ABSTRACT
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.
Subject(s)
Interleukin-1beta , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , NF-kappa B , Nuclear Proteins , Signal Transduction , Trans-Activators , Trophoblasts , Animals , Trophoblasts/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Trans-Activators/metabolism , Trans-Activators/genetics , Rats , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Signal Transduction/physiology , NF-kappa B/metabolism , Female , Myocytes, Smooth Muscle/metabolism , Interleukin-1beta/metabolism , Pregnancy , Coculture Techniques , Rats, Sprague-Dawley , Cells, Cultured , Cell Plasticity/physiology , CalponinsABSTRACT
Enrichment of angiomotin (AMOT) in the ectoplacental cone of E7.5 murine placenta prompted our investigation on the role of AMOT in trophoblast differentiation. We show here that AMOT levels increased in mouse placenta during gestation and also upon induction of differentiation in trophoblast stem cell ex vivo. Proteomic data unravelling AMOT-interactome in trophoblast cells indicated a majority of AMOT interactors to be involved in protein translation. In-depth analysis of AMOT-interactome led to identification of eukaryotic translation initiation factor 4A (eIF4A) as the most plausible AMOT interactor. Loss of function of AMOT enhanced, whereas, gain in function resulted in decline of global protein synthesis in trophoblast cells. Bioinformatics analysis evaluating the potential energy of AMOT-eIF4A binding suggested a strong AMOT-eIF4A interaction using a distinct groove encompassing amino acid residue positions 238 to 255 of AMOT. Co-immunoprecipitation of AMOT with eIF4A reaffirmed AMOT-eIF4A association in trophoblast cells. Deletion of 238 to 255 amino acids of AMOT resulted in abrogation of AMOT-eIF4A interaction. In addition, 238 to 255 amino acid deletion of AMOT was ineffective in eliciting AMOT's function in reducing global protein synthesis. Interestingly, AMOT-dependent sequestration of eIF4A dampened its loading to the m7 -GTP cap and hindered its interaction with eIF4G. Furthermore, enhanced AMOT expression in placenta was associated with intrauterine growth restriction in both rats and humans. These results not only highlight a hitherto unknown novel function of AMOT in trophoblast cells but also have broad biological implications as AMOT might be an inbuilt switch to check protein synthesis in developmentally indispensable trophoblast cells.
Subject(s)
Angiomotins/biosynthesis , Eukaryotic Initiation Factor-4A/biosynthesis , Protein Biosynthesis/physiology , Trophoblasts/metabolism , Angiomotins/chemistry , Angiomotins/genetics , Animals , Cells, Cultured , Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4A/genetics , Female , Hep G2 Cells , Humans , Mice , Placenta/cytology , Placenta/metabolism , Pregnancy , Protein Structure, Tertiary , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Trophoblast stem cells (TSCs), the precursors of trophoblast cells of placenta, possess the potential to differentiate into various trophoblastic subtypes in vitro. Establishment of extraembryonic trophoblastic lineage is preceded by the "outside versus inside" positional information in preimplantation embryos, critically synchronized by the Hippo components. Abundant expression of Hippo effector YAP in TSCs and differentiated cells with paucity of information on Hippo regulation of TSC proliferation/differentiation led us test the hypothesis that Hippo dynamics is one of the regulators of TSC proliferation/differentiation. METHODS: Blastocyst-derived murine TSCs were used. Dynamics of Hippo components were analyzed using immunofluorescence, western blotting, immunoprecipitation, qRT-PCR. Interaction studies were performed using full-length and deletion constructs. BrdU incorporation assay, flow cytometry-based polyploidy analysis and confocal microscopy were used to decipher the underlying mechanism. RESULTS: YAP translocates to the nucleus in TSCs and utilizes its WW2 domain to interact with the PPQY motif of the stemness factor, CDX2. YAP limits TSC proliferation with associated effect on CDX2 target CyclinD1. Trophoblast giant cells (TGC) differentiation is associated with cytoplasmic retention of YAP, heightened pYAPSer127, decrease in the level of the core Hippo component, LATS1, which thereby impedes LATS1-LIMK2 association. Decreased LATS1-LIMK2 complex formation in TGCs was associated with elevated pLIMK2Thr505 as well as its target pCOFILINSer3. Precocious overexpression of LATS1 during trophoblast differentiation decreased TGC marker, Prl2c2, diminished pLIMK2Thr505 and inactive COFILIN (pCOFILINSer3) while COFILIN-phosphatase, CHRONOPHIN remained unchanged. LATS1 overexpression inhibited trophoblast endoreduplication with smaller-sized TGC-nuclei, lower ploidy level and disintegrated actin filaments. Inhibition of LIMK2 activity recapitulated the effects of LATS1 overexpression in trophoblast cells. CONCLUSION: These results unveil a multilayered regulation of trophoblast self-renewal and differentiation by the Hippo components.
Subject(s)
Cell Self Renewal , Giant Cells , Protein Serine-Threonine Kinases , Trophoblasts , YAP-Signaling Proteins , Actin Depolymerizing Factors , Animals , Cell Differentiation , Cell Proliferation , Female , Giant Cells/cytology , Mice , Pregnancy , Protein Serine-Threonine Kinases/genetics , YAP-Signaling Proteins/geneticsABSTRACT
Murine trophoblast stem cells (TSCs) have shaped placental research by providing resources for investigating trophoblast subtype specialization. Trophoblast giant cells (TGCs) are large polyploid cells, which undergo repetitive rounds of DNA replication without intervening mitosis by a process called endoreduplication. Endocrine and paracrine functions of TGCs aid in maternal adaptations to pregnancy. Here, we describe a protocol for in vitro differentiation of murine TSCs to TGCs together with the genotypic as well as phenotypic characterization of the endoreduplicated TGCs. For complete details on the use and execution of this protocol, please refer to Basak and Ain (2022).