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alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.

DiPersio CM, Hodivala-Dilke KM, Jaenisch R, Kreidberg JA, Hynes RO - J. Cell Biol. (1997)

Bottom Line: Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa.Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins.However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, and Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.

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α3β1 is required for postadhesion spreading of mouse  keratinocytes on laminin-5. (A and B) Primary keratinocytes from  neonatal mice heterozygous (A) or homozygous (B) for the α3 mutation were subcultured on HEK-secreted, laminin-5–rich  ECM (see Materials and Methods) and then photographed after  1.5 h. (C–H) Primary keratinocytes from wild-type (C, E, and G)  or α3- (D, F, and H) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde  and stained with Giemsa. (E and F) Fibronectin was included  with laminin-5 in the substrate. (G and H) Cells were treated with  the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.
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Figure 9: α3β1 is required for postadhesion spreading of mouse keratinocytes on laminin-5. (A and B) Primary keratinocytes from neonatal mice heterozygous (A) or homozygous (B) for the α3 mutation were subcultured on HEK-secreted, laminin-5–rich ECM (see Materials and Methods) and then photographed after 1.5 h. (C–H) Primary keratinocytes from wild-type (C, E, and G) or α3- (D, F, and H) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde and stained with Giemsa. (E and F) Fibronectin was included with laminin-5 in the substrate. (G and H) Cells were treated with the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.

Mentions: We subcultured primary mouse keratinocytes on laminin5-rich ECM prepared from human keratinocyte cultures, as described in the Materials and Methods section; this ECM supports α3β1-mediated cell attachment (Carter et al., 1991; Weitzman et al., 1993). Both α3-positive cells (in this case from a mouse heterozygous for the α3- mutation) and α3- cells attached to laminin-5–rich ECM within 15 min. After attachment, α3-positive keratinocytes spread rapidly on the laminin-5–rich ECM (Fig. 9 A). In contrast, α3- keratinocytes remained unspread for up to 2 h following attachment to this matrix (Fig. 9 B). The same results were obtained in spreading assays using purified laminin-5 (Fig. 9, C and D). α3- keratinocytes were able to spread when fibronectin was present in the substrate with laminin-5 (Fig. 9, E and F), demonstrating that α3β1-deficient cells were not generally deficient in the ability to spread. The antibody GoH3, which blocks cell adhesion mediated by α6 integrins (Sonnenberg et al., 1987), only slightly inhibited attachment, and did not affect spreading, of wild-type keratinocytes on laminin-5 (Fig. 9 G). In contrast, the same concentration of GoH3 completely inhibited attachment of α3β1-deficient cells to laminin-5 (Fig. 9 H). Therefore, while α6β4 and α3β1 are each sufficient for attachment of mouse keratinocytes to laminin-5, α3β1 is essential for postattachment cell spreading on this ligand, consistent with previous, integrin-blocking studies using human foreskin keratinocytes (Xia et al., 1996).


alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.

DiPersio CM, Hodivala-Dilke KM, Jaenisch R, Kreidberg JA, Hynes RO - J. Cell Biol. (1997)

α3β1 is required for postadhesion spreading of mouse  keratinocytes on laminin-5. (A and B) Primary keratinocytes from  neonatal mice heterozygous (A) or homozygous (B) for the α3 mutation were subcultured on HEK-secreted, laminin-5–rich  ECM (see Materials and Methods) and then photographed after  1.5 h. (C–H) Primary keratinocytes from wild-type (C, E, and G)  or α3- (D, F, and H) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde  and stained with Giemsa. (E and F) Fibronectin was included  with laminin-5 in the substrate. (G and H) Cells were treated with  the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.
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Related In: Results  -  Collection

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Figure 9: α3β1 is required for postadhesion spreading of mouse keratinocytes on laminin-5. (A and B) Primary keratinocytes from neonatal mice heterozygous (A) or homozygous (B) for the α3 mutation were subcultured on HEK-secreted, laminin-5–rich ECM (see Materials and Methods) and then photographed after 1.5 h. (C–H) Primary keratinocytes from wild-type (C, E, and G) or α3- (D, F, and H) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde and stained with Giemsa. (E and F) Fibronectin was included with laminin-5 in the substrate. (G and H) Cells were treated with the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.
Mentions: We subcultured primary mouse keratinocytes on laminin5-rich ECM prepared from human keratinocyte cultures, as described in the Materials and Methods section; this ECM supports α3β1-mediated cell attachment (Carter et al., 1991; Weitzman et al., 1993). Both α3-positive cells (in this case from a mouse heterozygous for the α3- mutation) and α3- cells attached to laminin-5–rich ECM within 15 min. After attachment, α3-positive keratinocytes spread rapidly on the laminin-5–rich ECM (Fig. 9 A). In contrast, α3- keratinocytes remained unspread for up to 2 h following attachment to this matrix (Fig. 9 B). The same results were obtained in spreading assays using purified laminin-5 (Fig. 9, C and D). α3- keratinocytes were able to spread when fibronectin was present in the substrate with laminin-5 (Fig. 9, E and F), demonstrating that α3β1-deficient cells were not generally deficient in the ability to spread. The antibody GoH3, which blocks cell adhesion mediated by α6 integrins (Sonnenberg et al., 1987), only slightly inhibited attachment, and did not affect spreading, of wild-type keratinocytes on laminin-5 (Fig. 9 G). In contrast, the same concentration of GoH3 completely inhibited attachment of α3β1-deficient cells to laminin-5 (Fig. 9 H). Therefore, while α6β4 and α3β1 are each sufficient for attachment of mouse keratinocytes to laminin-5, α3β1 is essential for postattachment cell spreading on this ligand, consistent with previous, integrin-blocking studies using human foreskin keratinocytes (Xia et al., 1996).

Bottom Line: Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa.Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins.However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, and Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.

Show MeSH
Related in: MedlinePlus