title: An intact connexin43 is required to enhance signaling and gene expression in osteoblast-like cells.
abstract: The cytoplasmic C-terminus of connexin43 (Cx43) interacts with numerous signaling complexes. We hypothesize that signal complex docking to the Cx43 C-terminus (CT) is required to propagate the molecules being shared by gap junctions. We have previously shown that Cx43 impacts the responsiveness of osteoblasts to FGF2 in a PKCδ- and ERK-dependent manner, converging on Runx2 activity. Here, we mapped the interaction domain of Cx43 and PKCδ to amino acids 243-302 of the Cx43 CT by GST pulldown assay. Using Runx2-responsive luciferase reporter assays, a Cx43 deletion construct (Cx43 S244Stop), which lacks the C-terminus (amino acids 244-382), failed to support the Cx43-dependent potentiation of transcription following FGF2 treatment in MC3T3 osteoblast-like cells. Similarly, overexpression of Cx43 S244Stop could not mimic the ability of the full length Cx43 to stimulate expression of osteoblast genes. In contrast to full length Cx43, overexpression of just the Cx43 CT (amino acids 236-382) inhibited both transcription from a Runx2 reporter and signaling via PKCδ and ERK. Inhibition of signaling by the CT did not occur in HeLa cells, which lack endogenous Cx43. In summary, the data support a model in which an intact Cx43 is required for both signal propagation/permeability (i.e., channel function) and local recruitment of signaling complexes to the CT (i.e., docking function) in order to mediate its cellular effects. Further, while the CT alone has channel independent activity, it is opposing to the effect of overexpression of the full length Cx43 channel in this cell context.
authors.affiliation: Department of Orthopedics, University of Maryland, School of Medicine, Baltimore, Maryland, 21201.
authors.forename: Carla
authors.initials: C
authors.lastname: Hebert
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Stains
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002450:Cell Communication / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology
chemical_list: D018031:Connexin 43; D016222:Fibroblast Growth Factor 2
keywords: BONE; CELL-TO-CELL COMMUNICATION; FIBROBLAST GROWTH FACTOR 2; GAP JUNCTION; PROTEIN KINASE C DELTA; SIGNAL TRANSDUCTION