Epidermal growth factor represses differentiation of mouse trophoblast stem cells into spongiotrophoblast cells via epidermal growth factor receptor.

The mouse placenta is composed of three different trophoblast layers that are occupied by particular trophoblast subtypes to maintain placental function and pregnancy. Accurate control of trophoblast differentiation is required for proper placental function; however, the molecular mechanisms underlying cell fate decisions in trophoblast stem cells remain poorly understood. Epidermal growth factor (EGF) signaling is involved in multiple biological processes including cell survival, proliferation, and differentiation. The effect of EGF on trophoblast function has been reported in various species; however, the role of EGF signaling in mouse trophoblast specification remains unclear. In this study, we aimed to elucidate the role of EGF signaling in mouse trophoblast differentiation using mouse trophoblast stem cells (mTSCs) in an in vitro culture system. EGF stimulation at the early stage of differentiation repressed mTSC differentiation into spongiotrophoblast cells (SpT). Gene deletion and inhibitor experiments showed that the effect of EGF exposure went through epidermal growth factor receptor (Egfr) activity in mTSCs. EGF stimuli induced acute downstream activation of MAPK/ERK, PI3K/AKT, and JNK pathways, and inhibition of the MAPK/ERK pathway, but not others, alleviated EGF-mediated repression of SpT differentiation. Moreover, expression of Mash2, a master regulator of SpT differentiation, was repressed by EGF stimulation, and MAPK/ERK inhibition counteracted this repression. The Mash2 overexpression recovered SpT marker expression, indicating that the decrease in Mash2 expression was due to abnormal SpT differentiation in EGF-treated mTSCs. Our findings suggest that the EGF-Egfr-MAPK/ERK-Mash2 axis is a core regulatory mechanism for the EGF-mediated repression of SpT differentiation.

N-Oleoyldopamine promotes the differentiation of mouse trophoblast stem cells into parietal trophoblast giant cells.

The placenta plays various roles in a healthy pregnancy, and abnormalities in the placenta result in adverse outcomes. Adequate differentiation of trophoblast subtypes is necessary for placental function, but the molecular mechanisms that determine trophoblast cell fate remain unclear. Here, we screened small molecular compound (SMC) libraries (1904 SMCs) to identify particular SMCs which regulate trophoblast differentiation in mouse trophoblast stem cells (mTSCs) to understand the molecular mechanisms underlying cell fate decision in trophoblast cells. The two-step screening revealed a novel effect of N-oleoyldopamine (OLDA), an endogenic vanilloid, to promote differentiation into parietal trophoblast giant cells (P-TGCs) and repress them into spongiotrophoblast cells in mTSCs. Analyses by gene deletion and inhibitor treatments indicated that transient receptor potential cation channel subfamily V member 3 (Trpv3), one of the candidates for targeting by OLDA, was involved in maintaining stem status and P-TGC differentiation in mTSCs. Finally, transcriptome analysis revealed that Fosl1, a key regulatory factor in differentiation into P-TGCs, was upregulated by OLDA treatment, suggesting that OLDA promoted the differentiation of mTSCs into P-TGCs via regulation of Fosl1 expression.

Linker histone variant H1T functions as a chromatin de-condenser on genic regions.

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. There are 11 variants in histone H1, and each variant has unique functions. Our previous study demonstrates that one of the H1 variants, H1T is mainly localized in the nucleolus and targets the rDNA repeat region. Moreover, H1T condenses the chromatin structures on rDNA to repress pre-rRNA expression. Although H1T is partially localized in the nucleoplasm area, the functions of H1T in the non-repeat genic region are unclear. In this study, we aimed to identify the target loci and the role of H1T in the genic region. Chromatin immunoprecipitation sequencing analysis showed that H1T is localized around the transcriptional start site and the chromatin structures of the region were relaxed. H1T knockdown and overexpression experiments revealed that H1T induced chromatin de-condensation and was associated with the increased expression of target genes. Moreover, we observed H1T co-localization with transcriptional factor SPZ1 on the genic region. Collectively, H1T has opposing roles in the genic region and in rDNA repeats; H1T functions to facilitate chromatin relaxation linked gene activation.

Extracellular glucose levels in cultures of undifferentiated mouse trophoblast stem cells affect gene expression during subsequent differentiation with replicable cell line-dependent variation.

