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1.  TIARP attenuates autoantibody-mediated arthritis via the suppression of neutrophil migration by reducing CXCL2/CXCR2 and IL-6 expression 
Scientific Reports  2016;6:38684.
TNFα-induced adipose-related protein (TIARP) is a six-transmembrane protein expressed on macrophages, neutrophils and synoviocytes. We reported recently that mice deficient in TIARP (TIARP−/−) spontaneously develop arthritis and are highly susceptible to collagen-induced arthritis (CIA) with enhanced interleukin (IL)-6 production. However, the effects of TIARP on neutrophils and fibroblast-like synoviocytes (FLS) have not been elucidated. We analyzed the roles of TIARP in K/BxN serum transfer model using TIARP−/− mice. Arthritis in TIARP−/− mice transferred with K/BxN serum was significantly exacerbated compared with WT mice. We characterized the differences in neutrophils between wild-type (WT) and TIARP−/− mice by DNA microarray. Overexpression of CXCR1 and CXCR2 was noted in TIARP−/− neutrophils. Neutrophils of TIARP−/− mice showed strong migration activity, which was markedly facilitated by CXCL2 in vitro and in vivo. Moreover, enhanced production of CXCL2 and IL-6 and cell proliferation was noted in TIARP−/− TNFα-stimulated FLS. Blockade of IL-6R significantly attenuated serum-transferred TIARP−/− arthritis with diminished neutrophil recruitment in joints. Our findings suggested that TIARP independently down-regulated CXCL2 and IL-6 production by FLS, and the expression of chemokine receptors (CXCR1 and CXCR2) in neutrophils, with resultant reduction of neutrophil migration into arthritic joints.
PMCID: PMC5171802  PMID: 27995997
2.  Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment 
Nature Communications  2016;7:13616.
Targeting self-renewal is an important goal in cancer therapy and recent studies have focused on Notch signalling in the maintenance of stemness of glioma stem cells (GSCs). Understanding cancer-specific Notch regulation would improve specificity of targeting this pathway. In this study, we find that Notch1 activation in GSCs specifically induces expression of the lncRNA, TUG1. TUG1 coordinately promotes self-renewal by sponging miR-145 in the cytoplasm and recruiting polycomb to repress differentiation genes by locus-specific methylation of histone H3K27 via YY1-binding activity in the nucleus. Furthermore, intravenous treatment with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system induces GSC differentiation and efficiently represses GSC growth in vivo. Our results highlight the importance of the Notch-lncRNA axis in regulating self-renewal of glioma cells and provide a strong rationale for targeting TUG1 as a specific and potent therapeutic approach to eliminate the GSC population.
Self-renewal of cancer stem cells can contribute to glioma progression. Here, the authors show that Notch1 activation in glioma stem cells induces expression of the lncRNA TUG1, which promotes self-renewal through the repression of differentiation genes, and that targeting TUG1 represses glioma growth in vivo.
PMCID: PMC5150648  PMID: 27922002
3.  Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis 
Pioglitazone (PGZ), a peroxisome proliferator-activated receptor γ agonist, which is known as a type 2 diabetes drug, inhibits cell proliferation in various cancer cell lines, including prostate carcinomas. This study focused on the effect of PGZ on prostate carcinogenesis using a transgenic rat for an adenocarcinoma of prostate (TRAP) model. Adenocarcinoma lesions as a percentage of overall lesions in the ventral prostate were significantly reduced by PGZ treatment in a dose-dependent manner. The number of adenocarcinomas per given area in the ventral prostate was also significantly reduced by PGZ treatment. The Ki67 labeling index in the ventral prostate was also significantly reduced by PGZ. Decreased cyclin D1 expression in addition to the inactivation of both p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)κB were detected in PGZ-treated TRAP rat groups. In LNCaP, a human androgen-dependent prostate cancer cell line, PGZ also inhibited cyclin D1 expression and the activation of both p38 MAPK and NFκB. The suppression of cultured cell growth was mainly regulated by the NFκB pathway as detected using specific inhibitors in both LNCaP and PC3, a human androgen-independent prostate cancer cell line. These data suggest that PGZ possesses a chemopreventive potential for prostate cancer.
