Molecular events in human placental trophoblast differentiation

The chorionic villi of the human placenta contain mononucleate cytotrophoblasts which differentiate into multinucleate syncytiotrophoblasts. Cytotrophoblasts and syncytiotrophoblasts are functionally different. Little is known at the molecular level regarding how this morphological and functional differentiation takes place. Cultured in serum-free media, cAMP analogs stimulated isolated human placental cytotrophoblasts to differentiate functionally without syncytialization. The cAMP-stimulated cells acquired the secretory activity of syncytiotrophoblasts. Northern blot analyses showed that cAMP regulates levels of mRNAs encoding proteins related to trophectoderm invasiveness: urokinase type plasminogen activator, plasminogen activator inhibitors type I and II. After 8-bromo-cAMP stimulation, mRNAs for $\alpha$-hCG, $\beta$-hCG, adrenodoxin and P450scc, were all elevated. An effect of cAMP on the mRNA levels of fos and actin was also demonstrated. These data suggest that cAMP plays a major role in coordinating trophoblastic functional differentiation. Differential effect of cycloheximide treatment on different mRNA responses provided evidence for a complex pattern of cAMP regulation. This indicates that trophoblastic strategies for cAMP control of differentiation are varied. Intracellular cAMP rose 3-fold between 48 and 72 hours after plating. This parallels the increase of secreted $\beta$-hCG and the morphological syncytialization, complimenting the hypothesis that cAMP plays a key role in coordinating expression of genes during cytotrophoblasts differentiation. A cytokeratin 8 cDNA clone was isolated during screening for CREB cDNA. Its mRNA level in cultured isolated cytotrophoblasts increased initially but was suppressed by 8-bromo-cAMP. Whether the interaction between intermediate filaments and DNA have importance in controlling differentiation remains to be determined. E-cadherin mRNA in isolated trophoblasts decreased progressively over the 96-hour culture period. During this period, non-syncytializing JEG-3 and BeWo cells showed no significant E-cadherin mRNA level change. BeWo cells underwent fusion when challenged with 8-bromo-cAMP and their E-cadherin mRNA levels decreased significantly. This suggests that down regulation of E-cadherin gene expression is correlated with syncytialization. These studies reveal that trophoblast differentiation is a complex phenomenon encompassing morphological and functional changes, and that cAMP regulates many aspects of this process