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Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Purpose:Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer. KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in bladder cancer. However, the molecular mechanisms of how KDM6A deficiency contributes to bladder cancer development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying KDM6A-mutated bladder cancer can help in designing new anticancer therapies.Experimental Design:We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the KDM6A mutation in muscle-invasive bladder cancer (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypic, pathologic, molecular, and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC.Results:We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased KDM6A expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient bladder cancer cell growth.Conclusions:Our findings provide insights into multistep carcinogenic processes of bladder cancer and suggest molecular targeted therapeutic approaches for patients with bladder cancer with KDM6A dysfunction.

Supplementary Information: Tables S1, S2, S3, S4 (gene set enrichment analyses); Figures S1, S2, S3, S4

Purpose: Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer. KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in bladder cancer. However, the molecular mechanisms of how KDM6A deficiency contributes to bladder cancer development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying KDM6A-mutated bladder cancer can help in designing new anticancer therapies. Experimental Design: We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the KDM6A mutation in muscle-invasive bladder cancer (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypic, pathologic, molecular, and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC. Results: We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased KDM6A expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient bladder cancer cell growth. Conclusions: Our findings provide insights into multistep carcinogenic processes of bladder cancer and suggest molecular targeted therapeutic approaches for patients with bladder cancer with KDM6A dysfunction.

Epigenetics is defined as heritable alterations in gene expression that are not caused by changes in DNA sequence. Such normal alterations are vital for appropriate differentiation and development. However, unfavorable epigenetic alterations are usually observed in human cancers and are closely linked to the generation of malignant phenotypes; they directly induce oncogenesis and cancer progression, and are indirectly involved in cancer through mutations that are frequently detected in malignancy-associated genes. Therefore, controlling epigenetic modifications is an interesting therapeutic approach for cancer. Indeed, many small molecules modulating epigenetic mechanisms such as DNA methylation and histone modifications have been reported to bring about promising anti- tumorigenic effects on some malignancies. In this chapter, we focus on epigenetics in cancer and describe small-molecule epigenetic modulators aimed at treating cancer.

Background Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotide+-dependent deacylases, which participates in modulation of cell cycle control, neurodegeneration, and tumorigenesis. SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated. Methods We assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit. Results Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy. Conclusions These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL. Electronic supplementary material The online version of this article (10.1186/s12885-018-4710-1) contains supplementary material, which is available to authorized users.

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignant T cell disease caused by human T cell leukemia virus-I (HTLV-1). Treatment outcomes for aggressive subtypes of ATL remain poor, with little improvement in overall survival since HTLV-1 was discovered. Therefore, new therapeutic strategies for ATL are required. STF-62247 induces autophagy and selectively kills renal cell carcinoma without apoptotic cell death. Here, we demonstrate that STF-62247 reduced cell viability and resulted in autophagosome accumulation and autophagy in leukemic cell lines (S1T, MT-2, and Jurkat). Interestingly, STF-62247 induced apoptosis in HTLV-1-infected cell lines (S1T and MT-2), as indicated by DNA fragmentation and caspase activation, but not in non-HTLV-1-infected Jurkat cells; a caspase inhibitor did not prevent this caspase-associated cell death. STF-62247 also increased nuclear endonuclease G levels. Furthermore, STF-62247 reduced cell viability and increased the number of apoptotic cells in peripheral blood mononuclear cells collected from patients with acute ATL, which has a poor prognosis. Therefore, STF-62247 may have novel therapeutic potential for ATL. This is the first evidence to demonstrate the cell growth-inhibitory effect of an autophagy inducer by caspase-dependent apoptosis and caspase-independent cell death via autophagy and endonuclease G in leukemic cells.

Germinal centers (GCs) in secondary lymphoid organs generate large numbers of apoptotic B cells that must be eliminated by phagocytes to prevent the development of autoimmune diseases. Although tingible body macrophages engulf apoptotic GC B cells, whether stromal cells are also involved in this process is unclear. In this study, we identified marginal reticular cells (MRCs) as novel nonprofessional phagocytes for the clearance of apoptotic GC B cells in the spleen. We used CD19eGFP (CD19creZ/EG) mice, which express enhanced GFP (eGFP) under the control of CD19cre expression, to track B cells in the GCs after immunization with NP-chicken γ globulin plus aluminum salt. We demonstrated that the MRC population, as determined by expression of podoplanin or Rankl, specifically showed an eGFP signal in the cytoplasm after immunization. These results suggest that MRCs contribute to the clearance of apoptotic B cells in GCs.

