Lecanda F, Towler D A, Ziambaras K, Cheng S L, Koval M, Steinberg T H, Civitelli R
Division of Bone and Mineral Diseases, Washington University School of Medicine, and Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.
Mol Biol Cell. 1998 Aug;9(8):2249-58. doi: 10.1091/mbc.9.8.2249.
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.
成骨细胞通过间隙连接组织成一个多细胞网络。在这些细胞中，间隙连接由连接蛋白43（Cx43）和连接蛋白45（Cx45）形成。与Cx45孔相比，Cx43间隙连接形成的孔对带负电荷的染料（如荧光素黄和钙黄绿素）具有更高的通透性。我们研究了通过操纵Cx43和Cx45的相对表达来改变间隙连接通讯是否会影响成骨细胞表型。在主要表达Cx43的细胞（ROS 17/2.8和MC3T3-E1）中转入Cx45，会降低染料转移以及骨钙素（OC）和骨唾液蛋白（BSP）的表达，这两种基因对骨基质形成和钙化至关重要。相反，在主要表达Cx45的细胞（UMR 106-01）中转入Cx43，会增加细胞间偶联以及OC和BSP的表达。启动子-荧光素酶构建体与连接蛋白表达载体的瞬时共转染表明，在ROS 17/2.8和MC3T3-E1细胞中，Cx45共转染会下调OC和BSP基因转录，同时染料偶联减少。相反，在UMR 106-01细胞中共转染Cx43会上调OC和BSP基因转录。其他特异性较低的成骨细胞启动子（如骨桥蛋白和骨连接素）的活性对间隙连接通讯变化的敏感性较低。因此，通过操纵成骨细胞中Cx43和Cx45的表达来改变间隙连接通透性，可能通过调节可在细胞间扩散的信号，改变成骨细胞特异性启动子的转录活性。一个相互通讯的细胞间网络对于充分展现分化的成骨细胞表型是必需的。
