start-ver=1.4 cd-journal=joma no-vol=17 cd-vols= no-issue=11 article-no= start-page=e0277307 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20221117 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption en-subtitle= kn-subtitle= en-abstract= kn-abstract=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(Delta 105)). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D-4, 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 D-4 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 D-4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1. en-copyright= kn-copyright= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=AndoAoi en-aut-sei=Ando en-aut-mei=Aoi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MizusawaYohei en-aut-sei=Mizusawa en-aut-mei=Yohei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=OnoMitsuaki en-aut-sei=Ono en-aut-mei=Mitsuaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HattoriTakako en-aut-sei=Hattori en-aut-mei=Takako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=HabutaMunenori en-aut-sei=Habuta en-aut-mei=Munenori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=OohashiToshitaka en-aut-sei=Oohashi en-aut-mei=Toshitaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=KubotaSatoshi en-aut-sei=Kubota en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Faculty of Medicine, Okayama University Medical School kn-affil= affil-num=3 en-affil=Faculty of Medicine, Okayama University Medical School kn-affil= affil-num=4 en-affil=Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=9 en-affil=Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= END start-ver=1.4 cd-journal=joma no-vol=13 cd-vols= no-issue=2 article-no= start-page=285 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220131 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice en-subtitle= kn-subtitle= en-abstract= kn-abstract=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, x10(6)), 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. en-copyright= kn-copyright= en-aut-name=XueRuizhi en-aut-sei=Xue en-aut-mei=Ruizhi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=LinWenfeng en-aut-sei=Lin en-aut-mei=Wenfeng kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SunJingkai en-aut-sei=Sun en-aut-mei=Jingkai kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KinoshitaRie en-aut-sei=Kinoshita en-aut-mei=Rie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=OchiaiKazuhiko en-aut-sei=Ochiai en-aut-mei=Kazuhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=FutamiJunichiro en-aut-sei=Futami en-aut-mei=Junichiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=WatanabeMasami en-aut-sei=Watanabe en-aut-mei=Masami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=SakaguchiMasakiyo en-aut-sei=Sakaguchi en-aut-mei=Masakiyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=TangZhengyan en-aut-sei=Tang en-aut-mei=Zhengyan kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=HuangPeng en-aut-sei=Huang en-aut-mei=Peng kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=NasuYasutomo en-aut-sei=Nasu en-aut-mei=Yasutomo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=KumonHiromi en-aut-sei=Kumon en-aut-mei=Hiromi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= affil-num=1 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Laboratory of Veterinary Hygiene, Nippon Veterinary and Life Science University kn-affil= affil-num=7 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=8 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=9 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=10 en-affil=Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=11 en-affil=Department of Urology, Xiangya Hospital, Central South University kn-affil= affil-num=12 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=13 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=14 en-affil=Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University kn-affil= en-keyword=Dkk3/REIC kn-keyword=Dkk3/REIC en-keyword=fibrous sheath kn-keyword=fibrous sheath en-keyword=knock-out kn-keyword=knock-out en-keyword=RNA-seq kn-keyword=RNA-seq en-keyword=spermiation kn-keyword=spermiation en-keyword=sperm motility kn-keyword=sperm motility END start-ver=1.4 cd-journal=joma no-vol=15 cd-vols= no-issue=10 article-no= start-page=e0240333 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201015 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=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 en-subtitle= kn-subtitle= en-abstract= kn-abstract=CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known thatFibroblast growth factor 10(Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in theFgf10-CRISPR F0 mice. However, how the lung phenotype in theFgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in theFgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functionalFgf10genotypes. Firstly, according to a previous report,Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of theFgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 +/- 6.2% in type I, 25.3 +/- 2.7% in type II, and 54.3 +/- 9.5% in type III (mean +/- standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in theFgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functionalFgf10genotypes. These data suggest theFgf10gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, theFgf10-CRISPR F0 mouse would present an ideal experimental system to explore it. en-copyright= kn-copyright= en-aut-name=HabutaMunenori en-aut-sei=Habuta en-aut-mei=Munenori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YasueAkihiro en-aut-sei=Yasue en-aut-mei=Akihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SuzukiKen-Ichi T. en-aut-sei=Suzuki en-aut-mei=Ken-Ichi T. