Publications by authors named "Hiroaki Honda"

82 Publications

Pathological Role of Pin1 in the Development of DSS-Induced Colitis.

Cells 2021 May 17;10(5). Epub 2021 May 17.

Department of Medical Chemistry, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Inflammatory bowel diseases (IBDs) are serious disorders of which the etiologies are not, as yet, fully understood. In this study, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein was shown to be dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Interestingly, knockout (KO) mice exhibited significant attenuation of DSS-induced colitis compared to wild-type (WT) mice, based on various parameters, including body weight, colon length, microscopic observation of the intestinal mucosa, inflammatory cytokine expression, and cleaved caspase-3. In addition, a role of Pin1 in inflammation was suggested because the percentage of M1-type macrophages in the colon was decreased in the KO mice while that of M2-type macrophages was increased. Moreover, KO mice showed downregulation of both and expression in the colon, both of which have been implicated in the development of colitis. Finally, oral administration of Pin1 inhibitor partially but significantly prevented DSS-induced colitis in mice, raising the possibility of Pin1 inhibitors serving as therapeutic agents for IBD.
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http://dx.doi.org/10.3390/cells10051230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155908PMC
May 2021

Progress of genome editing technology and developmental biology useful for radiation research.

J Radiat Res 2021 May;62(Supplement_1):i53-i63

Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.

Following the development of genome editing technology, it has become more feasible to create genetically modified animals such as knockout (KO), knock-in, and point-mutated animals. The genome-edited animals are useful to investigate the roles of various functional genes in many fields of biological science including radiation research. Nevertheless, some researchers may experience difficulty in generating genome-edited animals, probably due to the requirement for equipment and techniques for embryo manipulation and handling. Furthermore, after obtaining F0 generation, genome-edited animals generally need to be expanded and maintained for analyzing the target gene function. To investigate genes essential for normal birth and growth, the generation of conditional KO (cKO) animals in which a tissue- or stage-specific gene mutation can be introduced is often required. Here, we describe the basic principle and application of genome editing technology including zinc-finger nuclease, transcription-activator-like effector nuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas) systems. Recently advanced developmental biology methods have enabled application of the technology, especially CRISPR/Cas, to zygotes, leading to the prompt production of genome-edited animals. For pre-implantation embryos, genome editing via oviductal nucleic acid delivery has been developed as an embryo manipulation- or handling-free method. Examining the gene function at F0 generation is becoming possible by employing triple-target CRISPR technology. This technology, in combination with a blastocyst complementation method enables investigation of even birth- and growth-responsible genes without establishing cKO strains. We hope that this review is helpful for understanding and expanding genome editing-related technology and for progressing radiation research.
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http://dx.doi.org/10.1093/jrr/rraa127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114227PMC
May 2021

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.

Nat Commun 2021 03 23;12(1):1826. Epub 2021 Mar 23.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
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http://dx.doi.org/10.1038/s41467-021-22053-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988019PMC
March 2021

DEC1 deficiency results in accelerated osteopenia through enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3β signaling.

Metabolism 2021 05 17;118:154730. Epub 2021 Feb 17.

Department of Pharmacology, Nanjing Medical University, China.

Background: Human differentiated embryonic chondrocyte expressed gene 1 (DEC1) has been implicated in enhancing osteogenesis, a desirable outcome to counteract against deregulated bone formation such as retarded bone development, osteopenia and osteoporosis.

