Cellular interaction of integrin alpha3beta1 with laminin 5 promotes gap junctional communication - PubMed (original) (raw)

Cellular interaction of integrin alpha3beta1 with laminin 5 promotes gap junctional communication

P D Lampe et al. J Cell Biol. 1998.

Abstract

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin alpha3beta1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin alpha3beta1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking alpha3beta1-laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via alpha3beta1 promotes GJIC that integrates individual cells into synchronized epiboles.

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Figures

Figure 1

Immunostaining of human wounds identifies parallel changes in laminin 5, integrin α3β1, E-cadherin, and Cx43. Incisional wounds were prepared in normal human skin and then collected by punch biopsy at the indicated times (see Materials and Methods). Fixed cryostat sections (6 μm) were reacted with antibodies specific to components of cell as indicated for each panel. A–F are at the same lower magnification (bar, 100 μm) and G–I are at higher magnification (bar, 50 μm). (A) α3 chain of laminin 5 (mAb C2-5), 24 h wound. (B) Type VII collagen (mAb L3D), 24 h wound. (C) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. (D) Cx43 (PNRF), 24 h wound. (E) Integrin α3 chain (mAb P1B5), 53 h wound (the area within the small rectangle has been enlarged in the large rectangle inset to show the polarization of α3β1 to the basal plasma membrane in the wound bed). (F) Fibronectin (mAb P1H11), 53 h wound. (G) Cx43 (PNRF), 24 h wound. (H) E-cadherin (mAb HECD1), 24 h wound. (I) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. Different regions of the BM and epidermis were identified as follows: wound bed, unfilled arrowhead; normal epidermis distant to the wound, unfilled arrow; wound edge, filled arrow; epidermis adjacent to the wound bed, filled arrowhead.

Figure 2

Dye transfer in epibole cultures detects differences in GJIC in leading and following migratory keratinocytes. Explants of neonatal foreskin were grown on collagen for 5 d. Epibole outgrowth of keratinocytes formed a migratory tongue of keratinocytes on the collagen surface with a leading migratory edge and following cells. Lucifer yellow dye was microinjected into keratinocytes in the leading edge and following cells three to four rows back from the leading edge. Dye was allowed to transfer for ∼5 min. (A) Phase view of the epidermal tongue. Injected cells are denoted by arrows. (B) The corresponding Lucifer yellow fluorescence view. (C) Cx43 immunofluorescence of an adjacent area of the epibole culture. Note that cells at the leading edge expressed only perinuclear Cx43 (arrows) whereas following cells also assembled gap junctions (arrowheads). (D) Laminin 5 (C2-5) was increased in expression in keratinocytes at the leading edge of the epibole (arrows). (E) Integrin α3 chain (P1F2) was present in prominent protrusions of the plasma membrane of cells at the leading edge of the outgrowth (arrows) in contrast to staining at cell–cell junctions in the following cells (arrowheads).

Figure 3

Interaction of laminin 5 with HFKs promotes dye transfer better then collagen or fibronectin. (A) ECM regulation of dye transfer. HFKs containing calcein were mixed with DiI-labeled cells and plated on collagen, fibronectin, and laminin 5 as labeled. Note that cells were plated at a high cell density to ensure confluency. The top row shows the phase view and the bottom row calcein fluorescence of the same field. Cells adherent to laminin 5 transferred calcein dye to recipient cells more readily then cells on collagen or fibronectin. (B) Dye transfer assay. Phase, calcein (green), DiI (red), and an overlay of calcein and DiI (Overlay) fluorescence views are shown. Note that several cells in the overlay show punctate yellow fluorescence indicating dye transfer occurred. Five examples of cells that received calcein via dye transfer are marked by an arrow. Five examples of DiI-labeled cells that did not receive any calcein are marked by arrowheads. (C) Laminin 5 ECM or laminin 5–coated beads promote dye transfer. Calcein-labeled HFKs were plated on collagen, fibronectin, or laminin 5 at confluent cell densities as seen in A and dye transfer levels were compared (unfilled bars). Dye transfer was quantitated as the number of interfaces showing dye transfer over the total number of interfaces between calcein- and DiI-labeled cells (mean ± standard deviation). In an alternative approach, HFKs were attached to poly-

-lysine– coated surfaces and then laminin 5–, collagen-, or fibronectin-coated beads were added to the apical surface of the attached cells and dye transfer was quantitated (filled bars). Using either approach, laminin 5 promoted GJIC better then collagen or fibronectin.