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P, a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P, enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P-bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Background Although DNAM-1 is an activating receptor constitutively expressed on the majority of NK cells, CD8+ T cells, CD4+ T cells, monocytes, and platelets in human, several evidences demonstrated that a small population in B-lineage cells also expressed DNAM-1. However, the expression profile of DNAM-1 on B-lineage cells and its function remain obscure. Previous reports revealed that a considerable number of leukocytes including B cells in the peripheral blood conjugated to platelet. Thus, the proportion of DNAM-1+ B-lineage cells determined by flow cytometry analysis in the previous reports might be overestimated. Methods We examined whether platelets conjugate B cells and then analyzed the expression of DNAM-1 on the subpopulations of B-lineage cells according to their maturation stages after exclusion of platelet-conjugated B cells. We also assessed the involvement of DNAM-1 in IL-10 and antibody production from cultured B-lineage cells stimulated with CpG-ODN. Results Approximately 10% of human DNAM-1+ CD19+ B cells in the peripheral blood conjugated to platelets, resulting in the overestimation of the proportion of DNAM-1+ B cells. After exclusion of platelet-conjugating B cells, we show that DNAM-1 expression was detected on subpopulations of memory B cells, plasmablasts, and plasma cells and upregulated by stimulation with CpG-ODN. Moreover, DNAM-1 was involved in IL-10 and antibody productions by B cells after CpG-ODN stimulation. Conclusions DNAM-1 may be involved in B-lineage cell-mediated immune responses.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm with poor prognosis that develops after chronic infection with human T-cell leukemia virus type 1 (HTLV-1). Although AMP-activated protein kinase (AMPK) is a critical cellular energy sensor, it has recently become clear that AMPK can act as a tumor regulator. Here, we assessed the expression of AMPK in primary ATL cells and the effects of dorsomorphin, an AMPK inhibitor, on primary ATL cells and HTLV-1-infected T-cell lines. AMPK expression in acute and chronic ATL patients was significantly higher than in asymptomatic HTLV-1 carriers and healthy donors. Dorsomorphin induced apoptosis in peripheral blood mononuclear cells from ATL patients. Dorsomorphin also induced dose- and time-dependent apoptosis in HTLV-1-infected T-cell lines. Dorsomorphin increased the production of intracellular reactive oxygen species (ROS) and induced ataxia telangiectasia-mutated Ser1981 phosphorylation and p53 accumulation. These results indicated that dorsomorphin induces apoptosis via ROS-mediated DNA damage in HTLV-1-infected T-cell lines. Furthermore, dorsomorphin suppressed the growth of human ATL tumor xenografts in NOD/SCID mice. Together, these data suggest that AMPK could be a candidate therapeutic target for ATL and that dorsomorphin could be a therapeutic agent for ATL.

Marginal zone (MZ) B cells produce a first wave of antibodies for protection from blood-borne pathogens. However, the role of MZ B cells in inflammatory responses has not been elucidated. Here we show that MZ B cells produce pro-inflammatory cytokines, such as interleukin-6 (IL-6), and exacerbate systemic inflammatory responses to lipopolysaccharide (LPS). After intravenous injection of LPS or E. coli, mice deficient in MZ B cells or IL-6 only in MZ B cells have attenuated systemic inflammatory responses and prolonged survival compared with wild-type mice. LPS directly stimulates MZ B cells via Toll-like receptor 4 (TLR4) and MyD88 pathways for IL-6 production. Furthermore, TLR4 requires physical and functional association with Fcα/μR (CD351) for its oligomer formation, NF-κB signalling and IL-6 production from MZ B cells; this association is responsible for systemic inflammatory responses and endotoxic shock. These results reveal a pro-inflammatory role of MZ B cells in endotoxic shock., Marginal zone B cells are mostly characterized in the context of host defense against bacterial blood-borne pathogens. Here the authors show that TLR4 signaling in these cells requires Fcα/μR (CD351) and that they are a major source of IL-6 in a mouse model of sepsis.

