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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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Adhesion to laminin 5 promotes assembly of gap junctions but not transcriptional regulation of Cx43. (A) Northern  analysis of HFK cells plated on collagen or laminin 5. mRNA was  isolated from HFKs attached to collagen or laminin 5 and assayed by Northern blot with a cDNA probe homologous to Cx43  mRNA. The mRNA for Cx43 is 3.0 kb as expected. (B) Laminin  5 promotes assembly of Cx43 into Triton X-100–insoluble gap  junctions: effects of BFA. HFKs were attached to surfaces coated  with either laminin 5 or collagen for 2.5 h. Adherent HFKs were  solubilized with Laemmli sample buffer either before (Whole  cell) or after extraction with 1% Triton X-100 pretreatment (Triton insoluble). Triton extraction does not solubilize Cx43 that has  assembled into gap junctions. Whole cell and Triton insoluble cell  extracts were immunoblotted with anti-Cx43 antibody (mAb  3068). Cell extracts immunoblotted in the fifth through eighth  lanes were derived from twice as many cells as extracts in the first  through fourth lanes. Treatment with the protein trafficking inhibitor BFA inhibited the laminin 5–mediated assembly of gap  junctions.
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Figure 4: Adhesion to laminin 5 promotes assembly of gap junctions but not transcriptional regulation of Cx43. (A) Northern analysis of HFK cells plated on collagen or laminin 5. mRNA was isolated from HFKs attached to collagen or laminin 5 and assayed by Northern blot with a cDNA probe homologous to Cx43 mRNA. The mRNA for Cx43 is 3.0 kb as expected. (B) Laminin 5 promotes assembly of Cx43 into Triton X-100–insoluble gap junctions: effects of BFA. HFKs were attached to surfaces coated with either laminin 5 or collagen for 2.5 h. Adherent HFKs were solubilized with Laemmli sample buffer either before (Whole cell) or after extraction with 1% Triton X-100 pretreatment (Triton insoluble). Triton extraction does not solubilize Cx43 that has assembled into gap junctions. Whole cell and Triton insoluble cell extracts were immunoblotted with anti-Cx43 antibody (mAb 3068). Cell extracts immunoblotted in the fifth through eighth lanes were derived from twice as many cells as extracts in the first through fourth lanes. Treatment with the protein trafficking inhibitor BFA inhibited the laminin 5–mediated assembly of gap junctions.

Mentions: We determined if adhesion to collagen and laminin 5 had different effects on expression of mRNA encoding Cx43. Total RNA was purified from cells incubated on each substrate for 2.5 h, quantitated, and electrophoresed. After checking for equal loading with ethidium bromide staining, the gel was blotted and probed with a 32P-labeled cDNA for Cx43. No significant difference between cells plated on collagen or laminin 5 was observed (Fig. 4 A, Cx43 mRNA is ∼3 kb). Reprobing the same blot for changes in mRNA encoding the α3 chain of laminin 5 also showed essentially no difference (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)

Adhesion to laminin 5 promotes assembly of gap junctions but not transcriptional regulation of Cx43. (A) Northern  analysis of HFK cells plated on collagen or laminin 5. mRNA was  isolated from HFKs attached to collagen or laminin 5 and assayed by Northern blot with a cDNA probe homologous to Cx43  mRNA. The mRNA for Cx43 is 3.0 kb as expected. (B) Laminin  5 promotes assembly of Cx43 into Triton X-100–insoluble gap  junctions: effects of BFA. HFKs were attached to surfaces coated  with either laminin 5 or collagen for 2.5 h. Adherent HFKs were  solubilized with Laemmli sample buffer either before (Whole  cell) or after extraction with 1% Triton X-100 pretreatment (Triton insoluble). Triton extraction does not solubilize Cx43 that has  assembled into gap junctions. Whole cell and Triton insoluble cell  extracts were immunoblotted with anti-Cx43 antibody (mAb  3068). Cell extracts immunoblotted in the fifth through eighth  lanes were derived from twice as many cells as extracts in the first  through fourth lanes. Treatment with the protein trafficking inhibitor BFA inhibited the laminin 5–mediated assembly of gap  junctions.
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Figure 4: Adhesion to laminin 5 promotes assembly of gap junctions but not transcriptional regulation of Cx43. (A) Northern analysis of HFK cells plated on collagen or laminin 5. mRNA was isolated from HFKs attached to collagen or laminin 5 and assayed by Northern blot with a cDNA probe homologous to Cx43 mRNA. The mRNA for Cx43 is 3.0 kb as expected. (B) Laminin 5 promotes assembly of Cx43 into Triton X-100–insoluble gap junctions: effects of BFA. HFKs were attached to surfaces coated with either laminin 5 or collagen for 2.5 h. Adherent HFKs were solubilized with Laemmli sample buffer either before (Whole cell) or after extraction with 1% Triton X-100 pretreatment (Triton insoluble). Triton extraction does not solubilize Cx43 that has assembled into gap junctions. Whole cell and Triton insoluble cell extracts were immunoblotted with anti-Cx43 antibody (mAb 3068). Cell extracts immunoblotted in the fifth through eighth lanes were derived from twice as many cells as extracts in the first through fourth lanes. Treatment with the protein trafficking inhibitor BFA inhibited the laminin 5–mediated assembly of gap junctions.
Mentions: We determined if adhesion to collagen and laminin 5 had different effects on expression of mRNA encoding Cx43. Total RNA was purified from cells incubated on each substrate for 2.5 h, quantitated, and electrophoresed. After checking for equal loading with ethidium bromide staining, the gel was blotted and probed with a 32P-labeled cDNA for Cx43. No significant difference between cells plated on collagen or laminin 5 was observed (Fig. 4 A, Cx43 mRNA is ∼3 kb). Reprobing the same blot for changes in mRNA encoding the α3 chain of laminin 5 also showed essentially no difference (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