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.
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.
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama, Japan.
authors.forename: Nao
authors.initials: N
authors.lastname: Tomita
authors.forename: Takako
authors.initials: T
authors.lastname: Hattori
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Itoh
authors.forename: Eriko
authors.initials: E
authors.lastname: Aoyama
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yao
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamashiro
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002356:Cartilage / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D018878:Limb Buds / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D017451:Receptors, Somatomedin / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; C526318:CCN2 protein, mouse; D017451:Receptors, Somatomedin; C519530:insulin-like growth factor-1, mouse; D055513:Connective Tissue Growth Factor; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II
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