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR−/− mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR−/− mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18−/− and C3−/− mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

Although Th1 and Th2 cells are known to be involved in allergic inflammatory diseases, the molecular mechanisms underlying their differentiation are incompletely understood. In this study, we identified CD155 as a costimulatory molecule on CD4+ T cells. Importantly, CD155-mediated signaling induced Th1 development in both humans and mice, as evidenced by production of IFN-γ and upregulation of Tbx21 transcription; these effects were independent of IL-12 but dependent on NF-κB–induced autocrine IFN-γ that triggered positive feedback via STAT1 activation. Mice genetically deficient in CD155 or treated with anti-CD155 Ab exhibited attenuated Th1-type contact hypersensitivity. Thus, CD155 plays an important regulatory role in helper T cell differentiation and allergic diseases.

Fcα/μR (CD351) is an Fc receptor for both IgA and IgM, which is abundantly expressed in the small intestine. However, the role of Fcα/μR in the intestinal tissue is largely unknown. Here, we found that Fcα/μR is highly expressed on follicular dendritic cells (FDCs) in Peyer's patches (PP) in the small intestine. Fcα/μR-deficient mice on the (129 x C57BL/6) F1 background showed increased serum, but not fecal, IgA level in response to gut-oriented antigens. IgA + B cells were increased in PP, but not in the lamina propria, of Fcα/μR-deficient mice, which was attenuated after reduction of commensal microbiota by oral treatment with antibiotics. Analyses of bone marrow chimeric mice, in which either FDCs or blood cells or both lack the expression of Fcα/μR, suggested that FDCs, but not blood cells, were responsible for the increased serum IgA concentration in Fcα/μR-deficient mice. Moreover, Fcα/μR-deficient mice showed enhanced germinal center formation against commensal microbiota in PP. Thus, serum IgA production against gut-oriented antigens is negatively regulated by Fcα/μR on FDCs in the F1 mice.

Three-day-old rice seedlings treated with ethylene showed elongation of the 2nd and 3rd leaves. This ethylene-stimulated elongation was not observed in the presence of uniconazole-P or prohexadione, both gibberellin (GA) biosynthesis inhibitors, suggesting that GA was involved in the response. An analysis of endogenous GAs by GC-MS revealed that the GA1 level was reduced in the 3rd leaf in response to ethylene. Dose-response experiments showed that the responsiveness to GA1 was enhanced by ethylene. Feeding experiments of 14C-GA1 with ethylene-treated seedlings showed that ethylene may increase the conversion of GA1 to GA8. These results suggest that, in young seedlings of rice, ethylene stimulates leaf elongation by increasing the responsiveness to GA1 and the turnover of GA1.

Fc receptors play important roles for a wide array of immune responses. In contrast to the well-defined Fcγ and Fcε receptors, the molecular and functional characteristics of Fc receptors for IgA and IgM have remained incompletely understood for years. Recent progress has unveiled the characteristics of Fc receptors for IgA and IgM, including Fcα/μ receptor (Fcα/μR) (CD351), polymeric immunoglobulin receptor (poly-IgR), Fcα receptor (FcαRI) (CD89) and Fcμ receptor (FcμR). In this review, we summarize the molecular and functional characteristics of Fcα/μR in comparison with poly-IgR, FcμR and FcαRI, and focus particularly on the pro-inflammatory function of Fcα/μR expressed on marginal zone B cells in sepsis.

Physiological and molecular biological analysis of the dwarf barley (Hordeum vulgare L.) mutant brachytic 1 (brh1) was conducted. The root responses of brh1 to brassinolide were weaker than those of wild type, but the responses of leaf segments of dark-grown plants were not. Responses of brh1 to gibberellin A3 were similar to or slightly stronger than those of wild type. Endogenous levels of these hormones in young seedlings were not clearly different between brh1 and wild type. Skotomorphogeneses of brh1 were similar to those of wild type. Some of these physiological characteristics of brh1 resemble those of the dwarf rice (Oryza sativa L.) mutant daikoku (dwarf1; d1), whose dwarfism is caused by a mutation in the heterotrimeric G protein α (Gα) subunit. A database search indicated that the barley Gα gene is located near the locus where Brh1 has already been genetically mapped. Sequences of the Gα gene and cDNAs of five brh1 alleles contained substitutions and a deletion that lead to the production of abnormal Gα proteins. These results indicate that the phenotype of brh1, similarly to that of d1, is caused by mutations in an orthologous gene encoding Gα.

