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=23 cd-vols= no-issue=23 article-no= start-page=15311 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20221204 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Elevated Expression of CCN3 in Articular Cartilage Induces Osteoarthritis in Hip Joints Irrespective of Age and Weight Bearing en-subtitle= kn-subtitle= en-abstract= kn-abstract=Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1 beta than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1 beta in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnf alpha, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA. en-copyright= kn-copyright= en-aut-name=HiroseKazuki en-aut-sei=Hirose en-aut-mei=Kazuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KuwaharaMiho en-aut-sei=Kuwahara en-aut-mei=Miho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NakataEiji en-aut-sei=Nakata en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=TetsunagaTomonori en-aut-sei=Tetsunaga en-aut-mei=Tomonori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YamadaKazuki en-aut-sei=Yamada en-aut-mei=Kazuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SaigaKenta en-aut-sei=Saiga en-aut-mei=Kenta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=OzakiToshifumi en-aut-sei=Ozaki en-aut-mei=Toshifumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 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=9 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=10 ORCID= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=10 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= en-keyword=hip osteoarthritis kn-keyword=hip osteoarthritis en-keyword=cartilage kn-keyword=cartilage en-keyword=cellular communication network factor 3 (CCN3) kn-keyword=cellular communication network factor 3 (CCN3) en-keyword=senescence-associatedsecretory phenotype (SASP) kn-keyword=senescence-associatedsecretory phenotype (SASP) en-keyword=p16 kn-keyword=p16 en-keyword=ADAMTA4/5 kn-keyword=ADAMTA4/5 en-keyword=IL-6 kn-keyword=IL-6 en-keyword=TNFa kn-keyword=TNFa en-keyword=aging kn-keyword=aging en-keyword=Mankinscore kn-keyword=Mankinscore en-keyword=weight-bearing kn-keyword=weight-bearing en-keyword=non-weight-bearing kn-keyword=non-weight-bearing END start-ver=1.4 cd-journal=joma no-vol=23 cd-vols= no-issue=11 article-no= start-page=5887 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220524 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration en-subtitle= kn-subtitle= en-abstract= kn-abstract=Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described. en-copyright= kn-copyright= en-aut-name=KubotaSatoshi en-aut-sei=Kubota en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KawataKazumi en-aut-sei=Kawata en-aut-mei=Kazumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 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=3 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=cellular communication network factor kn-keyword=cellular communication network factor en-keyword=CCN2 kn-keyword=CCN2 en-keyword=CCN3 kn-keyword=CCN3 en-keyword=cartilage kn-keyword=cartilage en-keyword=fibrosis kn-keyword=fibrosis en-keyword=glycolysis kn-keyword=glycolysis END start-ver=1.4 cd-journal=joma no-vol=10 cd-vols= no-issue=5 article-no= start-page=1000 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220510 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction en-subtitle= kn-subtitle= en-abstract= kn-abstract=To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta. en-copyright= kn-copyright= en-aut-name=Uchida-FukuharaYoko en-aut-sei=Uchida-Fukuhara en-aut-mei=Yoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=FuShanqi en-aut-sei=Fu en-aut-mei=Shanqi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KondoSei en-aut-sei=Kondo en-aut-mei=Sei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KuwaharaMiho en-aut-sei=Kuwahara en-aut-mei=Miho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=FukuharaDaiki en-aut-sei=Fukuhara en-aut-mei=Daiki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=IslamMd Monirul en-aut-sei=Islam en-aut-mei=Md Monirul kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=KataokaKota en-aut-sei=Kataoka en-aut-mei=Kota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=EkuniDaisuke en-aut-sei=Ekuni en-aut-mei=Daisuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 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=10 ORCID= en-aut-name=MoritaManabu en-aut-sei=Morita en-aut-mei=Manabu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=IikegameMika en-aut-sei=Iikegame en-aut-mei=Mika kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=OkamuraHirohiko en-aut-sei=Okamura en-aut-mei=Hirohiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= affil-num=1 en-affil=Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Department of Preventive Dentistry, Okayama University Hospital kn-affil= affil-num=7 en-affil=Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=8 en-affil=Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=10 en-affil=Department of Biochemistry and Molecular Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=11 en-affil=Department of Preventive Dentistry, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=12 