MAP3K4 signaling regulates HDAC6 and TRAF4 coexpression and stabilization in trophoblast stem cells.

The morphogenesis of the hemochorial placenta is dependent upon the precise expansion and differentiation of trophoblast stem (TS) cells. SATB homeobox 1 (SATB1) and SATB2 are related proteins that have been implicated as regulators of some stem cell populations. SATB1 is highly expressed in TS cells, which prompted an investigation of SATB1 and the related SATB2 as regulators of TS cells. SATB1 and SATB2 were highly expressed in rat TS cells maintained in the stem state and rapidly declined following induction of differentiation. SATB proteins were also present within the rat placenta during early stages of its morphogenesis and disappeared as gestation advanced. Silencing Satb1 or Satb2 expression decreased TS cell self-renewal and increased differentiation, whereas ectopic expression of SATB proteins promoted TS cell expansion and blunted differentiation. Eomes, a key transcriptional regulator of TS cells, was identified as a target for SATB proteins. SATB knockdown decreased Eomes transcript levels and promoter activity, whereas SATB ectopic expression increased Eomes transcript levels and promoter activity. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation analyses demonstrated that SATB proteins physically associate with a regulatory site within the Eomes promoter. We conclude that SATB proteins promote TS cell renewal and inhibit differentiation. These actions are mediated in part by regulating the expression of the TS cell stem-associated transcription factor, EOMES.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase–inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase–inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Mouse androgenetic and parthenogenetic embryos, which have two sets of paternal and maternal genomes, respectively, are lethal until Day 9.5 of pregnancy, because the growth of the extraembryonic tissues including placenta is inadequate. These results suggest that both parental genomes are involved in placental development. However, the reason for placental deficiency in androgenetic and parthenogenetic embryos has been unclear. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage, have been used to elucidate the mechanism of differentiation and gene function in the development of the placenta. In this study, to characterize trophoblast lineage in androgenetic and parthenogenetic embryos, we examined TS cells from these embryos, and assessed their ability to differentiate into trophoblast lineage. The ovulated MII oocytes from B6D2F1 (C57BL/6 � DBA2) female mice were used for the uniparental embryo production. In order to produce parthenogenetic embryos, the oocytes were activated in SrCl2 and cytochalasin B. The androgenetic embryos were produced by in vitro fertilization using enucleated oocytes. These uniparental embryos were cultured for 3.5–4.5 days to develop into blastocysts. TS-like cell lines were established from these blastocysts, as described previously (Tanaka et al. 1998 Science 282, 2072–2075). TS-like cells were cultured without feeder cells in conditioned medium containing FGF4 and heparin to maintain the stem cell conditions. The expression of 5 TS cell maker genes (Eomes, CdX2, Fgfr2, Ap2a, and Errb) in TS-like cells was analyzed by RT-PCR and northern blotting. In addition, the localization of CDX2 was detected using immunostaining. To cause their differentiation into giant cells, TS-like cells were cultured for 6 days in TS medium without FGF4 and heparin. The giant cells were detected by the expression of 2 giant cell marker genes, Hand1 and Pl-1. TS cells from fertilized embryos between B6D2F1 male and female mice were also established as controls. Five TS cell marker genes were expressed both in androgenetic TS-like cells (ATS) and in parthenogenetic ones (PTS). The CDX2 gene was localized in the nucleus in both ATS and PTS; however, the number of CDX2-positive cells decreased in PTS. After 6 days of differentiation, Hand1 and Pl-1 were expressed and giant cells were detected in differentiated derivatives in ATS. On the other hand, giant cells were not detected in differentiated derivatives in PTS. These results suggest that parental genomes may regulate the gene expression independently to differentiate into trophoblast lineage. In conclusion, TS-like cells from uniparental embryos are useful tools to aid the understanding of the function of the parental genomes during placental development.

Many viruses are detrimental to pregnancy and negatively affect fetal growth and development. What is not well understood is how virus-induced inflammation impacts fetal-placental growth and developmental trajectories, particularly when inflammation occurs in early pregnancy during nascent placental and embryo development. To address this issue, we simulated a systemic virus exposure in early pregnant rats (gestational day 8.5) by administering the viral dsRNA mimic polyinosinic:polycytidylic acid (PolyI:C). Maternal exposure to PolyI:C induced a potent antiviral response and hypoxia in the early pregnant uterus, containing the primordial placenta and embryo. Maternal PolyI:C exposure was associated with decreased expression of the maternally imprinted genes Mest, Sfrp2, and Dlk1, which encode proteins critical for placental growth. Exposure of pregnant dams to PolyI:C during early pregnancy reduced fetal growth trajectories throughout gestation, concomitant with smaller placentas, and altered placental structure at midgestation. No detectable changes in placental hemodynamics were observed, as determined by ultrasound biomicroscopy. An antiviral response was not evident in rat trophoblast stem (TS) cells following exposure to PolyI:C, or to certain PolyI:C-induced cytokines including IL-6. However, TS cells expressed high levels of type I IFNR subunits (Ifnar1 and Ifnar2) and responded to IFN-⍺ by increasing expression of IFN-stimulated genes and decreasing expression of genes associated with the TS stem state, including Mest IFN-⍺ also impaired the differentiation capacity of TS cells. These results suggest that an antiviral inflammatory response in the conceptus during early pregnancy impacts TS cell developmental potential and causes latent placental development and reduced fetal growth.