Methods And Results: DEC1 knockout (KO) and the age-matched wild-type (WT) mice were tested for the impact of DEC1 deficiency on bone development and osteopenia as a function of age. DEC1 deficiency exhibited retarded bone development at the age of 4 weeks and osteopenic phenotype in both 4- and 24-week old mice. However, the osteopenia was more severe in the 24-week age groups. Mechanistically, DEC1 deficiency downregulated the expression of bone-enhancing genes such as Runx2 and β-catenin accompanied by upregulating DKK1, an inhibitor of the Wnt/β-catenin signaling pathway. Consistently, DEC1 deficiency favored the attenuation of the integrated PI3KCA/Akt/GSK3β signaling, a pathway targeting β-catenin for degradation. Likewise, the attenuation was greater in the 24-week age group. These changes, however, were reversed by in vivo treatment with lithium chloride, a stabilizer of β-catenin, and confirmed by gain-of-function study with DEC1 transfection into DEC1 KO bone marrow mesenchymal stem cells and loss-of-function study with siDEC1 lentiviral infection into the corresponding WT cells.

Conclusion: DEC1 is a positive regulator with a broad activity spectrum in both bone development and maintenance, and the osteopenic phenotype accelerated by DEC1 deficiency is achieved by enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3β signaling.
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http://dx.doi.org/10.1016/j.metabol.2021.154730DOI Listing
May 2021

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Blood 2021 02;137(7):908-922

Human Disease Models, Institute of Laboratory Animals, Tokyo Women's Medical University, Tokyo, Japan.

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.
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http://dx.doi.org/10.1182/blood.2019001044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918186PMC
February 2021

Jmjd3 Plays Pivotal Roles in the Proper Development of Early-Born Retinal Lineages: Amacrine, Horizontal, and Retinal Ganglion Cells.

Invest Ophthalmol Vis Sci 2020 09;61(11):43

Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Purpose: Trimethylation of histone H3 at lysine 27 (H3K27me3) is a critical mediator of transcriptional gene repression, and Jmjd3 and Utx are the demethylases specific to H3K27me3. Using an in vitro retinal explant culture system, we previously revealed the role of Jmjd3 in the development of rod bipolar cells; however, the roles of Jmjd3 in the development of early-born retinal cells are unknown due to limitations concerning the use of retinal explant culture systems. In this study, we investigated the roles of Jmjd3 in the development of early-born retinal cells.

Methods: We examined retina-specific conditional Jmjd3 knockout (Jmjd3-cKO) mice using immunohistochemistry and quantitative reverse transcription PCR and JMJD3 binding to a target locus by chromatin immunoprecipitation analysis.

Results: We observed reductions in amacrine cells (ACs) and horizontal cells (HCs), as well as lowered expression levels of several transcription factors involved in the development of ACs and HCs in the Jmjd3-cKO mouse retina. JMJD3 bound the promoter regions of these transcription factors. Notably, an elevated number of retinal ganglion cells (RGCs) was observed at embryonic stages, whereas RGCs were moderately reduced at later postnatal stages in the Jmjd3-cKO retina. We also observed reduced expression of Eomes, which is required for the maintenance of RGCs, as well as lower H3K27me3 level and lower JMJD3 binding in the promoter region of Eomes in RGC-enriched cells.

Conclusions: The results indicated that Jmjd3 has critical roles in the development of early-born retinal subtypes, and suggested biphasic roles of Jmjd3 in RGC production and maintenance.
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http://dx.doi.org/10.1167/iovs.61.11.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533738PMC
September 2020

Association of aberrant ASNS imprinting with asparaginase sensitivity and chromosomal abnormality in childhood BCP-ALL.

Blood 2020 11;136(20):2319-2333

Department of Pediatrics and.

Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.
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http://dx.doi.org/10.1182/blood.2019004090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702480PMC
November 2020

HHEX promotes myeloid transformation in cooperation with mutant ASXL1.

Blood 2020 10;136(14):1670-1684

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.
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http://dx.doi.org/10.1182/blood.2019004613DOI Listing
October 2020

Dual Regulation of Histone Methylation by mTOR Complexes Controls Glioblastoma Tumor Cell Growth via EZH2 and SAM.

Mol Cancer Res 2020 08 4;18(8):1142-1152. Epub 2020 May 4.

Division of Pathological Neuroscience, Department of Pathology, Tokyo Women's Medical University, Tokyo, Japan.