Figure 4

Adhesion to laminin 5 promotes assembly of gap junctions but not transcriptional regulation of Cx43. (A) Northern analysis of HFK cells plated on collagen or laminin 5. mRNA was isolated from HFKs attached to collagen or laminin 5 and assayed by Northern blot with a cDNA probe homologous to Cx43 mRNA. The mRNA for Cx43 is 3.0 kb as expected. (B) Laminin 5 promotes assembly of Cx43 into Triton X-100–insoluble gap junctions: effects of BFA. HFKs were attached to surfaces coated with either laminin 5 or collagen for 2.5 h. Adherent HFKs were solubilized with Laemmli sample buffer either before (Whole cell) or after extraction with 1% Triton X-100 pretreatment (Triton insoluble). Triton extraction does not solubilize Cx43 that has assembled into gap junctions. Whole cell and Triton insoluble cell extracts were immunoblotted with anti-Cx43 antibody (mAb 3068). Cell extracts immunoblotted in the fifth through eighth lanes were derived from twice as many cells as extracts in the first through fourth lanes. Treatment with the protein trafficking inhibitor BFA inhibited the laminin 5–mediated assembly of gap junctions.

Figure 5

Integrin α3β1 interaction with laminin 5 is sufficient to promote dye transfer. (A) Dye transfer in HFKs plated on immobilized antiintegrin antibodies. The indicated antiintegrin mAbs were immobilized on plastic, blocked with BSA, and used as an adhesive substrate for HFKs that had been labeled with either calcein or DiI. Adhesion and spreading occurred on all antibody ligands. The resulting dye transfer was quantitated as shown. (B) Dye transfer in integrin-transfected CHO cells. CHO cells transfected with human α2 or α3 cDNAs were plated on collagen and laminin 5, respectively. The top row shows the phase view and the bottom row calcein fluorescence. Calcein transfer was elevated in α3-transfected CHO cells plated on laminin 5. (C) Quantitation of dye transfer in integrin-transfected CHO cells. CHOs transfected with integrins α2, α3, and α6 were plated on collagen (filled bars) or laminin 5 (unfilled bars). Dye transfer was much higher on laminin 5 than collagen.

Figure 6

Protein trafficking mediates assembly of Triton insoluble gap junctions in α3-transfected CHO cells on laminin 5. (A) Western immunoblot for Cx43 of cell lysates from CHO cells plated on collagen or laminin 5 in the presence or absence of BFA for 1.5 h. As indicated, α3-transfected CHO cells plated on laminin 5 and α2-transfected CHO cells plated on collagen were either solubilized directly with Laemmli sample buffer (Whole cell) or after extraction with 1% Triton X-100 (Triton-insoluble) to enrich for gap junctional structures. The cell extracts were immunoblotted with anti-Cx43 rabbit antibody AT2. (B) Cx43 immunofluorescence. α3-transfected cells were plated on collagen or laminin 5, as indicated. The adherent cells were extracted with Triton X-100 before fixation and labeled with anti-Cx43 (PNRF) and FITC-conjugated secondary. Note the extensive punctate gap junctional fluorescence at cell–cell interfaces present in cells attached to laminin 5.

Figure 7

RhoA regulates dye transfer in cells adherent on laminin 5. (A) The FEPE1L8 human keratinocyte cell line was attached to surfaces coated with collagen, laminin 5, and fibronectin as indicated and dye transfer was quantitated (filled bars). (B) FEPE1L8 cells (hatched bars) were plated on laminin 5 in the presence or absence of LPA and Toxin B as indicated and dye transfer was quantitated. (C) FEPE1L8 cells transfected with a dominant negative Rho (DN-Rho; see Materials and Methods) were plated on collagen or laminin 5 in the presence or absence of LPA and Toxin B as indicated. Dye transfer was quantitated (unfilled bars). Note that LPA increases the extent of dye transfer whereas Toxin B–treated cells and the DN-Rho–transfected cells do not transfer dye well.

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Cellular interaction of integrin alpha3beta1 with laminin 5 promotes gap junctional communication - PubMed (original) (raw)