Gap junctional communication modulates gene expression in osteoblastic cells - PubMed (original) (raw)

Gap junctional communication modulates gene expression in osteoblastic cells

F Lecanda et al. Mol Biol Cell. 1998 Aug.

Free PMC article

Abstract

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.

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Figures

Figure 1

Figure 1

Steady-state mRNA levels of osteoblast products are altered by overexpression of chick Cx45 in ROS 17/2.8 osteoblastic cells. Cells stably expressing chick Cx45 (ROS/Cx45) or mock-transfected cells (ROS mock), as well as their parent cells (ROS 17/2.8), were grown to confluence in the same conditions, in the presence (+) or in the absence (−) of 10−8 M 1,25(OH)2D3, and poly-A RNA was separated on agarose gel and blotted onto nylon membranes. Membranes were sequentially hybridized with [32P]-labeled cDNA probes for (A) OC, BSP, Cx45, GAPDH, and (B) alkaline phosphatase (AP), ON, and OP. Between each hybridization step, the membrane was extensively washed and re-exposed to x-rays to ensure complete removal of the previous hybrizidation band.

Figure 2

Figure 2

Transient transfection of chick Cx45 decreases transcriptional activity of OC and BSP promoters in ROS 17/2.8 (A) and MC3T3-E1 (B) osteoblastic cells. Cells were cotransfected with either a chick Cx45 expression construct or its vector, and one of the promoter-luciferase constructs, OCLUC, BSPLUC, SV40LUC, or ONLUC, as indicated. Luciferase activity was measured 3 d after transfection. Data are representative of four different experiments and are expressed as the average ± SD of triplicate wells for each condition. *, p < 0.01 vs. vector- transfected cells (t test for unpaired samples).

Figure 3

Figure 3

Steady-state OC mRNA levels are increased by overexpression of rat Cx43 in UMR 106–01 osteoblastic cells. UMR 106–01 cells stably expressing rat Cx43 (UMR/Cx43) and their parent cells were grown to confluence in the same culture conditions, and poly-A RNA was separated on agarose gel and blotted onto nylon membranes. Membranes were sequentially hybridized with [32P]-labeled cDNA probes for OC, Cx43, and GAPDH, with extensive washes between each hybridization step.

Figure 4

Figure 4

Transient transfection of rat Cx43 increases transcriptional activity of OC and BSP promoters in UMR 106/01 and MC3T3-E1 cells. Cells were cotransfected with either a rat Cx43 expression construct or its vector, and one of the promoter–luciferase constructs, OCLUC, BSPLUC or SV40LUC, as indicated. Luciferase activity was measured 3 d after transfection. Data are representative of four (UMR 106–01) and six (MC3T3-E1) different experiments and are expressed as the average ± SD of triplicate wells for each condition. *, p < 0.01 vs. vector-transfected cells (t test for unpaired samples).

Figure 5

Figure 5

Transient transfection of Cx45 in ROS 17/2.8 and Cx43 in UMR 106–01 cells leads to cell surface expression of either connexin protein in a proportion of cells. Cells were seeded at low density on glass coverslips and incubated with either Cx45 or Cx43 expression constructs for 72 h. After fixation, cells were immunostained with anti-Cx45 (ROS 17/2.8, left) or anti-Cx43 (UMR 106–01, right) antibody, as indicated. Bar, 20 μm.

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References

    1. Beyer EC, Paul DL, Goodenough DA. Connexin family of the gap junction proteins. J Membr Biol. 1990;116:187–194. - PubMed
    1. Beyer EC, Steinberg TH. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J Biol Chem. 1991;266:7971–7974. - PubMed
    1. Boudreaux JM, Towler DA. Synergistic induction of osteocalcin gene expression. J Biol Chem. 1996;271:7508–7515. - PubMed
    1. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248–254. - PubMed
    1. Brown MEA, Arlot ME, Reeve J. Osteoblast density and the evolution of BMUs in vertebral osteoporosis. Bone. 1993;14:473–479. - PubMed

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