Connexin 31 (GJB3) Deficiency in Mouse Trophoblast Stem Cells Alters Giant Cell Differentiation and Leads to Loss of Oxygen Sensing1

Abstract

The nonphysiological placental oxidative environment has been implicated in many complications during human pregnancy. Oxygen tension can influence a broad spectrum of molecular changes leading to alterations in trophoblast cell lineage development. In this study, we report that mouse wild-type trophoblast stem cells (TSCs) react to low oxygen (3%) with an enhanced differentiation into the giant cell pathway, indicated by a downregulation of the early stem cell markers Eomes and Cdx2 as well as by a significant upregulation of Tfap2c and the differentiation markers Tpbpa and Prl3d1. Here we demonstrated that connexin 31/GJB3-deficient TSCs failed to stabilize HIF-1A under low oxygen, resulting in nonresponsiveness of different marker genes, such as Cdx2 and Eomes and Tfap2c and Tpbpa. Moreover, connexin 31-deficient TSCs revealed a shift in giant cell differentiation from Prl3d1 expressing parietal giant cells to Ctsq, Prl3b1, and Prl2c2-positive giant cells, probably sinusoidal and canal lining trophoblast giant cells. Thus, loss of connexin 31 led to different giant cell subtypes which bypass the progenitor regulators Tfap2c and Tpbpa under low oxygen conditions.

Connexin31.1 (Gjb5) Deficiency Blocks Trophoblast Stem Cell Differentiation and Delays Placental Development

“…The major finding of our study is that Cx31.1 has a directly opposed function to Cx31 on TSC differentiation and placental development. Cx31 deficiency enhances TSC differentiation into placental subpopulations [10,12,13], whereas the absence of Cx31.1 delays this process. Thus, both connexins are rather collectively balancing TSC fate by having opposite regulatory effects on differentiation processes than serving the same molecular functions by just having redundant properties.…”

“…Upon differentiation, Cx31-deficient TSC show an earlier temporal induction of the Ascl2 (Mash2) gene and subsequently the SPT marker gene Tpbpa, the syncytiotrophoblast marker Cx26 (Gjb2), and the TGC marker Prl3d1 (Pl-1, placental lactogen 1), compared to wild-type controls, which indicates an endogenous, enhanced differentiation of TSC [12]. A recent study analyzing markers for the different TGC populations, showed that Cx31-deficient TSC, in particular, reveal an upregulation of secondary TGC markers Prl2c2/Plf, Prl3b1/Pl-2, and Ctsq [13].…”

CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties

“…After 24 h of serum starvation, the cells were treated with 1 mg/ml g-rhCCN1, g-rhCCN3, or PBS/0.1% BSA as a solvent control for 24 h. Annexin V apoptosis assays were performed as described by Koch et al 25 using flow cytometry (FACSCalibur, Becton Dickinson) in combination with FITC-coupled annexin and propidium iodide (PI; BD Pharmingen).…”

Three-dimensional cultures of trophoblast stem cells autonomously develop vascular-like spaces lined by trophoblast giant cells

“…In the mature mouse placenta, the subtypes of TGCs on the venous side, Ch-TGCs and P-TGCs, are exposed to deoxygenated blood whereas SpATGCs and C-TGCs are in contact with oxygenated blood, and STGCs are bathed in diminishing concentrations of oxygen depending on the level within the labyrinth. Previous studies with trophoblast stem (TS) cells grown as adherent monolayers, showed that culturing in 3% oxygen, compared to the atmospheric levels ( $ 20%), increased expression of a P-TGC-specific gene (Prl3d1/Pl1α) but had no effect on other TGC-specific genes (Koch et al, 2012). The cellular response to low oxygen levels is regulated by a family of transcription factors, known as hypoxia inducible factors (Hifs).…”