Epigenetic regulation known for DNA methylation and histone modification is critical for securing proper gene expression and chromosomal function, and its aberration induces various pathologic conditions including cancer. Trimethylation of histone H3 on lysine 27 (H3K27me3) is known to suppress various genes related to cancer cell survival and the level of H3K27me3 may have an influence on tumor progression and malignancy. However, it remains unclear how histone methylation is regulated in response to genetic mutation and microenvironmental cues to facilitate the cancer cell survival. Here, we report a novel mechanism of the specific regulation of H3K27me3 by cooperatively two mTOR complexes, mTORC1 and mTORC2 in human glioblastoma (GBM). Integrated analyses revealed that mTORC1 upregulates the protein expression of enhancer of zeste homolog 2, a main component of polycomb repressive complex 2 which is known as H3K27-specific methyltransferase. The other mTOR complex, mTORC2, regulates production of S-adenosylmethionine, an essential substrate for histone methylation. This cooperative regulation causes H3K27 hypermethylation which subsequently promotes tumor cell survival both and xenografted mouse tumor model. These results indicate that activated mTORC1 and mTORC2 complexes cooperatively contribute to tumor progression through specific epigenetic regulation, nominating them as an exploitable therapeutic target against cancer. IMPLICATIONS: A dynamic regulation of histone methylation by mTOR complexes promotes tumor growth in human GBM, but at the same time could be exploitable as a novel therapeutic target against this deadly tumor.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0024DOI Listing
August 2020

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina.

Genes Cells 2020 Jun 14;25(6):402-412. Epub 2020 Apr 14.

Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Di- and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down-regulation of the H3K27 demethylase, Jumonji domain-containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α-positive rod ON-bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA-mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα-positive rod ON-bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina-specific knockout of Utx in mice, the in vivo effects of UTX down-regulation were examined. Again, the number of PKCα-positive rod ON-bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina-specific Utx and Jmjd3 double-knockout mice and found that although the number of rod ON-bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage-specific manner.
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http://dx.doi.org/10.1111/gtc.12767DOI Listing
June 2020

Deficiency Activates Inflammatory Pathways, Promotes M2 Macrophage Polarization, and Causes Bladder Cancer in Cooperation with Dysfunction.

Clin Cancer Res 2020 04 11;26(8):2065-2079. Epub 2020 Feb 11.

Field of Human Disease Models, Major in Advanced Life Sciences and Medicine, Institute of Laboratory Animals, Tokyo Women's Medical University, Shinjuku-ku, Tokyo, Japan.

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 -mutated bladder cancer can help in designing new anticancer therapies.

Experimental Design: We generated mice lacking in the urothelium and crossed them with mice heterozygous for , whose mutation/deletion significantly overlaps with the 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 deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to deficiency, efficiently suppressed -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 dysfunction.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2230DOI Listing
April 2020

Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas.

Cell Biochem Funct 2020 Apr 30;38(3):300-308. Epub 2019 Dec 30.

Division of Molecular Signaling and Biochemistry, Department of Health Improvement, Kyushu Dental University, Kitakyushu, Japan.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130Cas ) mice also exhibited a similar phenotype to c-Src mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src or p130Cas mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src and p130Cas osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src and p130Cas osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130Cas osteoclasts but not c-Src osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130Cas ) osteoclasts but not c-Src osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.
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http://dx.doi.org/10.1002/cbf.3476DOI Listing
April 2020

The loss of dopaminergic neurons in DEC1 deficient mice potentially involves the decrease of PI3K/Akt/GSK3β signaling.

Aging (Albany NY) 2019 12 28;11(24):12733-12753. Epub 2019 Dec 28.

Department of Pharmacology, Nanjing Medical University, Nanjing, China.

