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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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Immunostaining of human wounds identifies parallel changes in laminin 5, integrin α3β1, E-cadherin, and Cx43. Incisional  wounds were prepared in normal human skin and then collected by punch biopsy at the indicated times (see Materials and Methods).  Fixed cryostat sections (6 μm) were reacted with antibodies specific to components of cell as indicated for each panel. A–F are at the  same lower magnification (bar, 100 μm) and G–I are at higher magnification (bar, 50 μm). (A) α3 chain of laminin 5 (mAb C2-5), 24 h  wound. (B) Type VII collagen (mAb L3D), 24 h wound. (C) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. (D) Cx43 (PNRF),  24 h wound. (E) Integrin α3 chain (mAb P1B5), 53 h wound (the area within the small rectangle has been enlarged in the large rectangle  inset to show the polarization of α3β1 to the basal plasma membrane in the wound bed). (F) Fibronectin (mAb P1H11), 53 h wound.  (G) Cx43 (PNRF), 24 h wound. (H) E-cadherin (mAb HECD1), 24 h wound. (I) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h  wound. Different regions of the BM and epidermis were identified as follows: wound bed, unfilled arrowhead; normal epidermis distant  to the wound, unfilled arrow; wound edge, filled arrow; epidermis adjacent to the wound bed, filled arrowhead.
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Figure 1: Immunostaining of human wounds identifies parallel changes in laminin 5, integrin α3β1, E-cadherin, and Cx43. Incisional wounds were prepared in normal human skin and then collected by punch biopsy at the indicated times (see Materials and Methods). Fixed cryostat sections (6 μm) were reacted with antibodies specific to components of cell as indicated for each panel. A–F are at the same lower magnification (bar, 100 μm) and G–I are at higher magnification (bar, 50 μm). (A) α3 chain of laminin 5 (mAb C2-5), 24 h wound. (B) Type VII collagen (mAb L3D), 24 h wound. (C) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. (D) Cx43 (PNRF), 24 h wound. (E) Integrin α3 chain (mAb P1B5), 53 h wound (the area within the small rectangle has been enlarged in the large rectangle inset to show the polarization of α3β1 to the basal plasma membrane in the wound bed). (F) Fibronectin (mAb P1H11), 53 h wound. (G) Cx43 (PNRF), 24 h wound. (H) E-cadherin (mAb HECD1), 24 h wound. (I) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. Different regions of the BM and epidermis were identified as follows: wound bed, unfilled arrowhead; normal epidermis distant to the wound, unfilled arrow; wound edge, filled arrow; epidermis adjacent to the wound bed, filled arrowhead.

Mentions: We investigated the possible interdependence of changes in the expression and localization of the junctional components laminin 5, integrin α3β1, E-cadherin, and Cx43 in response to activation by injury of human epidermis (Fig. 1). Laminin 5, detected with mAb C2-5 against the α3 chain, was expressed in the normal BM distant to the wound (Fig. 1 A, unfilled arrow). Incisional wounding of the skin disrupts the normal BM in the wound bed and exposes dermal fibronectin (Fig. 1 F) and collagen (not shown) present throughout the dermis. By 24 h after wounding, laminin 5 was deposited into the provisional BM of the wound bed by migrating epithelial cells (Fig. 1 A, unfilled arrowhead). The wound edge is marked with filled arrows. Similar results were observed with staining with mAbs that interact with the β3 and γ2 chains of laminin 5 (results not shown). For comparison, type VII collagen, which has been reported to interact with laminin 5 (Chen et al., 1997; Rousselle et al., 1997), was expressed in the mature BM distant to (Fig. 1 B, unfilled arrow) and adjacent to the wound edge (filled arrowhead) but was absent from the provisional BM (unfilled arrowhead). Thus, laminin 5 does not depend on interactions with type VII collagen for localization to the provisional BM. Significantly, we detected a precursor form of the α3 chain of laminin 5 with mAb D2-1 (Fig. 1 C and a subsequent section at higher magnification in I). The precursor α3 chain is expressed in the cytoplasm of epithelial cells in the wound bed (Fig. 1, C and I, unfilled arrowheads) but is reduced or undetectable in the normal BM distant from the wound edge (Fig. 1, C and I, unfilled arrows). Most importantly, precursor α3 detected by mAb D2-1 is expressed in the BM immediately adjacent to the wound edge at 24 h (Fig. 1, C and I, filled arrowheads) and as early as 8 h after wounding (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)