Antibody exerts various immune responses via binding to Fc receptors expressed on immune cells. Although several reports have demonstrated that IgM prevents autoantibody production, the role of IgM Fc receptors is largely unknown. To analyze the involvement of Fcα/μR (CD351), an Fc receptor for IgM and IgA expressed on B cells and follicular dendritic cells (FDCs), in IgM-mediated suppression of autoantibody production, we generated mice deficient in Fcα/μR on the background of MRL/MpJ-Fas(lpr/lpr) (Fcamr(-/-)Fas(lpr/lpr)) mice. Fcamr(-/-)Fas(lpr/lpr) mice showed significantly lower titers of IgG autoantibodies against double strand (ds) DNA, histone and cardiolipin in the sera than did Fcamr(+/+)Fas(lpr/lpr) mice. Moreover, Fcamr(-/-)Fas(lpr/lpr) mice showed higher survival rate at the ages of 28, 32 and 40 weeks old, compared with Fcamr(+/+)Fas(lpr/lpr) mice. These results suggest that Fcα/μR enhances, rather than suppresses, autoantibody production.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1 and has a poor outcome. New molecular targets for the prevention and treatment of ATL are needed urgently. We previously reported high expression of Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting enzyme in NAD+ biosynthesis, in ATL cells. We found that peripheral blood mononuclear cells from patients with acute-type ATL expressed significantly higher levels of NAMPT protein than cells from healthy subjects. FK866, a NAMPT inhibitor, induced apoptosis of freshly isolated ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. However, a pan-caspase inhibitor failed to prevent this FK866-induced cell death, while FK866 increased the caspase-independent cell death mediator endonuclease G. Intriguingly, FK866 also activated autophagy, as demonstrated by increases in protein levels of autophagosome marker LC3-II. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased the growth of human ATL tumor xenografts in immunodeficient mice. We showed that NAMPT is highly expressed in primary ATL cells ex vivo, and that FK866 induces autophagy and caspase-dependent and -independent cell death pathways in vitro and has an anti-tumor activity in vivo. These results suggest a novel therapeutic strategy for patients with this fatal disease.

Obesity commonly occurs in postmenopausal women, increasing the risk of various diseases. Estrogen can prevent obesity by activating lipid metabolism and suppressing depressive behavior. However, the reasons for obesity in postmenopausal women are not clearly elucidated. To mimic the effect of estrogen decline in postmenopausal women, we analyzed the behavior and the lipid metabolism-related genes, PPARγ and CD36 in ovariectomized (OVX) mice. The OVX mice showed increased visceral fat mass and PPARγ and CD36 expression in the visceral fat. In contrast, they were not significantly affected in terms of physical activity and food intake. Further, subcutaneous supplementation of estrogen effectively suppressed the increase in subcutaneous and visceral fat mass in OVX mice. We conclude that obesity in postmenopausal women is unlikely to be caused by overeating and reduction in physical activity, and subcutaneous supplementation of estrogen is an effective strategy to prevent obesity in postmenopausal women., Highlights • We studied the effects of estrogen deficiency/supplementation on obesity in mice. • After ovariectomy, the study animals received transdermal injections of estrogen. • Exogenous 17β-estradiol appears to mediate the effects of menopause on obesity. • Obesity in the mice was caused solely by estrogen deficiency, not overeating. • Estrogen deficiency altered the mechanics of fat deposition in the mice.