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SatoKeita en-aut-sei=Sato en-aut-mei=Keita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KonoHitomi en-aut-sei=Kono en-aut-mei=Hitomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakayamaAyuko en-aut-sei=Takayama en-aut-mei=Ayuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=BandoTetsuya en-aut-sei=Bando en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MiyaishiSatoru en-aut-sei=Miyaishi en-aut-mei=Satoru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=OyadomariSeiichi en-aut-sei=Oyadomari en-aut-mei=Seiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=TanakaEiji en-aut-sei=Tanaka en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= affil-num=1 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School kn-affil= affil-num=3 en-affil=Department of Mathematical and Life Sciences, Hiroshima University kn-affil= affil-num=4 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Center for the Development of New Model Organisms, National Institute for Basic Biology kn-affil= affil-num=8 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=9 en-affil=Department of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=10 en-affil=Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University kn-affil= affil-num=11 en-affil=Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School kn-affil= affil-num=12 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= END start-ver=1.4 cd-journal=joma no-vol=74 cd-vols= no-issue=3 article-no= start-page=199 end-page=208 dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=202006 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=BandoTetsuya en-aut-sei=Bando en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OyadomariSeiichi en-aut-sei=Oyadomari en-aut-mei=Seiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=OchiaiKazuhiko en-aut-sei=Ochiai en-aut-mei=Kazuhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WatanabeMasami en-aut-sei=Watanabe en-aut-mei=Masami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KumonHiromi en-aut-sei=Kumon en-aut-mei=Hiromi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Division of Molecular Biology, Institute for Genome Research, University of Tokushima kn-affil= affil-num=4 en-affil=Department of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University kn-affil= affil-num=5 en-affil=Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University kn-affil= affil-num=7 en-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= en-keyword=Dkk3 knockout mouse kn-keyword=Dkk3 knockout mouse en-keyword=adrenal gland kn-keyword=adrenal gland en-keyword=glucose-regulated protein 78 kn-keyword=glucose-regulated protein 78 en-keyword=proteome kn-keyword=proteome en-keyword=endoplasmic reticulum stress kn-keyword=endoplasmic reticulum stress END start-ver=1.4 cd-journal=joma no-vol=61 cd-vols= no-issue=5 article-no= start-page=38 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=202005 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KanzakiYuki en-aut-sei=Kanzaki en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SatoKeita en-aut-sei=Sato en-aut-mei=Keita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HosokawaMio en-aut-sei=Hosokawa en-aut-mei=Mio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MatsumaeHiroshi en-aut-sei=Matsumae en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShiragaFumio en-aut-sei=Shiraga en-aut-mei=Fumio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MorizaneYuki en-aut-sei=Morizane en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= en-keyword=Kir7.1 kn-keyword=Kir7.1 en-keyword=KCNJ13 kn-keyword=KCNJ13 en-keyword=human-induced pluripotent cells kn-keyword=human-induced pluripotent cells en-keyword=retinal pigment epithelium kn-keyword=retinal pigment epithelium en-keyword=phagocytosis kn-keyword=phagocytosis END start-ver=1.4 cd-journal=joma no-vol=67 cd-vols= no-issue=3 article-no= start-page=153 end-page=164 dt-received= dt-revised= dt-accepted= dt-pub-year=2013 dt-pub=201306 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Improvement of the Efficacy of 5-aminolevulinic Acid-mediated Photodynamic Treatment in Human Oral Squamous Cell Carcinoma HSC-4 en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=YamamotoMasanao en-aut-sei=Yamamoto en-aut-mei=Masanao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KataseNaoki en-aut-sei=Katase en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=InoueKeiji en-aut-sei=Inoue en-aut-mei=Keiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=NagatsukaHitoshi en-aut-sei=Nagatsuka en-aut-mei=Hitoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=UtsumiKozo en-aut-sei=Utsumi en-aut-mei=Kozo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SasakiJunzo en-aut-sei=Sasaki en-aut-mei=Junzo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=2 