en-affil=Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=13 en-affil=Department of Oral Morphology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=maternal microbiome kn-keyword=maternal microbiome en-keyword=endochondral ossification kn-keyword=endochondral ossification en-keyword=fetal chondrocytes kn-keyword=fetal chondrocytes END start-ver=1.4 cd-journal=joma no-vol=15 cd-vols= no-issue=1 article-no= start-page=81 end-page=91 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=202114 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1 en-subtitle= kn-subtitle= en-abstract= kn-abstract= Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities. en-copyright= kn-copyright= en-aut-name=ElseoudiAbdellatif en-aut-sei=Elseoudi en-aut-mei=Abdellatif kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MizukawaTomomi en-aut-sei=Mizukawa en-aut-mei=Tomomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 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=4 ORCID= en-aut-name=KawataKazumi en-aut-sei=Kawata en-aut-mei=Kazumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TahaEman A. en-aut-sei=Taha en-aut-mei=Eman A. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu 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= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Advanced Research Center for Oral and Craniofacial Sciences, Dental School, Okayama University kn-affil= affil-num=8 en-affil=Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=FGF-1 kn-keyword=FGF-1 en-keyword=CCN2 kn-keyword=CCN2 en-keyword=Osteoarthritis kn-keyword=Osteoarthritis en-keyword=Chondrocytes kn-keyword=Chondrocytes en-keyword=Cartilage kn-keyword=Cartilage END start-ver=1.4 cd-journal=joma no-vol=21 cd-vols= no-issue=20 article-no= start-page=7556 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201013 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage en-subtitle= kn-subtitle= en-abstract= kn-abstract=Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-beta gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21. en-copyright= kn-copyright= en-aut-name=KuwaharaMiho en-aut-sei=Kuwahara en-aut-mei=Miho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KadoyaKoichi en-aut-sei=Kadoya en-aut-mei=Koichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KondoSei en-aut-sei=Kondo en-aut-mei=Sei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=FuShanqi en-aut-sei=Fu en-aut-mei=Shanqi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MiyakeYoshiko en-aut-sei=Miyake en-aut-mei=Yoshiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=OgoAyako en-aut-sei=Ogo en-aut-mei=Ayako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 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=7 ORCID= en-aut-name=FurumatsuTakayuki en-aut-sei=Furumatsu en-aut-mei=Takayuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=NakataEiji en-aut-sei=Nakata en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=SasakiTakako en-aut-sei=Sasaki en-aut-mei=Takako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=MinagiShogo en-aut-sei=Minagi en-aut-mei=Shogo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 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=13 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=14 ORCID= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Department of Biochemistry and Molecular Dentistry, 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 Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=9 en-affil=Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=10 en-affil=Department of Biochemistry, Faculty of Medicine, Oita University kn-affil= affil-num=11 en-affil=Department of Occlusal and Oral Functional Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=12 en-affil=Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=13 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=14 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= en-keyword=cellular communication network factor 3 kn-keyword=cellular communication network factor 3 en-keyword=CCN3 kn-keyword=CCN3 en-keyword=NOV kn-keyword=NOV en-keyword=primary chondrocytes kn-keyword=primary chondrocytes en-keyword=aging kn-keyword=aging en-keyword=oxidative stress kn-keyword=oxidative stress en-keyword=senescence kn-keyword=senescence en-keyword=p21 kn-keyword=p21 en-keyword=p53 kn-keyword=p53 en-keyword=SASP kn-keyword=SASP END start-ver=1.4 cd-journal=joma no-vol=21 cd-vols= no-issue=8 article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200416 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes en-subtitle= kn-subtitle= en-abstract= kn-abstract=To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes. en-copyright= kn-copyright= en-aut-name=HoshijimaMitsuhiro en-aut-sei=Hoshijima en-aut-mei=Mitsuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=AoyamaEriko en-aut-sei=Aoyama en-aut-mei=Eriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 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=5 ORCID= en-aut-name=KamiokaHiroshi en-aut-sei=Kamioka en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= en-keyword=cellular communication network factor 2 kn-keyword=cellular communication network factor 2 en-keyword=CCN2 kn-keyword=CCN2 en-keyword=CTGF kn-keyword=CTGF en-keyword=Rab14 kn-keyword=Rab14 