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.