Functional reorganization of monoamine transport systems during villous trophoblast differentiation: evidence of distinct differences between primary human trophoblasts and BeWo cells

Abstract

Background Three primary monoamines—serotonin, norepinephrine, and dopamine—play major roles in the placenta-fetal brain axis. Analogously to the brain, the placenta has transport mechanisms that actively take up these monoamines into trophoblast cells. These transporters are known to play important roles in the differentiated syncytiotrophoblast layer, but their status and activities in the undifferentiated, progenitor cytotrophoblast cells are not well understood. Thus, we have explored the cellular handling and regulation of monoamine transporters during the phenotypic transitioning of cytotrophoblasts along the villous pathway. Methods Experiments were conducted with two cellular models of syncytium development: primary trophoblast cells isolated from the human term placenta (PHT), and the choriocarcinoma-derived BeWo cell line. The gene and protein expression of membrane transporters for serotonin (SERT), norepinephrine (NET), dopamine (DAT), and organic cation transporter 3 (OCT3) was determined by quantitative PCR and Western blot analysis, respectively. Subsequently, the effect of trophoblast differentiation on transporter activity was analyzed by monoamine uptake into cells. Results We present multiple lines of evidence of changes in the transcriptional and functional regulation of monoamine transporters associated with trophoblast differentiation. These include enhancement of SERT and DAT gene and protein expression in BeWo cells. On the other hand, in PHT cells we report negative modulation of SERT, NET, and OCT3 protein expression. We show that OCT3 is the dominant monoamine transporter in PHT cells, and its main functional impact is on serotonin uptake, while passive transport strongly contributes to norepinephrine and dopamine uptake. Further, we show that a wide range of selective serotonin reuptake inhibitors affect serotonin cellular accumulation, at pharmacologically relevant drug concentrations, via their action on both OCT3 and SERT. Finally, we demonstrate that BeWo cells do not well reflect the molecular mechanisms and properties of healthy human trophoblast cells. Conclusions Collectively, our findings provide insights into the regulation of monoamine transport during trophoblast differentiation and present important considerations regarding appropriate in vitro models for studying monoamine regulation in the placenta.

Serotonin system in the human placenta – the knowns and unknowns

“…Thus, in one IHC study, prominent OCT3 staining was found on basolateral surface and in cytoplasm of cytotrophoblasts, while it was absent in syncytiotrophoblast in all three trimesters of pregnancy ( 82 ). This is consistent with OCT3 protein levels being down-regulated during spontaneous syncytialization of human term primary trophoblasts ( 95 ). However, in another study, OCT3 was localized to the basal (fetal-facing), but not the apical (maternal-facing) membrane of term syncytiotrophoblast ( 85 ).…”

“…Single-cell transcriptome analyses showed lower SERT mRNA levels in syncytiotrophoblast and cytotrophoblasts, both in the first trimester ( 88 ) and at term ( 87 ). On the other hand, recent study found that SERT mRNA levels were upregulated during spontaneous syncytialization of human primary trophoblasts, but SERT protein and activity levels were downregulated by syncytialization ( 95 ). Low levels of SERT mRNA and SERT protein were also detected in feto-placental endothelial cells ( 80 , 82 , 89 ), but functional analysis did not support significant SERT activity in these cells ( 89 ).…”

“…These choriocarcinoma-derived cells are commonly used in vitro models in human placenta research ( 99 ). Recently, however, concerns have been raised about their suitability for studying serotonin pathways ( 70 , 89 , 95 ). Suitable in vitro models for future studies would be primary trophoblasts, placental explants, and organoid trophoblast cultures ( 100 ), as well as primary feto-placental endothelial cells, Hofbauer cells, and other cell types.…”

“…Estrogen (17β-estradiol) decreased the activity of SERT in trophoblast-like BeWo cells, but increased the level of SERT protein and had no effect on the level of SERT mRNA ( 114 ). A discrepancy between the levels of SERT mRNA and SERT protein in human placenta was also observed in relation to the effects of gestational age ( 69 ) and trophoblast differentiation ( 95 ), indicating the presence of important regulatory mechanisms acting at the translational level.…”

Developmental expression of catecholamine system in the human placenta and rat fetoplacental unit

“…Interestingly, while the choriocarcinoma-derived cells express TH , the rate-limiting enzyme in dopamine synthesis, PHT cells do not. Likewise, BeWo and JEG-3 cells show DAT expression at both mRNA, protein, and functional levels, which is not seen in other in vitro models 31 . This observation is noteworthy given their isolation from brain metastatic choriocarcinoma 32 , 33 .…”