Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts.
Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required Ca(2+) and was >30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at approximately 15 min that matured by approximately 2-3 h; distinct gap junctional complexes were evident by approximately 3 h. Immunoblotting showed that HGF expressed beta-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor beta-glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers (>10(5)) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions.
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Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5S 3E2. keniv_ko@hotmail.com
Arora
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Lee
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McCulloch
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D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D017629:Gap Junctions / Q000378:metabolism / Q000502:physiology*; D005881:Gingiva / Q000166:cytology; D006801:Humans; D007700:Kinetics; D014018:Tissue Distribution; D015534:Trans-Activators; D051176:beta Catenin
C495270:CTNNB1 protein, human; D015820:Cadherins; D018031:Connexin 43; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D051176:beta Catenin