Abstract
Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.
Highlights
Homeostasis depends on close connections and intimate molecular exchanges among extracellular, intracellular and intercellular networks
TGF-β1 increases the number of dendritic processes of osteocytes in bone tissue and MLO-Y4 cell line To explore the effects of TGF-β1 on communications between osteocytes, we first detected the osteocyte morphological changes induced by recombined TGF-β1 in cortical bones from mouse femurs ex vivo and in the MLO-Y4 cell line in vitro
We found that the number of dendritic processes of osteocytes in cortical bone increased after TGF-β1 (10 ng/ml) treatment for 21 days
Summary
Homeostasis depends on close connections and intimate molecular exchanges among extracellular, intracellular and intercellular networks. Direct communication between cells through specialized intercellular channels is completed by transmembrane channels in the plasma membranes between adjacent cells[1]. These channels are called gap junctions (GJ) and comprise connexin (Cx) proteins. The role of these channels is to allow the Structurally, the 21 members of the connexin family have similar topological structures, including cytoplasmic N-, and C-terminal domains, along with four membrane spanning domains, two extracellular loops, and one intracellular loop[4,6]. There are 3 pannexin genes, namely, Panx[1], Panx[2], and Panx[3], which encode proteins strikingly similar to connexins in terms of structural topology
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