PMCID: PMC5187871  PMID: 27973395
peroxisome proliferator-activated receptor γ; pioglitazone; prostate; carcinogenesis; prostate cancer; nuclear factor κB
4.  4′-Modified Nucleoside Analogs: Potent Inhibitors Active against Entecavir-resistant Hepatitis B Virus 
Hepatology (Baltimore, Md.)  2015;62(4):1024-1036.
Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against HIV-1 and HBV. However, both viruses often acquire NRTI resistance, making it crucial to develop more potent agents that offer profound viral suppression. We report here that 4′-C-cyano-2-amino-2′-deoxyadenosine (CAdA) is a novel highly potent inhibitor of both HBV (IC50=0.4 nM) and HIV-1 (IC50=0.4 nM). In contrast, the approved anti-HBV NRTI entecavir (ETV) potently inhibits HBV (IC50=0.7 nM) but is much less active against HIV-1 (IC50=1,000 nM). Similarly, the highly potent HIV-1 inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) (IC50=0.3 nM) is less active against HBV (IC50=160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50=7.2 nM) and ETV-resistant HBV (IC50=69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 μM. Once daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (p=0.0005) in human-liver-chimeric/HBVETV-RL180M/S202G/M204V-infected mice, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4′-cyano of CAdA-triphosphate (TP), but not the longer 4′-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4′-substitutions of both NRTIs. The ETV-TP’s cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. Conclusion: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.
PMCID: PMC4589464  PMID: 26122273
5.  Ophiocordyceps formosana improves hyperglycemia and depression-like behavior in an STZ-induced diabetic mouse model 
A newly defined Cordyceps species, Ophiocordyceps formosana (O. formosana) has been implicated in multitudinous bioactivities, including lowering glucose and cholesterol levels and modulating the immune system. However, few literatures demonstrate sufficient evidence to support these proposed functions. Although the use of Cordyceps spp. has been previously addressed to improve insulin insensitivity and improve the detrimental symptoms of depression; its mechanistic nature remains unsettled. Herein, we reveal the effects of O. formosana in ameliorating hyperglycemia accompanied with depression.
Diabetes was induced in mice by employing streptozotocin(STZ), a chemical that is toxic to insulin-producing β cells of the pancreas. These streptozotocin (STZ)-induced diabetic mice showed combined symptoms of hyperglycemia and depressive behaviors. Twenty-four STZ-induced mice were randomly divided into 3 groups subjected to oral gavage with 100 μL solution of either PBS or 25 mg/mL Ophiocordyceps formosana extract (OFE) or 2 mg/mL rosiglitazone (Rosi, positive control group). Treatments were administered once per day for 28 days. An additional 6 mice without STZ induction were treated with PBS to serve as the control group. Insulin sensitivity was measured by a glucose tolerance test and levels of adiponectin in plasma and adipose tissue were also quantified. Behavioral tests were conducted and levels of monoamines in various brain regions relating to depression were evaluated.
HPLC analysis uncovered three major constituents, adenosine, D-mannitol and cordycepin, within O. formosana similar to other prestigious medicinal Cordyceps spp.. STZ-induced diabetic mice demonstrated decreased body weight and subcutaneous adipose tissue, while these symptoms were recovered in mice receiving OFE treatment. Moreover, the OFE group displayed improved insulin sensitivity and elevated adiponectin within the plasma and adipose tissue. The anti-depressive effect of OFE was observed in various depression-related behavior tests. Concurrently, neurotransmitters, like 5-HT and dopamine in the frontal cortex, striatum and hippocampus were found to be up-regulated in OFE-treated mice.
Our findings illustrated, for the first time, the medicinal merits of O. formosana on Type I diabetes and hyperglycemia-induced depression. OFE were found to promote the expression of adiponectin, which is an adipokine involved in insulin sensitivity and hold anti-depressive effects. In addition, OFE administration also displayed altered levels of neurotransmitters in certain brain regions that may have contributed to its anti-depressive effect. Collectively, this current study provided insights to the potential therapeutic effects of O. formosana extracts in regards to hyperglycemia and its depressive complications.
PMCID: PMC4995616  PMID: 27553852
Ophiocordyceps formosana; Insulin insensitivity; Depression
6.  Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells 
PLoS ONE  2016;11(7):e0159246.
Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.
