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

Lampe PD, Nguyen BP, Gil S, Usui M, Olerud J, Takada Y, Carter WG - J. Cell Biol. (1998)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Basic Sciences and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org

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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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-l-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.
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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-l-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.

Mentions: The possible interdependence of cell–substrate adhesion and GJIC was investigated in an in vitro model. Laminin 5, collagen, and fibronectin were chosen to examine their influence on GJIC. HFKs were assayed for GJIC by loading separate plates of adherent cells with either of two different fluorescent dyes, calcein or DiI, suspending the labeled cells with trypsin, mixing the cells in a 1:4 ratio (calcein/DiI), and then adhering the cells onto laminin 5, collagen, or fibronectin ligands. Calcein, which can be readily loaded into cells via its acetoxymethylester form, is hydrophilic and small enough to pass between cells via gap junctions, and DiI is a lipophilic molecule that marks cells that could function as dye acceptors (Lampe, 1994). Labeled HFKs plated at high density on either collagen- or laminin 5–coated surfaces attached within 1–2 min and spread within 5–20 min. After 30 min, no difference in the amount of attachment or spreading could be observed on either ligand. With fibronectin-coated plates, HFK attachment took 5–10 min. After 2.5 h of incubation, the HFKs had extensive cell–cell contacts on all three ECM ligands (Fig. 3 A, top). Note that in all cases these cells were plated in excess numbers so they would be essentially 100% confluent in order to ensure that extensive cell–cell interfaces formed. By comparison, HFKs plated on tissue culture plastic take >1 h to bind and much longer to spread (not shown). After plating on the different ECM ligands for 2.5 h, phase images (Fig. 3 A, top) and fluorescence images of calcein (Fig. 3 A, bottom) and DiI (not shown) were collected. A representative example of HFKs plated on laminin 5 is presented in color in Fig. 3 B to illustrate the dye transfer assay. Cells with bright green calcein fluorescence in these views represent cells initially loaded with calcein (donors) and cells with red DiI fluorescence are potential recipients of dye via transfer at a cell–cell interface seen in the phase image. The assignment of a cell as a recipient of dye via transfer rather than a poorly loaded or leaking donor was checked by digitally overlaying scanned images of red DiI and green calcein fluorescence. If a cell in contact with a calcein-loaded, DiI-negative cell contained both punctate red DiI and more diffuse green calcein yellow punctate fluorescence was produced in a recipient cell. This indicated that gap junction assembly and dye transfer had occurred (arrows). If a DiI-labeled cell adjacent to a calcein-loaded cell did not contain calcein, then dye transfer did not occur at that interface (arrowheads). The ratio of recipients to total cell interfaces was calculated for HFK attachment to collagen, fibronectin, and laminin 5 in at least three separate experiments and the results are shown in Fig. 3 C. Dye transfer was approximately two- and fivefold better on laminin 5 than on collagen and fibronectin, respectively. These results clearly and reproducibly indicated that adhesion to laminin 5 promoted GJIC when compared with collagen or fibronectin.


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

Lampe PD, Nguyen BP, Gil S, Usui M, Olerud J, Takada Y, Carter WG - J. Cell Biol. (1998)

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-l-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.
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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-l-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.
Mentions: The possible interdependence of cell–substrate adhesion and GJIC was investigated in an in vitro model. Laminin 5, collagen, and fibronectin were chosen to examine their influence on GJIC. HFKs were assayed for GJIC by loading separate plates of adherent cells with either of two different fluorescent dyes, calcein or DiI, suspending the labeled cells with trypsin, mixing the cells in a 1:4 ratio (calcein/DiI), and then adhering the cells onto laminin 5, collagen, or fibronectin ligands. Calcein, which can be readily loaded into cells via its acetoxymethylester form, is hydrophilic and small enough to pass between cells via gap junctions, and DiI is a lipophilic molecule that marks cells that could function as dye acceptors (Lampe, 1994). Labeled HFKs plated at high density on either collagen- or laminin 5–coated surfaces attached within 1–2 min and spread within 5–20 min. After 30 min, no difference in the amount of attachment or spreading could be observed on either ligand. With fibronectin-coated plates, HFK attachment took 5–10 min. After 2.5 h of incubation, the HFKs had extensive cell–cell contacts on all three ECM ligands (Fig. 3 A, top). Note that in all cases these cells were plated in excess numbers so they would be essentially 100% confluent in order to ensure that extensive cell–cell interfaces formed. By comparison, HFKs plated on tissue culture plastic take >1 h to bind and much longer to spread (not shown). After plating on the different ECM ligands for 2.5 h, phase images (Fig. 3 A, top) and fluorescence images of calcein (Fig. 3 A, bottom) and DiI (not shown) were collected. A representative example of HFKs plated on laminin 5 is presented in color in Fig. 3 B to illustrate the dye transfer assay. Cells with bright green calcein fluorescence in these views represent cells initially loaded with calcein (donors) and cells with red DiI fluorescence are potential recipients of dye via transfer at a cell–cell interface seen in the phase image. The assignment of a cell as a recipient of dye via transfer rather than a poorly loaded or leaking donor was checked by digitally overlaying scanned images of red DiI and green calcein fluorescence. If a cell in contact with a calcein-loaded, DiI-negative cell contained both punctate red DiI and more diffuse green calcein yellow punctate fluorescence was produced in a recipient cell. This indicated that gap junction assembly and dye transfer had occurred (arrows). If a DiI-labeled cell adjacent to a calcein-loaded cell did not contain calcein, then dye transfer did not occur at that interface (arrowheads). The ratio of recipients to total cell interfaces was calculated for HFK attachment to collagen, fibronectin, and laminin 5 in at least three separate experiments and the results are shown in Fig. 3 C. Dye transfer was approximately two- and fivefold better on laminin 5 than on collagen and fibronectin, respectively. These results clearly and reproducibly indicated that adhesion to laminin 5 promoted GJIC when compared with collagen or fibronectin.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Basic Sciences and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org

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.

Show MeSH
Related in: MedlinePlus