Here we study the effects of differentiated embryonic chondrocyte gene 1(DEC1) deficiency on midbrain dopaminergic(DA) neurons in the substantia nigra pars compacta(SNpc) through behavioral, histological and molecular analysis. We have found that compared to the age-matched WT mice, DEC1 deficient mice show a decrease in locomotor activity and motor coordination, which shows the main features of Parkinson's disease(PD). But there is no significant difference in spatial learning and memory skills between WT and DEC1 KO mice. Compared to the age-matched WT mice, DEC1 deficient mice exhibit the loss of DA neurons in the SNpc and reduction of dopamine and its metabolites in the striatum. The activated caspase-3 and TH/TUNEL cells increase in the SNpc of 6- and 12-month-old DEC1 KO mice compared to those of the age-matched WT mice. But we haven't found any NeuN/TUNEL cell increase in the hippocampus of the above two types of mice at the age of 6 months. Furthermore, DEC1 deficiency leads to a significant inhibition of PI3K/Akt/GSK3β signaling pathway. Additionally, LiCl could rescue the DA neuron loss of midbrain in the 6-month-old DEC1 KO mice. Taken together, the loss of DA neurons in the DEC1 deficient mice potentially involves the downregulation of PI3K/Akt/GSK3β signaling.
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http://dx.doi.org/10.18632/aging.102599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949058PMC
December 2019

Discrimination of Dormant and Active Hematopoietic Stem Cells by G Marker Reveals Dormancy Regulation by Cytoplasmic Calcium.

Cell Rep 2019 12;29(12):4144-4158.e7

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G marker (GM) mouse line that visualizes quiescent cells and identify a small population of active HSCs (GM), which are distinct from dormant HSCs (GM), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca]) are linked to dormancy of HSCs. These findings indicate that GM separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca]. This GM mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.
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http://dx.doi.org/10.1016/j.celrep.2019.11.061DOI Listing
December 2019

Accumulation of versican facilitates wound healing: Implication of its initial ADAMTS-cleavage site.

Matrix Biol 2020 05 26;87:77-93. Epub 2019 Oct 26.

Institute for Molecular Science of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan. Electronic address:

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSβ domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFβ-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.
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http://dx.doi.org/10.1016/j.matbio.2019.10.006DOI Listing
May 2020

Lysine-specific demethylase 1 accelerates oncogenesis in p53 heterozygous mice via transcriptional repression of the residual Trp53 allele.

Leuk Res 2019 07 23;82:29-32. Epub 2019 May 23.

Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, 3311-1, Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.leukres.2019.05.008DOI Listing
July 2019

Loss of Endothelium-Derived Wnt5a Is Associated With Reduced Pericyte Recruitment and Small Vessel Loss in Pulmonary Arterial Hypertension.

Circulation 2019 04;139(14):1710-1724

Division of Pulmonary and Critical Care Medicine (K.Y., E.A.S., M.E.O., A.N., V.A.d.J.P.), Stanford University, Palo Alto, CA.

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disorder of the pulmonary circulation associated with loss and impaired regeneration of microvessels. Reduced pericyte coverage of pulmonary microvessels is a pathological feature of PAH and is caused partly by the inability of pericytes to respond to signaling cues from neighboring pulmonary microvascular endothelial cells (PMVECs). We have shown that activation of the Wnt/planar cell polarity pathway is required for pericyte recruitment, but whether production and release of specific Wnt ligands by PMVECs are responsible for Wnt/planar cell polarity activation in pericytes is unknown.

Methods: Isolation of pericytes and PMVECs from healthy donor and PAH lungs was carried out with 3G5 or CD31 antibody-conjugated magnetic beads. Wnt expression profile of PMVECs was documented via quantitative polymerase chain reaction with a Wnt primer library. Exosome purification from PMVEC media was carried out with the ExoTIC device. Hemodynamic profile, right ventricular function, and pulmonary vascular morphometry were obtained in a conditional endothelium-specific Wnt5a knockout ( Wnt5a) mouse model under normoxia, chronic hypoxia, and hypoxia recovery.

