Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts

Despite their significance in wound healing, little is known about the mole cular determinants of cell-to-cell adhesion and gap junctional communicatio n in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcei n-labeled donor cells in suspension were added onto an established, Texas r ed dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required Ca2+ and was >30-fold stronger than cell-to-fibronectin adhesion a t 15 min. Electron micrographs showed rapid formation of adherens junction- like structures at similar to 15 min that matured by similar to 2-3 h; dist inct gap junctional complexes were evident by similar to 3 h. Immunoblottin g showed that HGF expressed beta-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent c ultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye trans fer in a cohort of Texas red dextran-labeled cells, but this was almost com pletely abolished by the gap junction inhibitor beta-glycyrrhetinic acid an d the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model all ows large numbers (>10(5)) of cells to form synchronous cell-to-cell contac ts, thereby enabling the simultaneous functional and molecular studies of a dherens junctions and gap junctions.