PMCID: PMC4943650  PMID: 27409080
7.  Preventive Effects of Fermented Brown Rice and Rice Bran against Prostate Carcinogenesis in TRAP Rats 
Nutrients  2016;8(7):421.
Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is considered to have the potential to prevent chemically-induced carcinogenesis in multiple organs of rodents. In the present study, we evaluated the possible chemopreventive effects of FBRA against prostate tumorigenesis. Six-week-old male rats of the transgenic rat for adenocarcinoma of prostate (TRAP) strain were fed diets containing 5% or 10% FBRA for 15 weeks. Animals were sacrificed at 21 weeks of age, and the ventral and lateral prostate were removed for histopathological evaluation and immunoblot analyses. FBRA decreased the incidence of adenocarcinoma in the lateral prostate and suppressed the progression of prostate carcinogenesis. Treatment with FBRA induced apoptosis and inhibited cell proliferation in histologically high-grade prostatic intraepithelial neoplasias. Phospho-AMP-activated kinase α (Thr172) was up-regulated in the prostate of rats fed the diet supplemented with FBRA. These results indicate that FBRA controls tumor growth by activating pathways responsive to energy deprivation and suggest that FBRA has translational potential for the prevention of human prostate cancer.
PMCID: PMC4963897  PMID: 27409632
fermented brown rice and rice bran with Aspergillus oryzae (FBRA); prostate cancer; transgenic rat
8.  Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors 
Cancer Science  2016;107(7):924-935.
Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT‐N (NIKKISO) after administration to the rat lung. MWCNT‐N was fractionated by passing it through a sieve with a pore size of 25 μm. The average lengths of the MWCNT were 4.2 μm before filtration and 2.6 μm in the flow‐through fraction; the length of the retained MWCNT could not be determined. For the present study, 10‐week‐old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT‐N before filtration, MWCNT‐N flow‐through and MWCNT‐N retained groups. Administration was by the trans‐tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo‐alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.
PMCID: PMC4946724  PMID: 27098557
Intratracheal instillation; lung tumors; malignant mesothelioma; multiwalled carbon nanotubes; rat
9.  Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system 
Scientific Reports  2016;6:27857.
cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism.
PMCID: PMC4904192  PMID: 27291420
10.  Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs 
The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell–specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte–specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte–specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell–regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell–specific transcriptional activity.
PMCID: PMC4767349  PMID: 26808502
11.  MafB deficiency accelerates the development of obesity in mice 
FEBS Open Bio  2016;6(6):540-547.
MafB, a transcription factor expressed selectively in macrophages, has important roles in some macrophage‐related diseases, especially in atherosclerosis. In this study, we investigated the mechanism by which hematopoietic‐specific MafB deficiency induces the development of obesity. Wild‐type and hematopoietic cell‐specific Mafb‐deficient mice were fed a high‐fat diet for 10 weeks. The Mafb‐deficient mice exhibited higher body weights and faster rates of body weight increase than control mice. The Mafb‐deficient mice also had a higher percentage of body fat than the wild‐type mice, due to increased adipocyte size and serum cholesterol levels. Reverse transcription‐PCR analysis showed a reduction in apoptosis inhibitor of macrophage (AIM) in Mafb‐deficient adipose tissue. AIM is known as an inhibitor of lipogenesis in adipocytes and is expressed in adipose tissue macrophages. Collectively, our data suggest that Mafb deficiency in hematopoietic cells accelerates the development of obesity.
PMCID: PMC4887969  PMID: 27419056
adipose tissue macrophages; apoptosis inhibitor of macrophage; MafB; obesity
12.  Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming 
PLoS ONE  2016;11(3):e0150715.
Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.
PMCID: PMC4778944  PMID: 26943822
13.  Pain-reducing anesthesia prevents oxidative stress in human term placenta 
Anesthesia is sometimes used for the reduction of maternal pain in normal human term labor, but whether the drugs affect oxidative stress remains unclear. The placenta serves as an interface between the maternal and fetal vasculature. In this study, we immunohistochemically analyzed two markers for oxidative stress, namely 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal–modified proteins (HNE), using placentas from 21 cases of normal tansvaginal delivery (V group), 20 Caesarean sections (C group), and 17 normal transvaginal deliveries with epidural anesthesia (E group). 8-OHdG staining in the nuclei of trophoblasts lining the chorionic villi was significantly stronger in the V group either compared with the C or E group (p<0.001), without significant differences in the C and E groups (p = 0.792). Moderate to intense staining by HNE of the intravascular serum of chorionic villi vasculature was frequently observed in the placentas from the V group, but less frequently of those in either C or E groups (p<0.001), nor the p value comparing the C and E groups was significant (p = 0.128) for HNE staining. Our results suggest that although the role of oxidative stress and its influences on fetal state in the placenta in labor remains unclear, it seems to be lessened by epidural anesthesia.
