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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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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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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.

Mentions: FEPE1L8 cells are a human keratinocyte cell line that has been immortalized by transfection with human papilloma virus 16. FEPE1L8 cells retain their ability to differentiate in response to calcium, they stratify in organotypic cultures, form focal adhesions, and express the same integrin profile as HFKs, but produce little laminin 5 (Kaur and Carter, 1992). The fact that these cells produce little laminin 5 makes them dependent upon exogenous ECM ligands for rapid adhesion. FEPE1L8 cells show a fivefold increase in GJIC on exogenous laminin 5 versus collagen and a sixfold increase versus fibronectin (Fig. 7 A). In addition, the increase in GJIC observed on laminin 5 could be largely inhibited if the Rho inhibitor Toxin B was included during the incubation (Fig. 7 B).


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)

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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Related In: Results  -  Collection

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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.
Mentions: FEPE1L8 cells are a human keratinocyte cell line that has been immortalized by transfection with human papilloma virus 16. FEPE1L8 cells retain their ability to differentiate in response to calcium, they stratify in organotypic cultures, form focal adhesions, and express the same integrin profile as HFKs, but produce little laminin 5 (Kaur and Carter, 1992). The fact that these cells produce little laminin 5 makes them dependent upon exogenous ECM ligands for rapid adhesion. FEPE1L8 cells show a fivefold increase in GJIC on exogenous laminin 5 versus collagen and a sixfold increase versus fibronectin (Fig. 7 A). In addition, the increase in GJIC observed on laminin 5 could be largely inhibited if the Rho inhibitor Toxin B was included during the incubation (Fig. 7 B).

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