Mechanisms in trophoblast differentiation and function

     The multinucleated syncytiotrophoblast (SynT) of the human placenta is formed by fusion of underlying proliferating cytotrophoblasts (CytT). The SynT, which covers the chorionic villi, is bathed in maternal blood and performs several essential functions to ensure growth and survival of the developing embryo. These include transport of O2 and nutrients and synthesis and secretion of protein and steroid hormones, including estrogen and progesterone. Synthesis of estrogens from C19-steroids is catalyzed by aromatase P450 (product of the hCYP19A1 gene). The ability of the human placenta to synthesize estrogens is vastly increased after the ninth week of gestation, in association with CytT invasion and enlargement of the uterine arterioles, increased blood flow and O2 availability to the floating chorionic villi. The exceptionally high levels of placental aromatase likely function to metabolize large amounts of C19-steroids produced by the human fetal adrenals (e.g. dehydroepiandrosterone), thus preventing conversion of these steroids to active androgens, which can masculinize the fetus. Biologically active estrogens and their metabolites formed by placental aromatase may also enhance angiogenesis and uteroplacental blood flow and reduce systemic vascular resistance.

     We have explored mechanisms that underlie human placental trophoblast differentiation and effects of O2 tension using the placenta-specific (PS) hCYP19A1/ aromatase promoter as a model. Trophoblast stem cells (TSC) and CytT do not express aromatase; however, when CytT fuse to form SynT, aromatase is markedly induced. By contrast, when CytT are cultured in a hypoxic (2% O2) environment, SynT differentiation and induction of hCYP19A1PS are prevented by increased binding of transcriptional repressors, MASH-2/ASCL2 (15) and USF1/2 (Fig. 2); these factors are degraded upon SynT differ­entia­tion in 20% O2. This likely allows assem­bly of an enhanceosome (Fig. 2) comprised of transcription factors Sp1, estrogen receptor α (ERα), estrogen-related receptor γ (ERRγ) and glial cells missing 1 (GCM1), which serves a crucial role in trophoblast develop­ment and SynT fusion. This complex also contains NRF2 (Fig. 2), which we found to be essential for O2-mediated aromatase mRNA induction in cultured trophoblasts.

       The SynT also may sustain pregnancy through production of immune modulators, which promote immune tolerance at the maternal-fetal interface (MFI)/decidua, to protect the hemi-allogeneic fetus from rejection by the maternal immune system. However, the underlying mechanisms have not been fully defined. Using human trophoblasts in primary culture, we observed that several genes involved in the induction and maintenance of immune tolerance were markedly upregulated during differentiation of CytT to SynT. These include: heme oxygenase I (HMOX1); programmed death-ligand 1 (PD-L1); kynurenine pathway components, indoleamine 2,3-dioxygenase (IDO1) and arylhydrocarbon receptor (AhR). Intriguingly, we discovered that the O2-regulated transcription factors, NRF2, C/EBPβ and PPARγ, were markedly induced when CytT fuse to form SynT. NRF2 knockdown blocked the induction of aromatase, as well as upregulation of C/EBPβ, PPARγ and the above-mentioned immune modulatory factors (Fig. 3). NRF2 defi­ciency has been implicated in preeclampsia (PE), a hypertensive disorder of pregnancy associated with shallow implantation, placental hypoxia and inflammation. Mice with a global KO of Nrf2 were reported to manifest increased placental inflam­matory factors, oxidative stress and susceptibility to preterm birth induction. Importantly, we recently found that placentas of Nrf2 KO mice at 12.5 dpc express decreased Pparγ, Hmox1 and Ahr mRNA.

       Based on these and other findings we are utilizing human trophoblasts in primary culture, human trophoblast stem cells and gene-targeted mice to test the hypothesis that the redox-regulated transcription factor, NRF2, together with C/EBPβ and PPARγ, serve as ‘key regulators’ of the profound biochemical, genetic and epigenetic changes that underlie production of immune modulators. These modulators act on immune cells within the decidua to maintain an anti-inflammatory milieu and protect the fetus from rejection by the maternal immune system to maintain pregnancy. We further propose that increased inflammation at term or preterm causes decreased placental expres­sion of immune modulators and a ‘break’ in tolerance leading to term or preterm labor or PE.