PMCID: PMC4788402  PMID: 27013783
oxidative stress; placenta; anesthesia; 8-hydroxy-2'-deoxyguanosine; 4-hydroxy-2-nonenal-modified proteins
14.  TIMP-1 as well as Microvessel Invasion and High Nuclear Grade Is a Significant Determinant Factor for Extension of Tumor Diameter in Localized RCC 
Journal of Oncology  2016;2016:5035127.
Objectives. To clarify what kind of pathological factor is necessary for the extension of tumor diameter in localized RCC, we studied localized RCC patients. Methods. We retrospectively reviewed medical records of 237 RCC patients in our institute who underwent nephrectomy. We performed immune histological analysis of MMP-2, MMP-9, TIMP-1, TIMP-2, and MT-MMP-1 for all samples. Results. Among the clinicopathological factors, multivariate analysis revealed nuclear grade; TIMP-2 and MT-MMP-1 were independent prognostic factors of localized RCC (risk ratio 1.50, p = 0.037, risk ratio 1.12, p = 0.008, and risk ratio 1.84, p = 0.045, resp.). By the multiple logistic regression analysis among pT1a versus pT1b, TIMP-1 was an independent factor (risk ratio 3.30, p = 0.010) whereas all pT1 versus pT2a and all pT1 + pT2a versus pT2b high nuclear grade (risk ratio 5.15, p = 0.0015) and Micro vessel invasion (MVI, risk ratio 3.08, p = 0.002) were independent factors. For all pT1 + pT2a versus pT2b, nuclear grade (risk ratio 3.39, p = 0.020) and MVI (risk ratio 2.91, p = 0.018) were independent factors. Conclusion. Higher expression of TIMP-1 is necessary for advancement tumor diameter from pT1a to pT1b, and a process of tumor diameter extension beyond pT1 and pT2a category needs presence of MVI and high nuclear grade.
PMCID: PMC4785260  PMID: 27019657
15.  β-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development 
PLoS ONE  2016;11(2):e0150010.
The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.
PMCID: PMC4763111  PMID: 26901059
16.  SCFFbxo22-KDM4A targets methylated p53 for degradation and regulates senescence 
Nature Communications  2016;7:10574.
Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCFFbxo22-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCFFbxo22 ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCFFbxo22-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22−/− mice are almost half the size of Fbxo22+/− mice owing to the accumulation of p53. These results indicate that SCFFbxo22-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.
Cellular senescence—the permanent cessation of cell proliferation—is a process that can be deregulated in cancer and other aging-related diseases. Here the authors demonstrate that the SCFFbxo22-KDM4A complex plays an essential role during senescence as an E3 ligase that targets methylated p53 for degradation.
PMCID: PMC4754341  PMID: 26868148
17.  Four-dimensional noise reduction using the time series of medical computed tomography datasets with short interval times: a static-phantom study 
PeerJ  2016;4:e1680.
Backgrounds. This study examines the hypothesis that four-dimensional noise reduction (4DNR) with short interval times reduces noise in cardiac computed tomography (CCT) using “padding” phases. Furthermore, the capability of reducing the reduction dose in CCT using this post-processing technique was assessed.
Methods. Using base and quarter radiation doses for CCT (456 and 114 mAs/rot with 120 kVp), a static phantom was scanned ten times with retrospective electrocardiogram gating, and 4DNR with short interval times (50 ms) was performed using a post-processing technique. Differences in the computed tomography (CT) attenuation, contrast-to-noise ratio (CNR) and spatial resolution with modulation transfer function in each dose image obtained with and without 4DNR were assessed by conducting a Tukey–Kramer’s test and non-inferiority test.