Results: Quantification of Wnt ligand expression in healthy PMVECs cocultured with pericytes demonstrated a 35-fold increase in Wnt5a, a known Wnt/planar cell polarity ligand. This Wnt5a spike was not seen in PAH PMVECs, which correlated with an inability to recruit pericytes in Matrigel coculture assays. Exosomes purified from media demonstrated an increase in Wnt5a content when healthy PMVECs were cocultured with pericytes, a finding that was not observed in exosomes of PAH PMVECs. Furthermore, the addition of either recombinant Wnt5a or purified healthy PMVEC exosomes increased pericyte recruitment to PAH PMVECs in coculture studies. Although no differences were noted in normoxia and chronic hypoxia, Wnt5a mice demonstrated persistent pulmonary hypertension and right ventricular failure 4 weeks after recovery from chronic hypoxia, which correlated with significant reduction, muscularization, and decreased pericyte coverage of microvessels.

Conclusions: We identify Wnt5a as a key mediator for the establishment of pulmonary endothelium-pericyte interactions, and its loss could contribute to PAH by reducing the viability of newly formed vessels. We speculate that therapies that mimic or restore Wnt5a production could help prevent loss of small vessels in PAH.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.037642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443444PMC
April 2019

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL.

Oncotarget 2018 Aug 3;9(60):31682-31696. Epub 2018 Aug 3.

Department of Pharmacology, Tokyo Women's Medical University, Tokyo, Japan.

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11bLy6CLy6G cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1β, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.
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http://dx.doi.org/10.18632/oncotarget.25849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114964PMC
August 2018

Expression of mutant Asxl1 perturbs hematopoiesis and promotes susceptibility to leukemic transformation.

J Exp Med 2018 06 11;215(6):1729-1747. Epub 2018 Apr 11.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

() is frequently mutated in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). Although loss of ASXL1 promotes hematopoietic transformation, there is growing evidence that mutations might confer an alteration of function. In this study, we identify that physiological expression of a C-terminal truncated Asxl1 mutant in vivo using conditional knock-in (KI) results in myeloid skewing, age-dependent anemia, thrombocytosis, and morphological dysplasia. Although expression of mutant Asxl1 altered the functions of hematopoietic stem cells (HSCs), it maintained their survival in competitive transplantation assays and increased susceptibility to leukemic transformation by co-occurring mutation or viral insertional mutagenesis. KI mice displayed substantial reductions in H3K4me3 and H2AK119Ub without significant reductions in H3K27me3, distinct from the effects of Asxl1 loss. Chromatin immunoprecipitation followed by next-generation sequencing analysis demonstrated opposing effects of wild-type and mutant Asxl1 on H3K4me3. These findings reveal that mutations confer HSCs with an altered epigenome and increase susceptibility for leukemic transformation, presenting a novel model for CHIP.
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http://dx.doi.org/10.1084/jem.20171151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987913PMC
June 2018

The enigma of monosomy 7.

Blood 2018 06 3;131(26):2891-2898. Epub 2018 Apr 3.

Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.

Since a report of some 50 years ago describing refractory anemia associated with group C monosomy, monosomy 7 (-7) and interstitial deletions of chromosome 7 (del(7q)) have been established as one of the most frequent chromosomal aberrations found in essentially all types of myeloid tumors regardless of patient age and disease etiology. In the last century, researchers sought recessive myeloid tumor-suppressor genes by attempting to determine commonly deleted regions (CDRs) in del(7q) patients. However, these efforts were not successful. Today, tumor suppressors located in 7q are believed to act in a haploinsufficient fashion, and powerful new technologies such as microarray comparative genomic hybridization and high-throughput sequencing allow comprehensive searches throughout the genes encoded on 7q. Among those proposed as promising candidates, 4 have been validated by gene targeting in mouse models. (sterile α motif domain 9) and (SAMD9-like) encode related endosomal proteins, mutations of which cause hereditary diseases with strong propensity to infantile myelodysplastic syndrome (MDS) harboring monosomy 7. Because MDS develops in -deficient mice over their lifetime, / are likely responsible for sporadic MDS with -7/del(7q) as the sole anomaly. (enhancer of zeste homolog 2) and (mixed lineage leukemia 3) encode histone-modifying enzymes; loss-of-function mutations of these are detected in some myeloid tumors at high frequencies. In contrast to /, loss of or likely contributes to myeloid tumorigenesis in cooperation with additional specific gene alterations such as of or genes involved in the p53/Ras pathway, respectively. Distinctive roles with different significance of the loss of multiple responsible genes render the complex nature of myeloid tumors carrying -7/del(7q).
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http://dx.doi.org/10.1182/blood-2017-12-822262DOI Listing
June 2018