Trophoblasts are the first cell type to be specified during embryogenesis, and they are essential for placental morphogenesis and function. Trophoblast stem (TS) cells are the progenitor cells for all trophoblast lineages; control of TS cell differentiation into distinct trophoblast subtypes is not well understood. Mice lacking the transcription factor OVO-like 2 (OVOL2) fail to produce a functioning placenta, and die around embryonic day 10.5, suggesting that OVOL2 may be critical for trophoblast development. Therefore, our objective was to determine the role of OVOL2 in mouse TS cell fate. We found that OVOL2 was highly expressed in mouse placenta and differentiating TS cells. Placentas and TS cells lacking OVOL2 showed poor trophoblast differentiation potential, including increased expression of stem-state associated genes (Eomes, Esrrb, Id2) and decreased levels of differentiation-associated transcripts (Gcm1, Tpbpa, Prl3b1, Syna). Ectopic OVOL2 expression in TS cells elicited precocious differentiation. OVOL2 bound proximate to the gene encoding inhibitor of differentiation 2 (ID2), a dominant negative helix-loop-helix protein, and directly repressed its activity. Overexpression of ID2 was sufficient to reinforce the TS cell stem state. Our findings reveal a critical role of OVOL2 as a regulator of TS cell differentiation and placental development, in-part by coordinating repression of ID2.

Little is known about the combined effect of fluoride (F)and arsenic (As) on bone metabolism. This study aims to explore the effect of co-exposure to F and As on the expressions of TNF receptor-associated factor 6 (TRAF-6), nuclear factor-kappa B (NF-κB), and the related factors in cell and animal experiments. With the rats exposed to different doses of F, As, and combined F-As, we found that F exposure doses were positively correlated with the protein expression of receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), TRAF-6, NF-κB, and nuclear factor of activated T cells (NFAT-c1) (P < 0.001). As exposure doses were negatively correlated with RANK, TRAF-6, NF-κB, and NFAT-c1 (P < 0.001). The effect of F and As interaction on the protein expression of RANKL, TRAF-6, NF-κB, and NFAT-c1 was significant in bone tissue (P < 0.05). In the cellular experiment, F could promote the mRNA expression of RANK, TRAF-6, and NFAT-c1. A higher concentration of As could inhibit the mRNA expression of Tartrate-resistant acid phosphatase (TRAP), RANK, TRAF-6, and NFAT-c1. The effect of F and As interaction on the mRNA expression of TRAP, RANK, TRAF-6, and NFATc1 in osteoclasts was significant (P < 0.001). In conclusion, the expression of TRAF-6 and NF-κB pathway was affected by F and As co-exposure in osteogenic differentiation, and As could antagonize the promoting effect of F on the expression of TRAF-6, TRAP, RANKL, RANK, NF-κB, and NFAT-c1 in these exposure levels. These results could provide a scientific basis for understanding the interaction of F and As in bone formation.

The oncogene c-Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c-Myc is significantly up-regulated by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/- mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63-linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c-Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c-Myc. Second, the K63-linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c-Myc and promotes c-Myc protein acetylation, which thereby enhances c-Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70-kDa-interacting protein-mediated c-Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c-Myc signaling is also primed in hepatitis B virus-transgenic mice, unveiling a critical role for a mechanism in inflammation-cancer transition. In clinical specimens, TRAF6 positively correlates with c-Myc at both the mRNA and protein levels, and high TRAF6 and c-Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c-Myc to promote human hepatocarcinogenesis. Consistently, curbing c-Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c-Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c-Myc signaling, with potential implications for HCC therapy.

