/metabolism , Uterine Neoplasms/pathologyABSTRACT
Corticotropin Releasing Hormone (CRH), a 41-amino acid peptide, is a major regulator of hypothalamic-pituitary-adrenal axis function. CRH also has important roles in several processes pertaining to pregnancy and parturition, including being a possible regulator of gestational length and predictor of pre-term birth. Regulation of the CRH promoter exhibits some tissue-specificities, the most well characterized example being glucocorticoids, which can stimulate placental CRH production but suppress hypothalamic CRH. In the last decade there has been growing interest in the role of epigenetic regulation of gene expression. Modification of the structure of chromatin is an example of epigenetic change affecting gene expression. We have found that inhibition of histone deacetylases results in an increase in CRH expression in the AtT20 pituitary cell line, but a decrease in CRH expression in the placenta. In this paper we review tissue specific differences in CRH gene expression, and discuss how epigenetic chromatin modification mechanisms can relate to tissue specific differences in expression of CRH.