Immunostaining of human wounds identifies parallel changes in laminin 5, integrin α3β1, E-cadherin, and Cx43. Incisional  wounds were prepared in normal human skin and then collected by punch biopsy at the indicated times (see Materials and Methods).  Fixed cryostat sections (6 μm) were reacted with antibodies specific to components of cell as indicated for each panel. A–F are at the  same lower magnification (bar, 100 μm) and G–I are at higher magnification (bar, 50 μm). (A) α3 chain of laminin 5 (mAb C2-5), 24 h  wound. (B) Type VII collagen (mAb L3D), 24 h wound. (C) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. (D) Cx43 (PNRF),  24 h wound. (E) Integrin α3 chain (mAb P1B5), 53 h wound (the area within the small rectangle has been enlarged in the large rectangle  inset to show the polarization of α3β1 to the basal plasma membrane in the wound bed). (F) Fibronectin (mAb P1H11), 53 h wound.  (G) Cx43 (PNRF), 24 h wound. (H) E-cadherin (mAb HECD1), 24 h wound. (I) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h  wound. Different regions of the BM and epidermis were identified as follows: wound bed, unfilled arrowhead; normal epidermis distant  to the wound, unfilled arrow; wound edge, filled arrow; epidermis adjacent to the wound bed, filled arrowhead.
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Related In: Results  -  Collection

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Figure 1: Immunostaining of human wounds identifies parallel changes in laminin 5, integrin α3β1, E-cadherin, and Cx43. Incisional wounds were prepared in normal human skin and then collected by punch biopsy at the indicated times (see Materials and Methods). Fixed cryostat sections (6 μm) were reacted with antibodies specific to components of cell as indicated for each panel. A–F are at the same lower magnification (bar, 100 μm) and G–I are at higher magnification (bar, 50 μm). (A) α3 chain of laminin 5 (mAb C2-5), 24 h wound. (B) Type VII collagen (mAb L3D), 24 h wound. (C) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. (D) Cx43 (PNRF), 24 h wound. (E) Integrin α3 chain (mAb P1B5), 53 h wound (the area within the small rectangle has been enlarged in the large rectangle inset to show the polarization of α3β1 to the basal plasma membrane in the wound bed). (F) Fibronectin (mAb P1H11), 53 h wound. (G) Cx43 (PNRF), 24 h wound. (H) E-cadherin (mAb HECD1), 24 h wound. (I) Precursor α3 chain of laminin 5 (mAb D2-1), 24 h wound. Different regions of the BM and epidermis were identified as follows: wound bed, unfilled arrowhead; normal epidermis distant to the wound, unfilled arrow; wound edge, filled arrow; epidermis adjacent to the wound bed, filled arrowhead.
Mentions: We investigated the possible interdependence of changes in the expression and localization of the junctional components laminin 5, integrin α3β1, E-cadherin, and Cx43 in response to activation by injury of human epidermis (Fig. 1). Laminin 5, detected with mAb C2-5 against the α3 chain, was expressed in the normal BM distant to the wound (Fig. 1 A, unfilled arrow). Incisional wounding of the skin disrupts the normal BM in the wound bed and exposes dermal fibronectin (Fig. 1 F) and collagen (not shown) present throughout the dermis. By 24 h after wounding, laminin 5 was deposited into the provisional BM of the wound bed by migrating epithelial cells (Fig. 1 A, unfilled arrowhead). The wound edge is marked with filled arrows. Similar results were observed with staining with mAbs that interact with the β3 and γ2 chains of laminin 5 (results not shown). For comparison, type VII collagen, which has been reported to interact with laminin 5 (Chen et al., 1997; Rousselle et al., 1997), was expressed in the mature BM distant to (Fig. 1 B, unfilled arrow) and adjacent to the wound edge (filled arrowhead) but was absent from the provisional BM (unfilled arrowhead). Thus, laminin 5 does not depend on interactions with type VII collagen for localization to the provisional BM. Significantly, we detected a precursor form of the α3 chain of laminin 5 with mAb D2-1 (Fig. 1 C and a subsequent section at higher magnification in I). The precursor α3 chain is expressed in the cytoplasm of epithelial cells in the wound bed (Fig. 1, C and I, unfilled arrowheads) but is reduced or undetectable in the normal BM distant from the wound edge (Fig. 1, C and I, unfilled arrows). Most importantly, precursor α3 detected by mAb D2-1 is expressed in the BM immediately adjacent to the wound edge at 24 h (Fig. 1, C and I, filled arrowheads) and as early as 8 h after wounding (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