Potential AhR-independent mechanisms of 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibition of human glioblastoma A172 cells migration.

The toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is generally believed to be mediated by aryl hydrocarbon receptor (AhR), but some evidence suggests that the effects of TCDD can also be produced through AhR-independent mechanisms. In previous experiments, we found that mainly AhR-dependent mechanism was involved in the migration inhibition of glioblastoma U87 cells by TCDD. Due to the heterogeneity of glioblastomas, not all tumor cells have significant AhR expression. The effects and mechanisms of TCDD on the migration of glioblastomas with low AhR expression are still unclear. We employed a glioblastoma cell line A172 with low AhR expression as a model, using wound healing and Transwell® assay to detect the effect of TCDD on cell migration. We found that TCDD can inhibit the migration of A172 cells without activating AhR signaling pathway. Further, after being pre-treated with AhR antagonist CH223191, the inhibition of TCDD on A172 cells migration was not changed, indicating that the effect of TCDD on A172 cells is not dependent on AhR activation. By transcriptome sequencing analysis, we propose dysregulation of the expression of certain migration-related genes, such as IL6, IL1B, CXCL8, FOS, SYK, and PTGS2 involved in cytokines, MAPK, NF-κB, and IL-17 signaling pathways, as potential AhR-independent mechanisms that mediate the inhibition of TCDD migration in A172 cells.

Effects of Habitual Dietary Change on the Gut Microbiota and Health of Silkworms.

Diet plays a crucial role in shaping the gut microbiota and overall health of animals. Traditionally, silkworms are fed fresh mulberry leaves, and artificial diets do not support good health. The aim of this study was to explore the relationship between the dietary transition from artificial diets to mulberry leaves and the effects on the gut microbiota and physiological changes in silkworms as a model organism. With the transition from artificial diets to mulberry leaves, the diversity of the silkworm gut microbiota increased, and the proportion of Enterococcus and Weissella, the dominant gut bacterial species in silkworms reared on artificial diets, decreased, whereas the abundance of Achromobacter and Rhodococcus increased. Dietary transition at different times, including the third or fifth instar larval stages, resulted in significant differences in the growth and development, immune resistance, and silk production capacity of silkworms. These changes might have been associated with the rapid adaptation of the intestinal microbiota of silkworms to dietary transition. This study preliminarily established a dietary transition-gut microbial model in silkworms based on the conversion from artificial diets to mulberry leaves, thus providing an important reference for future studies on the mechanisms through which habitual dietary changes affect host physiology through the gut microbiome.

The LIM Domain Protein BmFHL2 Inhibits Egg Production in Female Silkworm, Bombyx mori.

The female Bombyx mori accumulates a large amount of egg proteins, mainly Vg and 30K, during egg formation to provide nutrition for embryo development. The synthesis and transport of Vg have been extensively studied, particularly the regulation of Vg transcription induced by 20E; however, the mechanism of 30K protein synthesis is poorly studied. As a model organism of the order Lepidoptera, B. mori has high reproduction potential. In the present study, we found that the FHL2 homologous gene (BmFhl2) in B. mori is involved in inhibiting female egg formation by influencing the synthesis of 30K protein. Interference of BmFhl2 expression in silkworm females increased 30K protein synthesis, accelerated ovarian development, and significantly increased the number of eggs produced and laid; however, the 20E pathway was inhibited. The transcription levels of Vg and 30Kc19 were significantly downregulated following BmFhl2 overexpression in the silkworm ovarian cell line BmN. The Co-IP assay showed that the potential binding protein of BmFHL2 included three types of 30K proteins (30Kc12, 30Kc19, and 30Kc21). These results indicate that BmFHL2 participates in egg formation by affecting 30K protein in female B. mori.

An insight into sex-specific neurotoxicity and molecular mechanisms of DEHP: A critical review.

Di-2-Ethylhexyl Phthalate (DEHP) is often used as an additive in polyvinyl chloride (PVC) to give plastics flexibility, which makes DEHP widely used in food packaging, daily necessities, medical equipment, and other products. However, due to the unstable combination of DEHP and polymer, it will migrate to the environment in the materials and eventually contact the human body. It has been recorded that low-dose DEHP will increase neurotoxicity in the nervous system, and the human health effects of DEHP have been paid attention to because of the extensive exposure to DEHP and its high absorption during brain development. In this study, we review the evidence that DEHP exposure is associated with neurodevelopmental abnormalities and neurological diseases based on human epidemiological and animal behavioral studies. Besides, we also summarized the oxidative damage, apoptosis, and signal transduction disorder related to neurobehavioral abnormalities and nerve injury, and described the potential mechanisms of neurotoxicity caused by DEHP. Overall, we found exposure to DEHP during the critical developmental period will increase the risk of neurobehavioral abnormalities, depression, and autism spectrum disorders. This effect is sex-specific and will continue to adulthood and even have an intergenerational effect. However, the research results on the sex-dependence of DEHP neurotoxicity are inconsistent, and there is a lack of systematic mechanisms research as theoretical support. Future investigations need to be carried out in a large-scale population and model organisms to produce more consistent and convincing results. And we emphasize the importance of mechanism research, which can enhance the understanding of the environmental and human health risks of DEHP exposure.

