Abstract
Tssc3 is a maternally expressed/paternally silenced imprinted gene. Recent evidence suggests that the loss of TSSC3 results in placental overgrowth in mice. These findings showed that the TSSC3 gene functions as a negative regulator of placental growth. In this study, we describe the function of TSSC3 and its signaling pathway in mouse trophoblast stem (TS) cell differentiation. First of all, we tested Tssc3 expression levels in TS cells. TS cells expressed Tssc3, and its expression level was the highest from day 1 to 4 but was down-regulated at day 5 after the induction of differentiation. Overexpression of TSSC3 in TS cells up-regulated Gcm1 and Mash2, which are marker genes of mouse trophoblast differentiation. Down-regulation of TSSC3 by siRNA enhanced Pl1 and Tpbpa expression in TS cells cultured under stem cell conditions, suggesting the contribution of TSSC3 to the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts. TSSC3 activated the PI3K/AKT pathway through binding with phosphatidylinositol phosphate lipids and enhanced the activity of a promoter containing an E-box structure, which is the binding sequence of the Mash2 downstream target gene promoter. PI3K inhibitor suppressed the promoter activity induced by TSSC3. TSSC3 induced Sp1 translocation from cytoplasm to nucleus through the PI3K/AKT pathway. Nuclear Sp1 activated the Mash2 transcription by Sp1 binding with a consensus Sp1-binding motif. This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway.
Highlights
Tssc3 is a maternally expressed imprinted gene
Gcm1, which is known as a marker of labyrinth [14], was up-regulated 2 days after withdrawal of FGF/ conditioned medium (CM), followed by a decrease after day 4. These results indicated that TSSC3 was expressed in trophoblast stem (TS) cells, and the prominent upregulation of Tssc3 at days 1– 4 after withdrawal of FGF/CM was followed by a decrease of Tssc3, and withdrawal of FGF/CM from TS cell cultures resulted in the appearance of trophoblast giant cells, spongiotrophoblasts, and labyrinth trophoblasts concomitant with the disappearance of TS cells
The present study demonstrated that TS cells express Tssc3, and its expression level is the highest from day 1 to 4 after the withdrawal of FGF/CM, followed by a decline after day 5
Summary
Tssc is a maternally expressed imprinted gene. Results: TSSC3 regulates Mash transcription in TS cells through phosphorylation of AKT and Sp1 translocation from cytoplasm to nucleus. Nuclear Sp1 activated the Mash transcription by Sp1 binding with a consensus Sp1-binding motif This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway. MASH2, a member of the basic helix-loophelix (bHLH) transcription factor family (8 –10), is essential for differentiation into spongiotrophoblast cells, because the absence of MASH2 results in the loss of spongiotrophoblast layer, followed by abundant formation of trophoblast giant cells [11]. TSSC3 is expressed only in cytotrophoblasts in normal human placenta [21] These findings implicate TSSC3 in trophoblast differentiation and further resultant placental formation. TSSC3 regulates initial phases of trophoblast differentiation from TS upstream of MASH2 through AKT/Sp1 signaling
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