Mouse trophoblast stem cells (TSCs) have been established and maintained using hyperglycemic conditions (11 mM glucose) for no apparent good reason. Because glucose metabolites are used as resources for cellular energy production, biosynthesis, and epigenetic modifications, differences in extracellular glucose levels may widely affect cellular function. Since the hyperglycemic culture conditions used for TSC culture have not been fully validated, the effect of extracellular glucose levels on the properties of TSCs remains unclear. To address this issue, we investigated the gene expression of stemness-related transcription factors in TSCs cultured in the undifferentiated state under various glucose concentrations. We also examined the expression of trophoblast subtype markers during differentiation, after returning the glucose concentration to the conventional culture concentration (11 mM). As a result, it appeared that the extracellular glucose conditions in the stem state not only affected the gene expression of stemness-related transcription factors before differentiation but also affected the expression of marker genes after differentiation, with some line-to-line variation. In the TS4 cell line, which showed the largest glucose concentration-dependent fluctuations in gene expression among all the lines examined, low glucose (1 mM glucose, LG) augmented H3K27me3 levels. An Ezh2 inhibitor prevented these LG-induced changes in gene expression, suggesting the possible involvement of H3K27me3 in the changes in gene expression seen in LG. These results collectively indicate that the response of the TSCs to the change in the extracellular glucose concentration is cell line-dependent and a part of which may be epigenetically memorized.

A SOX9-B7x axis safeguards dedifferentiated tumor cells from immune surveillance to drive breast cancer progression.

How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ T cell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.

A case of laparoscopic distal gastrectomy and median arcuate ligament release for a gastric cancer patient with median arcuate ligament syndrome.

BACKGROUND: The median arcuate ligament syndrome (MALS) is a disease in which the celiac artery is compressed by the arcuate ligament and causes stenosis. If abdominal pain or an aneurysm is observed in the head of the pancreas, it is necessary to release the arcuate ligament, and recently laparoscopic surgery has been reported. However, the indication for treatment in asymptomatic cases is unknown. The treatment for asymptomatic MALS in patients with gastric cancer who are indicated for surgery is also novel. CASE PRESENTATION: A 70-year-old female was found with early gastric cancer in the middle body of the stomach. An enhanced CT scan showed no metastasis, but a gallstone and stenosis of the celiac artery due to the MALS were found. The patient underwent releasing median arcuate ligament after lymph node dissection. A median arcuate ligament was located on the ventral side of the left gastric artery stump, and the celiac artery was exposed when cutting it off. The operation time was 4 h and 59 min, and the bleeding was 6 ml. It took about 5 min to dissect the medial arcuate ligament. The postoperative course was satisfactory, and the patient was discharged 7 days after the operation. CT scan and 3-D CT angiography were performed about 2 months after the operation, and the findings revealed that the celiac artery's stenosis resolved. CONCLUSION: The patient underwent laparoscopic gastrectomy and simultaneously the median arcuate ligament release under an excellent visual field. Therefore, median arcuate ligament release may be considered if MALS is found in a gastrectomy case.

Kynurenine, 3-OH-kynurenine, and anthranilate are nutrient metabolites that alter H3K4 trimethylation and H2AS40 O-GlcNAcylation at hypothalamus-related loci.

Epigenetic mechanisms can establish and maintain mitotically stable patterns of gene expression while retaining the DNA sequence. These mechanisms can be affected by environmental factors such as nutrients. The importance of intracellular dosages of nutrient metabolites such as acetyl coenzyme A and S-adenosylmethionine, which are utilized as donors for post-translational modifications, is well-known in epigenetic regulation; however, the significance of indirect metabolites in epigenetic regulation is not clear. In this study, we screened for metabolites that function as epigenetic modulators. Because the expression of genes related to hypothalamic function is reportedly affected by nutritional conditions, we used a neural cell culture system and evaluated hypothalamic-linked loci. We supplemented the culture medium with 129 metabolites separately during induction of human-iPS-derived neural cells and used high-throughput ChIP-qPCR to determine the epigenetic status at 37 hypothalamus-linked loci. We found three metabolites (kynurenine, 3-OH-kynurenine, and anthranilate) from tryptophan pathways that increased H3K4 trimethylation and H2AS40 O-GlcNAcylation, resulting in upregulated gene expression at most loci, except those encoding pan-neural markers. Dietary supplementation of these three metabolites and the resulting epigenetic modification were important for stability in gene expression. In conclusion, our findings provide a better understanding of how nutrients play a role in epigenetic mechanisms.

Effect of Heat Accumulation on Femtosecond Laser Reductive Sintering of Mixed CuO/NiO Nanoparticles.

Direct laser-writing techniques have attracted attention for their use in two- and three-dimensional printing technologies. In this article, we report on a micropatterning process that uses femtosecond laser reductive sintering of mixed CuO/NiO nanoparticles. The writing speed, laser fluence, and incident total energy were varied to investigate the influence of heat accumulation on the micropatterns formed by these materials. Heat accumulation and the thermal history of the laser irradiation process significantly affected the material composition and the thermoelectric properties of the fabricated micropatterns. Short laser irradiation durations and high laser fluences decrease the amount of metal oxide in the micropatterns. Selective fabrication of p-type and n-type thermoelectric micropatterns was demonstrated to be possible with control of the reduction and reoxidization reactions through the control of writing speed and total irradiation energy.