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