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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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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).
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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).

Mentions: Parallel changes in cell junctional components were also observed in in vitro epibole cultures of skin (Fig. 2). Punches of human neonatal foreskin explanted onto collagen yielded outgrowths of primary keratinocytes that migrated as an integrated tongue of cells (Fig. 2 A). An assay of GJIC was performed by microinjection of Lucifer yellow into cells at both the leading edge of the tongue and the following cells (Fig. 2 B). In three separate experiments (with >10 injections/experiment), the cells at the leading edge did not transfer a detectable amount of dye to neighbors whereas the following cells transferred dye efficiently. Immunofluorescence of cells at the leading edge of the tongue localized Cx43 to the perinuclear cytoplasm (Fig. 2 C, arrows). In contrast, Cx43 assembled into apparent punctate gap junctions in cells three to four rows back from the leading edge (Fig. 2 C, arrowheads). The dramatic differences in transfer of dye and immunofluorescence established that cells at the leading edge of the migratory tongue did not assemble functional gap junctions whereas the following cells assembled gap junctions and transferred dye to neighboring cells.


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132974&req=5

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).
Mentions: Parallel changes in cell junctional components were also observed in in vitro epibole cultures of skin (Fig. 2). Punches of human neonatal foreskin explanted onto collagen yielded outgrowths of primary keratinocytes that migrated as an integrated tongue of cells (Fig. 2 A). An assay of GJIC was performed by microinjection of Lucifer yellow into cells at both the leading edge of the tongue and the following cells (Fig. 2 B). In three separate experiments (with >10 injections/experiment), the cells at the leading edge did not transfer a detectable amount of dye to neighbors whereas the following cells transferred dye efficiently. Immunofluorescence of cells at the leading edge of the tongue localized Cx43 to the perinuclear cytoplasm (Fig. 2 C, arrows). In contrast, Cx43 assembled into apparent punctate gap junctions in cells three to four rows back from the leading edge (Fig. 2 C, arrowheads). The dramatic differences in transfer of dye and immunofluorescence established that cells at the leading edge of the migratory tongue did not assemble functional gap junctions whereas the following cells assembled gap junctions and transferred dye to neighboring cells.

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