Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calciumpermeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca 2؉ transients (65 ؎ 9.4 nM above base line). Similar Ca 2؉ transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca 2؉ responses depended on influx of extracellular Ca 2؉ through mechanosensitive channels because both Ca 2؉ chelation and gadolinium chloride abolished the response and MnCl 2 quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca 2؉ release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca 2؉ transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn 2؉ after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca 2؉ with EGTA. Msp pretreatment reduced the amplitude of [Ca 2؉ ] i transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca 2؉ , Msp reduced Ca 2؉ efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca 2؉ release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca 2؉ depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca 2؉ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca 2؉ entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca 2؉ release from endoplasmic reticulum stores, and it inhibits subsequent Ca 2؉ influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.

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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