Background Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1, and has a poor outcome. New molecular targets for the prevention and treatment of ATL are urgently needed. We previously reported that Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, is highly expressed in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting. Methods Peripheral blood mononuclear cells from ATL patients were carried out in accordance with the guidelines of the Committees for Ethical Review of Research involving Human Subjects at Kagoshima University Hospital. Cell viability was evaluated in the S1T cell line derived from an ATL patient, MT-2 cell line derived from normal human leukocytes transformed by leukemic T-cells from an ATL patient, and primary ATL cells. Animal experiments were approved by the Animal Care and Use Committee of Rakuno Gakuen University in accordance with the Guide for the Care and Use of Laboratory Animals. Results Peripheral blood mononuclear cells from acute-type ATL patients expressed significantly higher NAMPT protein levels than cells from healthy controls. FK866, a NAMPT inhibitor, induced apoptosis in fresh ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by NAD+ depletion, activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. A pan-caspase inhibitor failed to prevent the FK866-induced cell death, while FK866 increased endonuclease G, a caspase-independent cell death mediator. Intriguingly, FK866 activated autophagy, revealed by increased LC3-II protein levels and autophagic flux. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased human ATL tumor xenograft growth in immunodeficient mice. Conclusions These results demonstrate that NAMPT inhibition induces autophagy and caspase-dependent and -independent cell death in ATL cells, suggesting a novel therapeutic strategy for patients with this fatal disease. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Fcα/μR (CD351) is an Fc receptor for both IgA and IgM and forms an atypical dimer that is resistant to reduction by 2-mercaptoethanol or boiling. We previously demonstrated that the cytoplasmic portion of Fcα/μR is required for dimer formation and for its efficient cell-surface expression. However, the biochemical nature of these phenomena has not been determined. By using a BW5147 mouse cell line expressing deletion mutants of the cytoplasmic region of Fcα/μR, we found that the region spanning amino acids 504–523 was required for efficient cell-surface expression, whereas the region spanning amino acids 481–490 was required for dimmer formation. Immunoblotting analyses of transfectants simultaneously expressing Flag-tagged Fcα/μR and hemagglutinin-tagged Fcα/μR suggested that Fcα/μR does not form homodimers. Instead, our data suggest that Fcα/μR forms heterodimers with an as-yet-unknown molecule with a molecular weight of 60–70 kDa.

SummaryNatural immunoglobulin M (IgM) is reactive to autoantigens and is believed to be important for autoimmunity. Blood pentameric IgM loaded with antigens forms a large immune complex (IC) that contains various elements, including apoptosis inhibitor of macrophage (AIM). Here we demonstrate that this IgM-AIM association contributes to autoantibody production under obese conditions. In mice fed a high-fat diet, natural IgM increased through B cell TLR4 stimulation. AIM associated with IgM and protected AIM from renal excretion, increasing blood AIM levels along with the obesity-induced IgM augmentation. Meanwhile, the AIM association inhibited IgM binding to the Fcα/μ receptor on splenic follicular dendritic cells, thereby protecting the IgM IC from Fcα/μ receptor-mediated internalization. This supported IgM-dependent autoantigen presentation to B cells, stimulating IgG autoantibody production. Accordingly, in obese AIM-deficient (AIM−/−) mice, the increase of multiple IgG autoantibodies observed in obese wild-type mice was abrogated. Thus, the AIM-IgM association plays a critical role in the obesity-associated autoimmune process.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (EedΔ/Δ) mice. EedΔ/Δ mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in EedΔ/Δ HSPCs and consistently, EedΔ/Δ HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other and these conflicting events would lead to abnormal differentiation and functional defect of EedΔ/Δ HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Follicular dendritic cells (FDCs) in lymphoid organs play an important role in the humoral immune response. However, because the isolation of FDCs is difficult due to their very small population size and fragility under mechanical and chemical stresses, the genetic and biochemical characteristics of FDCs remain unclear. Previously, we identified FDCs as ICAM-1+ cells in the CD45- non-hematopoietic cell fraction from naive mouse spleen after cell separation by means of digestion with a combination of enzymes. In the present study, using a new combination of enzymes, we found that FDCs are highly enriched in the CD45-ICAM-1+CD21/35+ cell fraction. CD45-ICAM-1+CD21/35+ cells in the mouse spleen retained an antigen administered in vivo for more than 7 days. Moreover, CD45-ICAM-1+CD21/35+ cells isolated from the spleen of mice administered with a cognate antigen enhanced the survival and proliferation of antigen-specific B cells in vitro. Our improved protocol for the isolation of naive FDCs will be useful for the analysis of FDCs in vitro and in vivo.

