IL1ß-NFκß-Myocardin signaling axis governs trophoblast-directed plasticity of vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) plasticity is fundamental in uterine spiral artery remodeling during placentation in Eutherian mammals. Our previous work showed that the invasion of trophoblast cells into uterine myometrium coincides with a phenotypic change of VSMCs. Here, we elucidate the mechanism by which trophoblast cells confer VSMC plasticity. Analysis of genetic markers on E13.5, E16.5, and E19.5 in the rat metrial gland, the entry point of uterine arteries, revealed that trophoblast invasion is associated with downregulation of MYOCARDIN, α-smooth muscle actin, and calponin1, and concomitant upregulation of Smemb in VSMCs. Myocardin overexpression or knockdown in VSMCs led to upregulation or downregulation of contractile markers, respectively. Co-culture of trophoblast cells with VSMCs decreased MYOCARDIN expression along with compromised expression of contractile markers in VSMCs. However, co-culture of trophoblast cells with VSMCs overexpressing MYOCARDIN inhibited their change in phenotype, whereas, overexpression of transactivation domain deleted MYOCARDIN failed to elicit this response. Furthermore, the co-culture of trophoblast cells with VSMCs led to the activation of NFκß signaling. Interestingly, despite producing IL-1ß, trophoblast cells possess only the decoy receptor, whereas, VSMCs possess the IL-1ß signaling receptor. Treatment of VSMCs with exogenous IL-1ß led to a decrease in MYOCARDIN and an increase in phosphorylation of NFκß. The effect of trophoblast cells in the downregulation of MYOCARDIN in VSMCs was reversed by blocking NFκß translocation to the nucleus. Together, these data highlight that trophoblast cells direct VSMC plasticity, and trophoblast-derived IL-1ß is a key player in downregulating MYOCARDIN via the NFκß signaling pathway.

Cellular Prion protein moonlights vascular smooth muscle cell fate: Surveilled by trophoblast cells.

Uterine spiral artery remodeling (uSAR) is a hallmark of hemochorial placentation. Compromised uSAR leads to adverse pregnancy outcomes. Salient developmental events involved in uSAR are active areas of research and include (a) trophendothelial cell invasion into the spiral arteries, selected demise of endothelial cells; (b) de-differentiation of vascular smooth muscle cells (VSMC); and (c) migration and/or death of VSMCs surrounding spiral arteries. Here we demonstrated that cellular prion (PRNP) is expressed in the rat metrial gland, the entry point of spiral arteries with the highest expression on E16.5, the day at which trophoblast invasion peaks. PRNP is expressed in VSMCs that drift away from the arterial wall. RNA interference of Prnp functionally restricted migration and invasion of rat VSMCs. Furthermore, PRNP interacted with two migration-promoting factors, focal adhesion kinase (FAK) and platelet-derived growth factor receptor-ß (PDGFR-ß), forming a ter-molecular complex in both the metrial gland and A7r5 cells. The presence of multiple putative binding site of odd skipped related-1 (OSR1) transcription factor on the Prnp promoter was observed using in silico promoter analysis. Ectopic overexpression of OSR1 increased, and knockdown of OSR1 decreased expression of PRNP in VSMCs. Coculture of VSMCs with rat primary trophoblast cells decreased the levels of OSR1 and PRNP. Interestingly, PRNP knockdown led to apoptotic death in ~9% of VSMCs and activated extrinsic apoptotic pathways. PRNP interacts with TRAIL-receptor DR4 and protects VSMCs from TRAIL-mediated apoptosis. These results highlight the biological functions of PRNP in VSMC cell-fate determination during uteroplacental development, an important determinant of healthy pregnancy outcome.

Tipping the balance toward stemness in trophoblast: Metabolic programming by Cox6B2.