A paralogous pair of mammalian host restriction factors form a critical host barrier against poxvirus infection.

PLoS Pathog 2018 02 15;14(2):e1006884. Epub 2018 Feb 15.

Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

Host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. Human SAMD9, a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (VACV) K1 and C7. A paralog of SAMD9, SAMD9L, is also encoded by some mammals, while only one of two paralogs is retained by others. Here, we show that SAMD9L functions similarly to SAMD9 as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. In mice, which naturally lack SAMD9, overcoming SAMD9L restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. While a VACV deleted of both K1 and C7 (vK1L-C7L-) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in SAMD9L-/- mice. In humans, both SAMD9 and SAMD9L are poxvirus restriction factors, although the latter requires interferon induction in many cell types. While knockout of SAMD9 with Crispr-Cas9 was sufficient for abolishing the restriction for vK1L-C7L- in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. Both paralogs are antagonized by VACV K1, C7 and C7 homologs from diverse mammalian poxviruses, but mouse SAMD9L is resistant to the C7 homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in SAMD9/SAMD9L genes serves as a barrier for cross-species poxvirus transmission.
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http://dx.doi.org/10.1371/journal.ppat.1006884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831749PMC
February 2018

Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Carcinogenesis 2017 05;38(5):570-578

Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.
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http://dx.doi.org/10.1093/carcin/bgw208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872566PMC
May 2017

Acquired expression of in mice induces dysplastic myelopoiesis mimicking chronic myelomonocytic leukemia.

Blood 2017 04 16;129(15):2148-2160. Epub 2017 Feb 16.

Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.

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 gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant in CMML pathogenesis, we generated conditional knockin mice for that express wild-type in a steady state and inducibly express , a CMML-associated mutant. 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 hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. , a gene encoding a GTPase that is upregulated by , enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, , a gene encoding a transcription factor, was found to cooperate with and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of -bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant -induced CMML and propose a possible molecular targeting therapy for mutant -carrying CMML patients.
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http://dx.doi.org/10.1182/blood-2016-06-724658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391621PMC
April 2017

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression.

Proc Natl Acad Sci U S A 2016 09 30;113(37):10370-5. Epub 2016 Aug 30.

Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan;

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.
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http://dx.doi.org/10.1073/pnas.1600070113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027452PMC
September 2016

Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency.

Sci Rep 2016 07 19;6:29454. Epub 2016 Jul 19.

Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

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 (Eed(Δ/Δ)) mice. Eed(Δ/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 Eed(Δ/Δ) HSPCs, and consistently, Eed(Δ/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 Eed(Δ/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.
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http://dx.doi.org/10.1038/srep29454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949429PMC
July 2016

Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells.

Proc Natl Acad Sci U S A 2016 06 25;113(24):E3394-402. Epub 2016 May 25.

Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15 Nishi-7, Sapporo 060-0815, Japan;

Invariant natural killer T (iNKT) cells are a subset of innate-like T cells that act as important mediators of immune responses. In particular, iNKT cells have the ability to immediately produce large amounts of IFN-γ upon activation and thus initiate immune responses in various pathological conditions. However, molecular mechanisms that control IFN-γ production in iNKT cells are not fully understood. Here, we report that basic helix-loop-helix transcription factor family, member e40 (Bhlhe40), is an important regulator for IFN-γ production in iNKT cells. Bhlhe40 is highly expressed in stage 3 thymic iNKT cells and iNKT1 subsets, and the level of Bhlhe40 mRNA expression is correlated with Ifng mRNA expression in the resting state. Although Bhlhe40-deficient mice show normal iNKT cell development, Bhlhe40-deficient iNKT cells show significant impairment of IFN-γ production and antitumor effects. Bhlhe40 alone shows no significant effects on Ifng promoter activities but contributes to enhance T-box transcription factor Tbx21 (T-bet)-mediated Ifng promoter activation. Chromatin immunoprecipitation analysis revealed that Bhlhe40 accumulates in the T-box region of the Ifng locus and contributes to histone H3-lysine 9 acetylation of the Ifng locus, which is impaired without T-bet conditions. These results indicate that Bhlhe40 works as a cofactor of T-bet for enhancing IFN-γ production in iNKT cells.
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http://dx.doi.org/10.1073/pnas.1604178113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914147PMC
June 2016

Identification of cooperative genes for E2A-PBX1 to develop acute lymphoblastic leukemia.

Cancer Sci 2016 Jul 13;107(7):890-8. Epub 2016 Jun 13.

Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.

E2A-PBX1 is a chimeric gene product detected in t(1;19)-bearing acute lymphoblastic leukemia (ALL) with B-cell lineage. To investigate the leukemogenic process, we generated conditional knock-in (cKI) mice for E2A-PBX1, in which E2A-PBX1 is inducibly expressed under the control of the endogenous E2A promoter. Despite the induced expression of E2A-PBX1, no hematopoietic disease was observed, strongly suggesting that additional genetic alterations are required to develop leukemia. To address this possibility, retroviral insertional mutagenesis was used. Virus infection efficiently induced T-cell, B-cell, and biphenotypic ALL in E2A-PBX1 cKI mice. Inverse PCR identified eight retroviral common integration sites, in which enhanced expression was observed in the Gfi1, Mycn, and Pim1 genes. In addition, it is of note that viral integration and overexpression of the Zfp521 gene was detected in one tumor with B-cell lineage; we previously identified Zfp521 as a cooperative gene with E2A-HLF, another E2A-involving fusion gene with B-lineage ALL. The cooperative oncogenicity of E2A-PBX1 with overexpressed Zfp521 in B-cell tumorigenesis was indicated by the finding that E2A-PBX1 cKI, Zfp521 transgenic compound mice developed B-lineage ALL. Moreover, upregulation of ZNF521, the human counterpart of Zfp521, was found in several human leukemic cell lines bearing t(1;19). These results indicate that E2A-PBX1 cooperates with additional gene alterations to develop ALL. Among them, enhanced expression of ZNF521 may play a clinically relevant role in E2A fusion genes to develop B-lineage ALL.
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http://dx.doi.org/10.1111/cas.12945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946715PMC
July 2016

The Histone Demethylase FBXL10 Regulates the Proliferation of Spermatogonia and Ensures Long-Term Sustainable Spermatogenesis in Mice.

Biol Reprod 2016 Apr 16;94(4):92. Epub 2016 Mar 16.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10(DeltaJ/DeltaJ)). In histological analysis, testis sections from 10-wk-old Fbxl10(DeltaJ/DeltaJ) mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10(DeltaJ/DeltaJ) mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10(DeltaJ/DeltaJ) GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10(DeltaJ/DeltaJ) GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10(DeltaJ/DeltaJ) GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10(DeltaJ/DeltaJ) mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle.
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http://dx.doi.org/10.1095/biolreprod.115.135988DOI Listing
April 2016

WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

FASEB J 2016 Apr 14;30(4):1534-47. Epub 2015 Dec 14.

*Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada; Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois, USA; Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; and Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Osaka, Japan

Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.
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http://dx.doi.org/10.1096/fj.15-280313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799500PMC
April 2016