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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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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.
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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.

Mentions: We investigated which integrins mediate signals from laminin 5 that increase assembly and communication of gap junctions. To this end, antibodies to different integrins and control proteins were immobilized on culture dishes. Soluble integrin antibodies (Symington et al., 1993) and beads coated with antibodies to specific integrins have been shown to elicit cellular responses (e.g., focal adhesion kinase phosphorylation) normally initiated by ligand–integrin interaction (Miyamoto et al., 1995). A mixture of calcein- and DiI-loaded HFKs was plated on the dishes and dye transfer was recorded after 2–3 h of incubation. Cells readily adhered to the antibody-coated dishes if the antibody was to an extracellular portion of an integrin receptor and a confluent monolayer was produced. The best dye transfer was observed when cells were plated on immobilized mAbs against the integrin α3 (P1B5, P1F2; Fig. 5 A). Antibodies specific for α2 integrin (P4B4, P1H5), a collagen adhesion receptor, did not enhance dye transfer. An antibody to α6 (G0H3), a receptor for laminin 5, supported dye transfer at about the same level as antibodies to α2. In controls, if nuclear protein-specific antibodies or antibodies that do not react with the cells were used, very little cell attachment and hence no dye transfer was observed (not shown).


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)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132974&req=5

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.
Mentions: We investigated which integrins mediate signals from laminin 5 that increase assembly and communication of gap junctions. To this end, antibodies to different integrins and control proteins were immobilized on culture dishes. Soluble integrin antibodies (Symington et al., 1993) and beads coated with antibodies to specific integrins have been shown to elicit cellular responses (e.g., focal adhesion kinase phosphorylation) normally initiated by ligand–integrin interaction (Miyamoto et al., 1995). A mixture of calcein- and DiI-loaded HFKs was plated on the dishes and dye transfer was recorded after 2–3 h of incubation. Cells readily adhered to the antibody-coated dishes if the antibody was to an extracellular portion of an integrin receptor and a confluent monolayer was produced. The best dye transfer was observed when cells were plated on immobilized mAbs against the integrin α3 (P1B5, P1F2; Fig. 5 A). Antibodies specific for α2 integrin (P4B4, P1H5), a collagen adhesion receptor, did not enhance dye transfer. An antibody to α6 (G0H3), a receptor for laminin 5, supported dye transfer at about the same level as antibodies to α2. In controls, if nuclear protein-specific antibodies or antibodies that do not react with the cells were used, very little cell attachment and hence no dye transfer was observed (not shown).

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