Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell–cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987–995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249–2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

ABSTRACT

Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. In order to better understand the genetic etiology of osteosarcoma, we performed a multi-stage genome-wide association study (GWAS) consisting of 941 cases and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: rs1906953 at 6p21.3, in the glutamate receptor metabotropic 4 [GRM4] gene (P = 8.1 ×10-9), and rs7591996 and rs10208273 in a gene desert on 2p25.2 (P = 1.0 ×10-8 and 2.9 ×10-7). These two susceptibility loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.