Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132974&req=5

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.

Mentions: CHO cells were treated with BFA to test whether the increased gap junctional assembly observed when cells were plated on laminin 5 was dependent on protein trafficking. Equal numbers of α2- and α3-transfected CHO cells were plated on collagen or laminin 5 for 1 h in the presence or absence of BFA. The level of total Cx43 protein did not vary when the α2-transfected CHO cells were plated on collagen or α3-transfected CHO cells were plated on laminin 5 for 1 h as determined by immunoblot analysis (Fig. 6 A, first and third lanes). BFA treatment also did not significantly affect the level of total Cx43 protein (Fig. 6 A, second and fourth lanes), but did apparently affect the phosphorylation/migration of Cx43 as has been reported previously (Musil and Goodenough, 1991; Laird et al., 1995). In contrast, densitometry of the Western blots indicated that the α3-transfected CHO cells plated on laminin 5 had 10-fold more Triton-insoluble Cx43 than α2-transfected CHO cells plated on collagen (Fig. 6 A, compare the fifth and seventh lanes). Again, this increase in the Triton insolubility of Cx43 appeared to be at least partially dependent upon protein trafficking since BFA eliminated 46% of the increase. BFA also reduced the fraction of cells transferring dye from the control value of 0.94 to 0.35. To confirm that the increase in Triton insolubility was due to increased gap junction assembly, α3-CHO cells were attached to laminin 5 or collagen, extracted with Triton, and then processed for immunofluorescence with an anti-Cx43 antibody. Cells on both ligands showed some perinuclear labeling. However, typical punctate gap junctional staining was clearly evident when the cells were plated on laminin 5 whereas little was observed on collagen (Fig. 6 B). The results in Figs. 5 and 6 indicate that interaction of integrin α3 with laminin 5 in transfected CHO cells is sufficient to promote GJIC and assembly of Cx43 into Triton-insoluble gap junctions. This implies that increased GJIC on laminin 5 may result from increased assembly of gap junctions. However, increased GJIC could also result from changes in the channel properties of the Cx43 complexes that are already at the cell surface or increased trafficking of a protein other than Cx43 necessary for gap junction formation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: CHO cells were treated with BFA to test whether the increased gap junctional assembly observed when cells were plated on laminin 5 was dependent on protein trafficking. Equal numbers of α2- and α3-transfected CHO cells were plated on collagen or laminin 5 for 1 h in the presence or absence of BFA. The level of total Cx43 protein did not vary when the α2-transfected CHO cells were plated on collagen or α3-transfected CHO cells were plated on laminin 5 for 1 h as determined by immunoblot analysis (Fig. 6 A, first and third lanes). BFA treatment also did not significantly affect the level of total Cx43 protein (Fig. 6 A, second and fourth lanes), but did apparently affect the phosphorylation/migration of Cx43 as has been reported previously (Musil and Goodenough, 1991; Laird et al., 1995). In contrast, densitometry of the Western blots indicated that the α3-transfected CHO cells plated on laminin 5 had 10-fold more Triton-insoluble Cx43 than α2-transfected CHO cells plated on collagen (Fig. 6 A, compare the fifth and seventh lanes). Again, this increase in the Triton insolubility of Cx43 appeared to be at least partially dependent upon protein trafficking since BFA eliminated 46% of the increase. BFA also reduced the fraction of cells transferring dye from the control value of 0.94 to 0.35. To confirm that the increase in Triton insolubility was due to increased gap junction assembly, α3-CHO cells were attached to laminin 5 or collagen, extracted with Triton, and then processed for immunofluorescence with an anti-Cx43 antibody. Cells on both ligands showed some perinuclear labeling. However, typical punctate gap junctional staining was clearly evident when the cells were plated on laminin 5 whereas little was observed on collagen (Fig. 6 B). The results in Figs. 5 and 6 indicate that interaction of integrin α3 with laminin 5 in transfected CHO cells is sufficient to promote GJIC and assembly of Cx43 into Triton-insoluble gap junctions. This implies that increased GJIC on laminin 5 may result from increased assembly of gap junctions. However, increased GJIC could also result from changes in the channel properties of the Cx43 complexes that are already at the cell surface or increased trafficking of a protein other than Cx43 necessary for gap junction formation.

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