Neurosteroidal oestrogen has been proposed to play important roles in a variety of reproductive behaviours. Aromatase, a key enzyme in oestrogen synthesis, is localised in neural nuclei of specific brain regions and is developmentally regulated, with a transient expression peak at the perinatal period. The brain-specific promoter of the aromatase gene was analysed aiming to determine the transcriptional control mechanisms that could help explain the spatiotemporal expression. We previously reported that a 202-bp sequence, which is upstream from the transcriptional initiation site, is essential for the basal transcriptional activity. The 202-bp upstream region of brain-specific exon 1 comprises at least three types of cis-acting elements: aro-AI (Arom-Aα), aro-AII (Arom-Aβ) and aro-B (Arom-B). To identify the binding proteins for the cis-acting elements, a yeast one-hybrid screen was performed with these cis-element sequences using a mouse foetal cDNA library. Lhx2, a LIM-homeodomain protein, was identified as one of the aro-B binding proteins. The identification was further confirmed using the gel shift assay, which demonstrated binding competition of nuclear proteins to the aro-B element with a typical Lhx2-binding element. In addition, a chromatin immunoprecipitation assay with an anti-Lhx2 antibody demonstrated that Lhx2 bound to the aro-B site in vivo. A reporter assay of the brain-specific promoter demonstrated increased Lhx2-dependent promoter activity. Furthermore, the time-dependent increase in aromatase mRNA in primary cultured foetal neurones was suppressed by an small-interfering RNA-mediated knockdown of Lhx2 expression. These results show that Lhx2 is involved in the transcriptional regulation of aromatase in the rodent brain.

Cytokinins (CKs) are thought to play important roles in fruit development, especially cell division. However, the mechanisms and regulation of CK activity have not been well investigated. This study analysed CK concentrations and expression of genes involved in CK metabolism in developing tomato (Solanum lycopersicum) ovaries. The concentrations of CK ribosides and isopentenyladenine and the transcript levels of the CK biosynthetic genes SlIPT3, SlIPT4, SlLOG6, and SlLOG8 were high at anthesis and decreased immediately afterward. In contrast, trans-zeatin concentration and the transcript levels of the CK biosynthetic genes SlIPT1, SlIPT2, SlCYP735A1, SlCYP735A2, and SlLOG2 increased after anthesis. The expression of type-A response regulator genes was high in tomato ovaries from pre-anthesis to early post-anthesis stages. These results suggest that the CK signal transduction pathway is active in the cell division phase of fruit development. This study also investigated the effect of CK application on fruit set and development. Application of a synthetic CK, N-(2-chloro-pyridin-4-yl)-N’-phenylurea (CPPU), to unpollinated tomato ovaries induced parthenocarpic fruit development. The CPPU-induced parthenocarpic fruits were smaller than pollinated fruits, because of reduction of pericarp cell size rather than reduced cell number. Thus, CPPU-induced parthenocarpy was attributable to the promotion of cell division, not cell expansion. Overall, the results provide evidence that CKs are involved in cell division during development of tomato fruit.

EED mutants impair polycomb repressive complex 2 in myelodysplastic syndrome and related neoplasms

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide(+)-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, owing to its ability to deacetylate numerous substrates, such as histone and NF-κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family and degradation of SIRT1 in the nucleus. Furthermore, SIRT1 knockdown by SIRT1-specific small interfering RNA caused apoptosis via activation of caspase-3 and PARP in MT-2 cells, HTLV-1-related cell line. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.

CD300a is a member of CD300 family molecules consisting of seven genes on human chromosome 17 and nine genes in mouse chromosome 11. CD300a has a long cytoplasmic region containing the consensus immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence. Upon crosslinking with antibodies against CD300a, CD300a mediates an inhibitory signal in myeloid cells. However, the ligand for CD300a has not been identified and the physiological role of CD300a remained unclear. Here, we demonstrate that the chimeric fusion protein of CD300a extracellular domain with the Fc portion of human IgG specifically bound phosphatidylserine (PS), which is exposed on the outer leaflet of the plasma membrane of apoptotic cells. PS binding to CD300a induced SHP-1 recruitment by CD300a in mast cells in response to LPS. These results indicated that CD300a is a new PS receptor.