Emodin inhibits U87 glioblastoma cells migration by activating aryl hydrocarbon receptor (AhR) signaling pathway.

Aryl hydrocarbon receptor (AhR) is a ligand-activated receptor to mediates the biological reactions of many environmental and natural compounds, which is highly expressed in glioblastoma. Although it has been reported that AhR agonist emodin can suppress some kinds of tumors, its inhibitory effect on glioblastoma migration and its relationship with AhR remain unclear. Based on the complexity of tumor pathogenesis and the tissue specificity of AhR, we hope can further understand the effect of emodin on glioblastoma and explore its mechanism. We found that the inhibitory effect of emodin on the migration of U87 glioblastoma cells increased with time, and the cell migration ability was inhibited by about 25% after 36 h exposure. In this process, emodin promoted the expression of the tumor suppressor IL24 by activating the AhR signaling pathway. Reducing the expression of AhR or IL24 by interfering RNA could block or relieve the inhibitory effect of emodin on the U87 cells migration, which indicates the inhibition of emodin on the migration of glioblastoma is mediated by the AhR-IL24 axis. Our data proved the AhR-IL24 signal axis is an important pathway for emodin to inhibit the migration of glioblastoma, and the AhR signaling pathway can be used as a key target to research the regulation effect and its mechanism of compounds on glioblastoma migration.

Rutaecarpine Inhibits U87 Glioblastoma Cell Migration by Activating the Aryl Hydrocarbon Receptor Signaling Pathway.

Glioblastoma is the most frequent and aggressive primary astrocytoma in adults. The high migration ability of the tumor cells is an important reason for the high recurrence rate and poor prognosis of glioblastoma. Recently, emerging evidence has shown that the migration ability of glioblastoma cells was inhibited upon the activation of aryl hydrocarbon receptor (AhR), suggesting potential anti-tumor effects of AhR agonists. Rutaecarpine is a natural compound with potential tumor therapeutic effects which can possibly bind to AhR. However, its effect on the migration of glioblastoma is unclear. Therefore, we aim to explore the effects of rutaecarpine on the migration of human glioblastoma cells U87 and the involvement of the AhR signaling pathway. The results showed that: (i) compared with other structural related alkaloids, like evodiamine and dehydroevodiamine, rutaecarpine was a more potent AhR activator, and has a stronger inhibitory effect on the glioblastoma cell migration; (ii) rutaecarpine decreased the migration ability of U87 cells in an AhR-dependent manner; (iii) AhR mediated the expression of a tumor suppressor interleukin 24 (IL24) induced by rutaecarpine, and AhR-IL24 axis was involved in the anti-migratory effects of rutaecarpine on the glioblastoma. Besides IL24, other candidates AhR downstream genes both associated with cancer and migration were proposed to participate in the migration regulation of rutaecarpine by RNA-Seq and bioinformatic analysis. These data indicate that rutaecarpine is a naturally-derived AhR agonist that could inhibit the migration of U87 human glioblastoma cells mostly via the AhR-IL24 axis.

The nuclear receptor corepressor NCoR1 regulates hematopoiesis and leukemogenesis in vivo.

Enhanced understanding of normal and malignant hematopoiesis pathways should facilitate the development of effective clinical treatment strategies for hematopoietic malignancies. Nuclear receptor corepressor 1 (NCoR1) has been implicated in transcriptional repression and embryonic organ development, but its role in hematopoiesis is yet to be fully elucidated. Here, we showed that hematopoietic-specific loss of NCoR1 leads to expansion of the hematopoietic stem cell (HSC) pool due to aberrant cell cycle entry of long-term HSCs under steady-state conditions. Moreover, NCoR1-deficient HSCs exhibited normal self-renewal capacity but severely impaired lymphoid-differentiation potential in competitive hematopoietic-reconstitution assays. Transcriptome analysis further revealed that several hematopoiesis-associated genes are regulated by NCoR1. In addition, NCoR1 deficiency in hematopoietic cells delayed the course of leukemia and promoted leukemia cell differentiation in an MLL-AF9-induced mouse model. NCoR1 and its partner, histone deacetylase 3, can modulate histone acetylation and gene transcription through binding the promoter regions of myeloid-differentiation genes. Our collective results support the critical involvement of NCoR1 in normal and malignant hematopoiesis in vivo.

Ring finger protein 31-mediated atypical ubiquitination stabilizes forkhead box P3 and thereby stimulates regulatory T-cell function.

The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.