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=3 en-affil= kn-affil=Department of Oral Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=4 en-affil= kn-affil=Department of Urology, Kochi University Medical School affil-num=5 en-affil= kn-affil=Department of Oral Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=6 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=7 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=8 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences en-keyword=5-aminolevulinic acid kn-keyword=5-aminolevulinic acid en-keyword=protoporphyrin IX kn-keyword=protoporphyrin IX en-keyword=oncology kn-keyword=oncology en-keyword=photodynamic therapy kn-keyword=photodynamic therapy en-keyword=apoptosis kn-keyword=apoptosis END start-ver=1.4 cd-journal=joma no-vol=7 cd-vols= no-issue=11 article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2012 dt-pub=20121126 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation en-subtitle= kn-subtitle= en-abstract= kn-abstract=Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. en-copyright= kn-copyright= en-aut-name=KobuchiHirotsugu en-aut-sei=Kobuchi en-aut-mei=Hirotsugu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MoriyaKoko en-aut-sei=Moriya en-aut-mei=Koko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OginoTetsuya en-aut-sei=Ogino en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=InoueKeiji en-aut-sei=Inoue en-aut-mei=Keiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShuinTaro en-aut-sei=Shuin en-aut-mei=Taro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YasudaTatsuji en-aut-sei=Yasuda en-aut-mei=Tatsuji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=UtsumiKozo en-aut-sei=Utsumi en-aut-mei=Kozo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=UtsumiToshihiko en-aut-sei=Utsumi en-aut-mei=Toshihiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Dept Cell Chem, Grad Sch Med Dent & Pharmaceut Sci affil-num=2 en-affil= kn-affil=Yamaguchi Univ, Grad Sch Med affil-num=3 en-affil= kn-affil=Okayama Prefectural Univ, Dept Nursing Sci, Fac Hlth & Welf Sci affil-num=4 en-affil= kn-affil=Okayama Univ, Dept Cytol & Histol, Grad Sch Med Dent & Pharmaceut Sci affil-num=5 en-affil= kn-affil=Kochi Med Sch, Dept Urol affil-num=6 en-affil= kn-affil=Kochi Med Sch, Dept Urol affil-num=7 en-affil= kn-affil=Okayama Univ, Dept Cell Chem, Grad Sch Med Dent & Pharmaceut Sci affil-num=8 en-affil= kn-affil=Okayama Univ, Dept Cytol & Histol, Grad Sch Med Dent & Pharmaceut Sci affil-num=9 en-affil= kn-affil=Yamaguchi Univ, Grad Sch Med END start-ver=1.4 cd-journal=joma no-vol=1206 cd-vols= no-issue= article-no= start-page=1 end-page=12 dt-received= dt-revised= dt-accepted= dt-pub-year=2008 dt-pub=20080623 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=alpha-lipoic acid suppresses 6-hydroxydoparnine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1 en-subtitle= kn-subtitle= en-abstract= kn-abstract=

We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. in addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.

en-copyright= kn-copyright= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ShiosakaMasahiko en-aut-sei=Shiosaka en-aut-mei=Masahiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OginoTetsuya en-aut-sei=Ogino en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=OkimuraYuya en-aut-sei=Okimura en-aut-mei=Yuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=UtsumiToshihiko en-aut-sei=Utsumi en-aut-mei=Toshihiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SatoEisuke F. en-aut-sei=Sato en-aut-mei=Eisuke F. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=AkagiReiko en-aut-sei=Akagi en-aut-mei=Reiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=InoueMasayasu en-aut-sei=Inoue en-aut-mei=Masayasu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=UtsumiKozo en-aut-sei=Utsumi en-aut-mei=Kozo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=SasakiJunzo en-aut-sei=Sasaki en-aut-mei=Junzo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= affil-num=1 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=2 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=3 en-affil= kn-affil=Department of Pathological Research, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=4 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=5 en-affil= kn-affil=Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University affil-num=6 en-affil= kn-affil=Department of Biochemistry and Molecular Pathology Osaka City University Medical School affil-num=7 en-affil= kn-affil=Faculty of Health and Welfare Science, Okayama Prefectural University affil-num=8 en-affil= kn-affil=Department of Biochemistry and Molecular Pathology Osaka City University Medical School affil-num=9 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences affil-num=10 en-affil= kn-affil=Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences en-keyword=apoptosis kn-keyword=apoptosis en-keyword=glutathione kn-keyword=glutathione en-keyword=gamma-glutamylcysteine synthetase; heme oxygenase-1 kn-keyword=gamma-glutamylcysteine synthetase; heme oxygenase-1 en-keyword=6-hydroxydopamine kn-keyword=6-hydroxydopamine en-keyword=alpha-lipoic acid kn-keyword=alpha-lipoic acid en-keyword=Nrf2 kn-keyword=Nrf2 END