研究者詳細 - 藤田　洋史

Protrusion of KCNJ13 Gene Knockout Retinal Pigment Epithelium Due to Oxidative Stress–Induced Cell Death

システイニルロイコトリエン受容体CysLTR1は破骨細胞分化と骨吸収に必須ではない

藤田洋史, 服部高子, 久保田聡

日本骨代謝学会学術集会プログラム抄録集 41回 162 - 162 2023年7月

Yuki Kanzaki, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Yuki Morizane, Hideyo Ohuchi

Investigative Opthalmology & Visual Science 63 ( 12 ) 29 - 29 2022年11月

Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption

Hirofumi Fujita, Aoi Ando, Yohei Mizusawa, Mitsuaki Ono, Takako Hattori, Munenori Habuta, Toshitaka Oohashi, Satoshi Kubota, Hideyo Ohuchi

PLOS ONE 17 ( 11 ) e0277307 - e0277307 2022年11月

掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1^Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D₄, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D₄ and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D₄-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice. 国際誌

Ruizhi Xue, Wenfeng Lin, Hirofumi Fujita, Jingkai Sun, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Masami Watanabe, Hideyo Ohuchi, Masakiyo Sakaguchi, Zhengyan Tang, Peng Huang, Yasutomo Nasu, Hiromi Kumon

Genes 13 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

DOI： 10.3390/genes13020285

UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions. 国際誌

Hirofumi Fujita, Munenori Habuta, Takako Hattori, Satoshi Kubota, Hiromi Kumon, Hideyo Ohuchi

Biochemical and biophysical research communications 566 184 - 189 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

DOI： 10.1016/j.bbrc.2021.06.013

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung

副腎髄質におけるUCP1タンパク質発現のUCP1レポーターマウスを用いた検証(UCP1-reporter mice reveal UCP1 protein expression in the adrenal medulla)

Fujita Hirofumi, Habuta Munenori, Hattori Takako, Kubota Satoshi, Kumon Hiromi, Ohuchi Hideyo

The Journal of Physiological Sciences 71 ( Suppl.1 ) 105 - 105 2021年8月

Munenori Habuta, Akihiro Yasue, Ken-ichi T. Suzuki, Hirofumi Fujita, Keita Sato, Hitomi Kono, Ayuko Takayama, Tetsuya Bando, Satoru Miyaishi, Seiichi Oyadomari, Eiji Tanaka, Hideyo Ohuchi

PLOS ONE 15 ( 10 ) e0240333 - e0240333 2020年10月

Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland.

Hirofumi Fujita, Tetsuya Bando, Seiichi Oyadomari, Kazuhiko Ochiai, Masami Watanabe, Hiromi Kumon, Hideyo Ohuchi

Acta medica Okayama 74 ( 3 ) 199 - 208 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.

DOI： 10.18926/AMO/59950

KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells. 国際誌

Yuki Kanzaki, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Fumio Shiraga, Yuki Morizane, Hideyo Ohuchi

Investigative ophthalmology & visual science 61 ( 5 ) 38 - 38 2020年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

DOI： 10.1167/iovs.61.5.38

Metabolic abnormalities in adult T-cell leukemia/lymphoma and induction of specific leukemic cell death using photodynamic therapy. 査読

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla. 査読

Habuta M, Fujita H, Sato K, Bando T, Inoue J, Kondo Y, Miyaishi S, Kumon H, Ohuchi H

Cell and tissue research 379 ( 1 ) 157 - 167 2019年10月

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye. 査読

Kato M, Sato K, Habuta M, Fujita H, Bando T, Morizane Y, Shiraga F, Miyaishi S, Ohuchi H

Biochemistry and biophysics reports 19 100665 2019年9月

Glutathione accelerates osteoclast differentiation and inflammatory bone destruction. 査読国際誌

Fujita H, Ochi M, Ono M, Aoyama E, Ogino T, Kondo Y, Ohuchi H

Free radical research 53 ( 2 ) 1 - 11 2019年3月

Oka T, Mizuno H, Sakata M, Fujita H, Yoshino T, Yamano Y, Utsumi K, Masujima T, Utsunomiya A

Scientific reports 8 ( 1 ) 14979 2018年10月

Expression analysis of Dickkopf-related protein 3 (Dkk3) suggests its pleiotropic roles for a secretory glycoprotein in adult mouse

Junji Inoue, Hirofumi Fujita, Tetsuya Bando, Yoichi Kondo, Hiromi Kumon, Hideyo Ohuchi

JOURNAL OF MOLECULAR HISTOLOGY 48 ( 1 ) 29 - 39 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.

DOI： 10.1007/s10735-016-9703-2

抗酸化物質グルタチオンは炎症性骨破壊を促進する

藤田洋史, 大野充昭, 青山絵理子, 大内淑代

日本骨代謝学会学術集会プログラム抄録集 34回 206 - 206 2016年7月

Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma

Hirofumi Fujita, Keisuke Nagakawa, Hirotsugu Kobuchi, Tetsuya Ogino, Yoichi Kondo, Keiji Inoue, Taro Shuin, Toshihiko Utsumi, Kozo Utsumi, Junzo Sasaki, Hideyo Ohuchi

CANCER RESEARCH 76 ( 7 ) 1837 - 1846 2016年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies.

DOI： 10.1158/0008-5472.CAN-15-1484

マウス頭蓋冠におけるリポポリサッカリドによる骨破壊とグルタチオンの効果

藤田洋史, 大野充昭, 青山絵理子, 荻野哲也, 近藤洋一, 大内淑代

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1LBA049] - [1LBA049] 2015年12月

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

Hirofumi Fujita, Masanao Yamamoto, Tetsuya Ogino, Hirotsugu Kobuchi, Naoko Ohmoto, Eriko Aoyama, Takashi Oka, Tohru Nakanishi, Keiji Inoue, Junzo Sasaki

CELL BIOCHEMISTRY AND FUNCTION 32 ( 1 ) 77 - 86 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright (c) 2013 John Wiley & Sons, Ltd.

DOI： 10.1002/cbf.2974

Improvement of the Efficacy of 5-aminolevulinic Acid-mediated Photodynamic Treatment in Human Oral Squamous Cell Carcinoma HSC-4

Masanao Yamamoto, Hirofumi Fujita, Naoki Katase, Keiji Inoue, Hitoshi Nagatsuka, Kozo Utsumi, Junzo Sasaki, Hideyo Ohuchi

ACTA MEDICA OKAYAMA 67 ( 3 ) 153 - 164 2013年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OKAYAMA UNIV MED SCHOOL

Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum.

DOI： 10.18926/AMO/50408