Results. For the base dose, by using 4DNR, the CNR was improved from 1.18 ± 0.15 to 2.08 ± 0.20 (P = 0.001), while the CT attenuation and spatial resolution of the image of 4DNR did not were significantly inferior to those of reference image (P < 0.001). CNRs of the quarter-dose image in 4DNR also improved to 1.28 ± 0.11, and were not inferior to those of the non-4DNR images of the base dose (P < 0.001).
Conclusions. 4DNR with short interval times significantly reduced noise. Furthermore, applying this method to CCT would have the potential of reducing the radiation dose by 75%, while maintaining a similar image noise level.
PMCID: PMC4756736  PMID: 26893966
Image quality; Computed tomography; Radiation dose; Temporal noise reduction; Cardiac CT
18.  Overexpression of RORγt Enhances Pulmonary Inflammation after Infection with Mycobacterium Avium 
PLoS ONE  2016;11(1):e0147064.
Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial disease in humans. The role of Th17 immunity in the pathogenesis of intracellular bacteria, such as MAC, is not currently understood. Transcription factor RAR-related orphan receptor gamma t (RORγt) is known as the master regulator for Th17 cell development. Here, we investigated the role of RORγt in host responses against MAC infection. Wild-type (WT) mice and RORγt-overexpressing mice were infected with MAC via intratracheal inoculation. Systemic MAC growth was not different between WT mice and RORγt-overexpressing mice. However, neutrophilic pulmonary inflammation following MAC infection was enhanced in RORγt-overexpressing mice compared with that in WT mice. The cytokine expression shifted toward a Th17 phenotype in the lungs of RORγt-overexpressing mice following MAC infection; the levels of IL-6 and IL-17 were significantly higher in the lung of these mice than in WT mice. In addition to the increase in IL-17 single-positive T cells, T cells producing both IL-17 and interferon-γ were elevated in the lung of RORγt-overexpressing mice following MAC infection. These findings suggest that RORγt overexpression-mediated Th17 bias contributes to local inflammation rather than systemic responses, by regulating neutrophil recruitment into the sites of infection during MAC infection.
PMCID: PMC4718649  PMID: 26784959
19.  Connexin 32 dysfunction promotes ethanol-related hepatocarcinogenesis via activation of Dusp1-Erk axis 
Oncotarget  2015;7(2):2009-2021.
There is abundant epidemiological evidence that heavy alcohol intake contributes to hepatocellular carcinoma (HCC) development. Previous reports indicated that connexin 32 (Cx32), which is a major hepatocyte gap junction protein, is down-regulated in chronic liver disease and has a protective role in hepatocarcinogenesis. However, functions of Cx32 in alcohol-related hepatocarcinogenesis have not been clarified. To evaluate them, 9-week-old Cx32 dominant negative transgenic (Tg) rats and their wild-type (Wt) littermates were given 1 % or 5 % ethanol (EtOH) or water ad libitum, for 16 weeks after an intraperitoneal injection of diethylnitrosamine (200 mg/kg). EtOH significantly increased the incidence and multiplicity of HCC and total tumors in a dose-dependent manner in Tg rats, but not in Wt rats. Although the number and area of glutathione S-transferase placental form (GST-P) positive foci were not significantly different between the groups, EtOH increased the Ki-67 labeling indices in GST-P positive foci only in Tg rats. EtOH up-regulated phosphorylated Erk1/2 with decrease of the Erk1/2 inhibitor, dual specificity protein phosphatase 1 (Dusp1) in whole livers of Tg and Wt rats. Immunofluorescence staining and quantitative RT-PCR revealed that EtOH significantly increased nucleolar localization of phosphorylated Erk1/2 and contrastingly reduced Dusp1 protein and mRNA expression in GST-P positive foci and HCC of Tg rats as compared to those of Wt rats. These findings suggest that Cx32 dysfunction like in chronic liver disease promoted EtOH-associated hepatocarcinogenesis through dysregulation of Erk-Dusp1 signaling.
PMCID: PMC4811513  PMID: 26655499
connexin 32; alcohol; hepatocarcinogenesis; Erk; Dusp1
20.  Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies 
Experimental Animals  2016;65(2):175-187.
The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module (“Kibo”) on the International Space Station. The CBEF provides “space-based controls” by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.