Metabolic effector(s) driving cell fate is an emerging concept in stem cell biology. Here we showed that Cytochrome C Oxidase Subunit 6B2 (Cox6B2) is essential to maintain the stemness of trophoblast stem (TS) cells. RNA interference of Cox6b2 resulted in decreased mitochondrial Complex IV activity, ATP production, and oxygen consumption rate in TS cells. Furthermore, depletion of Cox6b2 in TS cells led to decreased self-renewal capacity indicated by compromised BrdU incorporation, Ki67 staining, and decreased expression of TS cell genetic markers. As expected, the consequence of Cox6b2 knockdown was the induction of differentiation. TS cell stemness factor CDX2 transactivates Cox6b2 promoter in TS cells. In differentiated cells, Cox6b2 is post-transcriptionally regulated by two microRNAs, miR-322-5p and miR-503-5p, leading to its downregulation as demonstrated by the gain-in or loss of function of these miRNAs. Cox6b2 transcripts gradually rise in placental trophoblast gestation progresses in both mice and rats with predominant expression in labyrinthine trophoblast. Cox6b2 expression is compromised in the growth-restricted placenta of rats with reciprocal up-regulation of miR-322-5p and miR-503-5p. These data highlight the importance of Cox6B2 in the regulation of TS cell state and uncompromised placental growth.

Harnessing Autophagic Network Is Essential for Trophoblast Stem Cell Differentiation.

Differentiation of trophoblast stem (TS) cells into various cell lineages of the placenta during mammalian development is accompanied by dynamic changes in its proteome for exerting the highly specialized functions of various cell subtypes. In the present study, we demonstrate that the autophagic machinery, which includes proteins for initiation, vesicle nucleation, and autophagosome maturation are robustly upregulated during differentiation of TS cells. Interestingly, basal levels of autophagy were detectable in the developing mouse placenta as well as TS cells. However, autophagic flux was actively triggered by induction of differentiation evident from LC3 maturation. Formation of Beclin1, Vps34, and PIK3R4 ternary complex at the phagophore assembly site that is typically known to induce autophagy was also enhanced during differentiation. Degradation of the p62/SQSTM1 cargo protein and its colocalization with LC3, a mature autophagosome marker, was most prevalent in the trophoblast giant cells (TGCs) and negligible in other trophoblast cells at day 6 of differentiation. Furthermore, disruption of autophagy by impairing lysosomal fusion in TS cells before induction of differentiation led to a decrease in the giant cell and spongiotrophoblast cell markers Prl3d1, Prl2c2, Prl4a1, and Tpbpα upon differentiation. In addition, inhibition of autophagy was associated with a decrease in nuclear size of TGCs. Taken together, these data highlight that autophagy is a necessary prelude in commitment of trophoblast differentiation from the multipotent TS cells probably by regulating protein turnover at the onset of differentiation.

Regulation of Transcription by Circular RNAs.

Circular RNAs (circRNAs) are a class of noncoding RNA that are present in wide variety of cells in various tissue types across species. They are non-polyadenylated, single-stranded, covalently closed RNAs. CircRNAs are more stable than other RNAs due to lack of 5' or 3' end leading to resistance to exonuclease digestion. The length of circRNAs varies from 1 to 5 exons with retention of introns in mature circRNAs with ~25% frequency. They are primarily found in the cytosol within the cell although the mechanism of their nuclear export remains elusive. However, there is a subpopulation of circRNAs that remain in the nucleus and regulate RNA-Pol-II-mediated transcription. Bioinformatic approaches mining RNA sequencing data enabled genome-wide identification of circRNAs. In mammalian genome over 20% of the expressed genes in cells and tissues can produce these transcripts. Owing to their abundance, stability, and diverse expression profile, circRNAs likely play a pivotal role in regulatory pathways controlling lineage determination, cell differentiation, and function of various cell types. Yet, the impact of circRNA-mediated regulation on various cell transcriptome remains largely unknown. In this chapter, we will review the regulatory effects of circRNAs in the transcription of their own or other genes. Also, we will discuss the association of circRNAs with miRNAs and RNA-binding proteins (RBPs), with special reference to Drosophila circMbl and their role as an "mRNA trap," which might play a role in its regulatory potential transcriptionally or posttranscriptionally.