en-keyword=yeast two-hybrid kn-keyword=yeast two-hybrid en-keyword=chondrocyte kn-keyword=chondrocyte en-keyword=ER stress kn-keyword=ER stress en-keyword=aggrecan kn-keyword=aggrecan END start-ver=1.4 cd-journal=joma no-vol=21 cd-vols= no-issue=5 article-no= start-page=1564 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200225 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes en-subtitle= kn-subtitle= en-abstract= kn-abstract=Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed. en-copyright= kn-copyright= en-aut-name=KubotaSatoshi en-aut-sei=Kubota en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=IshikawaTakanori en-aut-sei=Ishikawa en-aut-mei=Takanori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KawataKazumi en-aut-sei=Kawata en-aut-mei=Kazumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 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=4 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= en-keyword=retrotransposon kn-keyword=retrotransposon en-keyword=endogenous retrovirus kn-keyword=endogenous retrovirus en-keyword=chondrocyte kn-keyword=chondrocyte en-keyword=cartilage kn-keyword=cartilage en-keyword=skeletal development kn-keyword=skeletal development END start-ver=1.4 cd-journal=joma no-vol=241 cd-vols= no-issue=2 article-no= start-page=161 end-page=173 dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190301 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Circadian production of melatonin in cartilage modifies rhythmic gene expression en-subtitle= kn-subtitle= en-abstract= kn-abstract= Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan, and Sox9, but inhibited Col10a1 expression in primary BALB/c mouse chondrocytes. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2, and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1, and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock. en-copyright= kn-copyright= en-aut-name=FuShanqi en-aut-sei=Fu en-aut-mei=Shanqi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KuwaharaMiho en-aut-sei=Kuwahara en-aut-mei=Miho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=UchidaYoko en-aut-sei=Uchida en-aut-mei=Yoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KondoSei en-aut-sei=Kondo en-aut-mei=Sei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HayashiDaichi en-aut-sei=Hayashi en-aut-mei=Daichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShimomuraYuji en-aut-sei=Shimomura en-aut-mei=Yuji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakagakiAsami en-aut-sei=Takagaki en-aut-mei=Asami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MaruyamaYusuke en-aut-sei=Maruyama en-aut-mei=Yusuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=IkegameMika en-aut-sei=Ikegame en-aut-mei=Mika kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=HattoriAtsuhiko en-aut-sei=Hattori en-aut-mei=Atsuhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 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=12 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=13 ORCID= affil-num=1 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=9 en-affil=Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University kn-affil= affil-num=10 en-affil=Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=11 en-affil=Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University kn-affil= affil-num=12 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=13 en-affil=Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= END start-ver=1.4 cd-journal=joma no-vol=115 cd-vols= no-issue=5 article-no= start-page=854 end-page=865 dt-received= dt-revised= dt-accepted= dt-pub-year=2014 dt-pub=201405 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=CCN2 as a Novel Molecule Supporting Energy Metabolism of Chondrocytes en-subtitle= kn-subtitle= en-abstract= kn-abstract=CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a few genes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals. en-copyright= kn-copyright= en-aut-name=Maeda-UematsuAya en-aut-sei=Maeda-Uematsu en-aut-mei=Aya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=KawakiHarumi en-aut-sei=Kawaki en-aut-mei=Harumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KawataKazumi en-aut-sei=Kawata en-aut-mei=Kazumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MiyakeYoshiaki en-aut-sei=Miyake en-aut-mei=Yoshiaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 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=6 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=MoritaniNorifumi en-aut-sei=Moritani en-aut-mei=Norifumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=LyonsKaren M. en-aut-sei=Lyons en-aut-mei=Karen M. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=IidaSeiji en-aut-sei=Iida en-aut-mei=Seiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=2 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=3 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=4 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=5 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Orthopaed Surg affil-num=6 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=7 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=8 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Oral & Maxillofacial Reconstruct Surg affil-num=9 en-affil= kn-affil=Univ Calif Los Angeles, Sch Med, Dept Orthoped Surg affil-num=10 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Oral & Maxillofacial Reconstruct Surg affil-num=11 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent en-keyword=CCN2 kn-keyword=CCN2 en-keyword=CTGF kn-keyword=CTGF en-keyword=CARTILAGE kn-keyword=CARTILAGE en-keyword=CHONDROCYTES kn-keyword=CHONDROCYTES en-keyword=METABOLISM kn-keyword=METABOLISM END start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue=3 article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2013 dt-pub=20130328 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Cartilage-Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth en-subtitle= kn-subtitle= en-abstract= kn-abstract=Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth. en-copyright= kn-copyright= en-aut-name=TomitaNao en-aut-sei=Tomita en-aut-mei=Nao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=ItohShinsuke en-aut-sei=Itoh en-aut-mei=Shinsuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=AoyamaEriko en-aut-sei=Aoyama en-aut-mei=Eriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YaoMayumi en-aut-sei=Yao en-aut-mei=Mayumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=YamashiroTakashi en-aut-sei=Yamashiro en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Biochem & Mol Dent affil-num=2 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Biochem & Mol Dent affil-num=3 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Biochem & Mol Dent affil-num=4 en-affil= kn-affil=Okayama Univ, Sch Dent, Biodent Res Ctr affil-num=5 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Biochem & Mol Dent affil-num=6 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Orthodont, Grad Sch Med Dent & Pharmaceut Sci affil-num=7 en-affil= kn-affil=Okayama Univ, Sch Dent, Dept Biochem & Mol Dent END start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue=8 article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2013 dt-pub=20130812 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes en-subtitle= kn-subtitle= en-abstract= kn-abstract=To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage. en-copyright= kn-copyright= en-aut-name=ItohShinsuke en-aut-sei=Itoh en-aut-mei=Shinsuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=TomitaNao en-aut-sei=Tomita en-aut-mei=Nao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=AoyamaEriko en-aut-sei=Aoyama en-aut-mei=Eriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YutaniYasutaka en-aut-sei=Yutani en-aut-mei=Yasutaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=YamashiroTakashi en-aut-sei=Yamashiro en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=2 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=3 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=4 en-affil= kn-affil=Okayama Univ, Sch Dent, Biodental Res Ctr affil-num=5 en-affil= kn-affil=Yutani Orthopaed Clin affil-num=6 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Orthodont affil-num=7 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent END start-ver=1.4 cd-journal=joma no-vol=279 cd-vols= no-issue=19 article-no= start-page=3597 end-page=3584 dt-received= dt-revised= dt-accepted= dt-pub-year=2012 dt-pub=201210 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes en-subtitle= kn-subtitle= en-abstract= kn-abstract=To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2CCN2 and CCN2CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 x 10-9 m for CCN2 and CCN2, and 1.95 x 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent proteinCCN2 and HaloCCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3. en-copyright= kn-copyright= en-aut-name=HoshijimaMitsuhiro en-aut-sei=Hoshijima en-aut-mei=Mitsuhiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 ORCID= en-aut-name=AoyamaEriko en-aut-sei=Aoyama en-aut-mei=Eriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NishidaTakashi en-aut-sei=Nishida en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YamashiroTakashi en-aut-sei=Yamashiro en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TakigawaMasaharu en-aut-sei=Takigawa en-aut-mei=Masaharu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=2 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=3 en-affil= kn-affil=Okayama Univ, Sch Dent, Biodent Res Ctr affil-num=4 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent affil-num=5 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Orthodont & Dentofacial Orthoped affil-num=6 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Biochem & Mol Dent en-keyword=ACAN kn-keyword=ACAN en-keyword=CCN2 kn-keyword=CCN2 en-keyword=CTGF kn-keyword=CTGF en-keyword=CCN3 kn-keyword=CCN3 en-keyword=NOV kn-keyword=NOV en-keyword=chondrocyte kn-keyword=chondrocyte en-keyword=dimerization kn-keyword=dimerization END start-ver=1.4 cd-journal=joma no-vol= cd-vols= no-issue= article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2000 dt-pub=20000930 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=軟骨由来の慢性関節リウマチ関連抗原RA-A47に関する研究 en-subtitle= kn-subtitle= en-abstract= kn-abstract= en-copyright= kn-copyright= en-aut-name= en-aut-sei= en-aut-mei= kn-aut-name=服部高子 kn-aut-sei=服部 kn-aut-mei=高子 aut-affil-num=1 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 END