PMCID: PMC4873486  PMID: 26822934
habitat cage unit; microgravity; mouse; spaceflight
21.  Th17-biased RORγt Transgenic Mice Become Susceptible to a Viral Model for Multiple Sclerosis 
In a viral model for multiple sclerosis (MS), Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), both immune-mediated tissue damage (immunopathology) and virus persistence have been shown to cause pathology. T helper (Th) 17 cells are a Th cell subset, whose differentiation requires the transcription factor retinoic acid-related orphan receptor (ROR) γt, secrete pro-inflammatory cytokines, including IL-17, and can antagonize Th1 cells. Although Th17 cells have been shown to play a pathogenic role in immune-mediated diseases or a protective role in bacterial and fungal infections, their role in viral infections is unclear. Using newly established Th17-biased RORγt Tg mice, we tested whether Th17 cells could play a pathogenic or protective role in TMEV-IDD by contributing to immunopathology and/or by modulating anti-viral Th1 immune responses. While TMEV-infected wild-type littermate C57BL/6 mice are resistant to TMEV-IDD, RORγt Tg mice developed inflammatory demyelinating lesions with virus persistence in the spinal cord. TMEV-infected RORγt Tg mice had higher levels of IL-17, lower levels of interferon-γ, and fewer CD8+ T cells, without alteration in overall levels of anti-viral lymphoproliferative and antibody responses, compared with TMEV-infected wild-type mice. This suggests that a Th17-biased “gain-of-function” mutation could increase susceptibility to virus-mediated demyelinating diseases.
PMCID: PMC4258441  PMID: 25046854
Picornaviridae; CNS Disease; Demyelinating disease; Viral CNS infections; CD8-positive lymphocytes; Disease susceptibility; Inflammation
22.  Identification, localization, and functional analysis of the homologues of mouse CABS1 protein in porcine testis 
Experimental Animals  2016;65(3):253-265.
Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.
PMCID: PMC4976239  PMID: 26960363
porcine; calcium-binding protein; CABS1; transmembrane; testis; acrosome; epididymal sperm
23.  Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice 
Experimental Animals  2016;65(3):319-327.
In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.
PMCID: PMC4976246  PMID: 27053096
bicistronic; cre-loxP; CRISPR/Cas9; pancreatic β cells; mouse
24.  Chemopreventive effect of resveratrol and apocynin on pancreatic carcinogenesis via modulation of nuclear phosphorylated GSK3β and ERK1/2 
Oncotarget  2015;6(40):42963-42975.
Despite progress in clinical cancer medicine in multiple fields, the prognosis of pancreatic cancer has remained dismal. Recently, chemopreventive strategies using phytochemicals have gained considerable attention as an alternative in the management of cancer. The present study aimed to evaluate the chemopreventive effects of resveratrol (RV) and apocynin (AC) in N-Nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamster. RV- and AC-treated hamsters showed significant reduction in the incidence of pancreatic cancer with a decrease in Ki-67 labeling index in dysplastic lesions. RV and AC suppressed cell proliferation of human and hamster pancreatic cancer cells by inhibiting the G1 phase of the cell cycle with cyclin D1 downregulation and inactivation of AKT-GSK3β and ERK1/2 signaling. Further, decreased levels of GSK3βSer9 and ERK1/2 phosphorylation and cyclin D1 expression in the nuclear fraction were observed in cells treated with RV or AC. Nuclear expression of phosphorylated GSK3βSer9 was also decreased in dysplastic lesions and adenocarcinomas of hamsters treated with RV or AC in vivo. These results suggest that RV and AC reduce phosphorylated GSK3βSer9 and ERK1/2 in the nucleus, resulting in inhibition of the AKT-GSK3β and ERK1/2 signaling pathways and cell cycle arrest in vitro and in vivo. Taken together, the present study indicates that RV and AC have potential as chemopreventive agents for pancreatic cancer.
PMCID: PMC4767484  PMID: 26556864
resveratrol; apocynin; pancreatic carcinogenesis; GSK3β; hamster
25.  A Sulfated Glycosaminoglycan Linkage Region Is a Novel Type of Human Natural Killer-1 (HNK-1) Epitope Expressed on Aggrecan in Perineuronal Nets 
PLoS ONE  2015;10(12):e0144560.
Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.
PMCID: PMC4686076  PMID: 26659409

Results 1-25 (98)