The chromatin remodeling subunit Baf200 promotes normal hematopoiesis and inhibits leukemogenesis.

BACKGROUND: Adenosine triphosphate (ATP)-dependent chromatin remodeling SWI/SNF-like BAF and PBAF complexes have been implicated in the regulation of stem cell function and cancers. Several subunits of BAF or PBAF, including BRG1, BAF53a, BAF45a, BAF180, and BAF250a, are known to be involved in hematopoiesis. Baf200, a subunit of PBAF complex, plays a pivotal role in heart morphogenesis and coronary artery angiogenesis. However, little is known on the importance of Baf200 in normal and malignant hematopoiesis. METHODS: Utilizing Tie2-Cre-, Vav-iCre-, and Mx1-Cre-mediated Baf200 gene deletion combined with fetal liver/bone marrow transplantation, we investigated the function of Baf200 in fetal and adult hematopoiesis. In addition, a mouse model of MLL-AF9-driven leukemogenesis was used to study the role of Baf200 in malignant hematopoiesis. We also explored the potential mechanism by using RNA-seq, RT-qPCR, cell cycle, and apoptosis assays. RESULTS: Tie2-Cre-mediated loss of Baf200 causes perinatal death due to defective erythropoiesis and impaired hematopoietic stem cell expansion in the fetal liver. Vav-iCre-mediated loss of Baf200 causes only mild anemia and enhanced extramedullary hematopoiesis. Fetal liver hematopoietic stem cells from Tie2-Cre + , Baf200 f/f or Vav-iCre + , Baf200 f/f embryos and bone marrow hematopoietic stem cells from Vav-iCre + , Baf200 f/f mice exhibited impaired long-term reconstitution potential in vivo. A cell-autonomous requirement of Baf200 for hematopoietic stem cell function was confirmed utilizing the interferon-inducible Mx1-Cre mouse strain. Transcriptomes analysis revealed that expression of several erythropoiesis- and hematopoiesis-associated genes were regulated by Baf200. In addition, loss of Baf200 in a mouse model of MLL-AF9-driven leukemogenesis accelerates the tumor burden and shortens the host survival. CONCLUSION: Our current studies uncover critical roles of Baf200 in both normal and malignant hematopoiesis and provide a potential therapeutic target for suppressing the progression of leukemia without interfering with normal hematopoiesis.

NCoR1 restrains thymic negative selection by repressing Bim expression to spare thymocytes undergoing positive selection.

Thymocytes must pass both positive and negative selections to become mature T cells. Negative selection purges thymocytes whose T-cell receptors (TCR) exhibit high affinity to self-peptide MHC complexes (self pMHC) to avoid autoimmune diseases, while positive selection ensures the survival and maturation of thymocytes whose TCRs display intermediate affinity to self pMHCs for effective immunity, but whether transcriptional regulation helps conserve positively selected thymocytes from being purged by negative selection remains unclear. Here we show that the specific deletion of nuclear receptor co-repressor 1 (NCoR1) in T cells causes excessive negative selection to reduce mature thymocyte numbers. Mechanistically, NCoR1 protects positively selected thymocytes from negative selection by suppressing Bim expression. Our study demonstrates a critical function of NCoR1 in coordinated positive and negative selections in the thymus.Thymocytes are screened by two processes, termed positive and negative selections, which are permissive only for immature thymocytes with intermediate avidity to the selecting ligands. Here the authors show that the nuclear receptor NCoR1 suppresses Bim1 to inhibit negative selection and promote thymocyte survival.

USP21 prevents the generation of T-helper-1-like Treg cells.

FOXP3+ Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) myeloid cells curtail CD4 T cell response by inducible nitric oxide synthase in murine hepatitis.

Myeloid-derived suppressor cells (MDSCs) play an important role in maintaining immune tolerance in response to tumors and inflammatory diseases. Several liver MDSCs have been described in hepatitis in humans and mouse models. Although all the murine MDSCs are CD11b(+)Gr-1(+), their true phenotype and mechanism of suppression remain elusive. This study revealed that SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) monocytic cells but not the other liver-infiltrating, CD11b(+)Gr-1(+) subsets could suppress CD4 T cell responses. Their suppressive activity was remarkably effective even at a ratio of 1:50 when co-cultured with CD4 T cells. Mechanistically, the suppression was dependent on nitric oxide production by inducible nitric oxide synthase (iNOS). Furthermore, the suppressive function by these liver MDSCs was found to require direct contact with activated CD4 T cells. Adoptive transfer experiments demonstrate that these liver MDSCs can dramatically ameliorate concanavalin A (Con A)-induced fulminant hepatitis in mice. Finally, MDSC-mediated suppression in vivo was dependent on iNOS expression. Altogether, SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) cells represent authentic MDSCs in the inflammatory liver and may function to minimize collateral damage caused by an overzealous CD4 T cell response following hepatitis infection.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.