New Insights for Ets2 Function in Trophoblast using Lentivirus-Mediated Gene Knockdown in Trophoblast Stem Cells

Proper placentation is fundamental to successful pregnancy. Placenta arises from differentiation of trophoblast stem (TS) cells during development. Despite being recognized as the counterpart of ES cells in placental development, the role of regulatory miRNAs in TS cell differentiation remains inadequately explored. Here, we have identified complete repertoire of microRNAs present in mouse trophoblast cells in proliferative and differentiated state. We demonstrated that two miRNA clusters, -290 and -322, displayed reciprocal expression during trophoblast differentiation. Loss of miR-290 cluster members or gain in miR-322 cluster members led to differentiation of TS cells. The trophoblast stemness factor, CDX2, transactivated the miR-290 cluster and Cyclin D1 MiR-290 cluster members repressed cell cycle repressors, P21, P27, WEE1, RBL2, and E2F7, in TS cells. MiR-322 cluster members repressed the cell cycle activators, CYCLIN D1, CYCLIN E1, CDC25B, and CDX2, to induce differentiation. Taken together, our findings highlight the importance of posttranscriptional regulation by conserved miRNA clusters that form a regulatory network with CDX2, cell cycle activators, and repressors in equipoising TS cell self-renewal and differentiation.

How does activation of aryl hydrocarbon receptor (AHR) signaling affect human trophoblast cell development and differentiation? AHR activation alters gene expression without impairing the ability of trophoblast cells to maintain a stem cell state or differentiate into essential cell types, such as extravillous trophoblast (EVT) cells or syncytiotrophoblast (ST), while promoting the production of 2-methoxy estradiol (2ME), which may impact placental development. The placenta serves both as a nutrient delivery system and a protective barrier against environmental toxins. AHR signaling is known to mediate cellular responses to environmental pollutants, potentially affecting trophoblast cell function, but the specific impacts of AHR activation on these cells were not fully understood. This study utilized an in vitro model of human trophoblast stem (TS) cells to investigate the downstream effects of AHR activation. The study focused on both undifferentiated TS cells and cells undergoing differentiation. Human TS cells were used as a model system. Researchers examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in TS cells maintained in their stem state and in TS cells induced to differentiate into EVT cells or ST. The study assessed changes in gene expression, particularly focusing on CYP1A1 and CYP1B1, as well as the production of 2ME. AHR activation stimulated the expression of CYP1A1 and CYP1B1, key genes associated with AHR signaling, in both undifferentiated and differentiating trophoblast cells. While AHR activation did not impact the ability of the cell to remain in a stem state or differentiate, it increased the production of 2ME, which may influence placentation. These effects were dependent on AHR signaling. n/a. This study was conducted in vitro, which may not fully replicate in vivo conditions. Further research is needed to confirm whether these findings apply to placental development in humans. The results suggest that AHR signaling activated by environmental pollutants could have a significant impact on placental development through mechanisms involving AHR activation. These findings may have broader implications for understanding how environmental factors affect fetal development. This work was funded by the National Institutes of Health: ES028957, HD020676, ES029280, HD105734, HD112559, and the Sosland Foundation. The authors declare no conflicts of interest.

STUDY QUESTIONHow does activation of AHR signaling affect human trophoblast cell development and differentiation?SUMMARY ANSWERAHR activation leads to altered gene expression but does not hinder the ability of trophoblast cells to remain in a stem cell state or differentiate into essential cell types, such as extravillous trophoblast cells (EVT) or syncytiotrophoblast (ST). It also promotes the production of 2 methoxy estradiol (2ME), a compound that could influence placental development.WHAT IS KNOWN ALREADYThe placenta serves both as a nutrient delivery system and a protective barrier against environmental toxins. AHR signaling is known to mediate cellular responses to environmental pollutants, potentially affecting trophoblast cell functions, but the specific impacts of AHR activation on these cells were not fully understood.STUDY DESIGN, SIZE, DURATIONThis study utilized an in vitro model of human trophoblast stem (TS) cells to investigate the downstream effects of AHR activation. The study focused on both undifferentiated TS cells and cells undergoing differentiation.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman trophoblast stem (TS) cells were used as the model system. Researchers examined the effects of TCDD exposure in both TS cells maintained in their stem state and those induced to differentiate into EVT or ST. The study assessed changes in gene expression, particularly focusing on CYP1A1 and CYP1B1, as well as the production of 2ME.MAIN RESULTS AND THE ROLE OF CHANCEAHR activation stimulated the expression of CYP1A1 and CYP1B1, key genes associated with AHR signaling, in both undifferentiated and differentiating trophoblast cells. While AHR activation did not impact the cells ability to remain in a stem state or differentiate, it increased the production of 2ME, which may influence placental function. These effects were dependent on AHR signaling.LIMITATIONS, REASONS FOR CAUTIONThis study was conducted in vitro, which may not fully replicate human conditions. Further research is needed to confirm whether these findings apply to actual placental development in humans.WIDER IMPLICATIONS OF THE FINDINGSThe results suggest that AHR signaling activated by environmental pollutants could have a subtle but significant impact on placental development through mechanisms involving AHR activation. These findings may have broader implications for understanding how environmental factors affect fetal development.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the National Institutes of Health: ES028957, HD020676, ES029280, HD105734 and the Sosland Foundation. The authors declare no conflicts of interest.