title: ODDD-linked Cx43 mutants reduce endogenous Cx43 expression and function in osteoblasts and inhibit late stage differentiation.
abstract: BACKGROUND
  Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD.
  
  
  METHODS
  We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin.
  
  
  RESULTS
  We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation.
  
  
  CONCLUSIONS
  Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.
authors.affiliation: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McLachlan
authors.forename: Isabelle
authors.initials: I
authors.lastname: Plante
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Dan
authors.initials: D
authors.lastname: Tong
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Kidder
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Bernier
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
mesh_terms: D000818:Animals; D001848:Bone Diseases, Developmental / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism*; D004195:Disease Models, Animal; D017629:Gap Junctions / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D013997:Time Factors
chemical_list: D018031:Connexin 43