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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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Distributions of α6β4 and laminin-5 in the developing skin of normal and α3- embryos. Frozen sections from mouse embryonic skin at days E15.5 (A–F) or E17.5 (G–L) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit (A, C, and G) or GoH3 monoclonal antibody against the α6 integrin subunit (E,  I, and K), and anti–laminin-5 serum (B, D, H, and F, J, L, respectively). Control sections were from wild-type embryos (A–D) or heterozygous embryos (G and H). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more  stratified regions (C and D), but not in less stratified regions (A and B); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3- embryos at E15.5 (E and F) and E17.5 (I and J), arrowheads point to areas of laminin-5 staining in areas of  disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. (K and L)  Higher magnification of α3- skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive  basement membrane, marked by arrowheads. e, epidermis; d, dermis. Bars: (shown in J for A–J) 50 μm; and (in L for K and L) 50 μm.
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Figure 7: Distributions of α6β4 and laminin-5 in the developing skin of normal and α3- embryos. Frozen sections from mouse embryonic skin at days E15.5 (A–F) or E17.5 (G–L) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit (A, C, and G) or GoH3 monoclonal antibody against the α6 integrin subunit (E, I, and K), and anti–laminin-5 serum (B, D, H, and F, J, L, respectively). Control sections were from wild-type embryos (A–D) or heterozygous embryos (G and H). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more stratified regions (C and D), but not in less stratified regions (A and B); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3- embryos at E15.5 (E and F) and E17.5 (I and J), arrowheads point to areas of laminin-5 staining in areas of disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. (K and L) Higher magnification of α3- skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive basement membrane, marked by arrowheads. e, epidermis; d, dermis. Bars: (shown in J for A–J) 50 μm; and (in L for K and L) 50 μm.

Mentions: To compare developmental expression of laminin-5 and its receptors α3β1 and α6β4, we first examined expression of these proteins in wild-type or heterozygous mice. α3β1 was expressed in the embryonic epidermis as early as E9.5 and remained restricted to basal keratinocytes, in a basolateral distribution, throughout development (Fig. 1 B and data not shown). The relative distributions of α6β4 and laminin-5 were determined by double-label immunofluorescence using a monoclonal antibody against the β4 subunit (346-11A; Kennel et al., 1989) and anti–laminin-5 serum (Fig. 7, A–D, G, and H). Laminin-5 was not detected in the unstratified skin of E11.5 embryos (data not shown). At E15.5, the degree of stratification varied throughout the embryo, and the appearance of laminin-5 in the basement membrane zone coincided roughly with increased epidermal stratification; anti–laminin-5 did not stain the basement membrane in less stratified regions (Fig. 7 B), but clearly stained the basement membrane in more stratified regions (Fig. 7 D). The distribution of β4 at E15.5 was similar; in less stratified regions it was present in a basolateral distribution within the basal keratinocyte layer (Fig. 7 A), similar to the distribution of α3β1 (not shown), and in more stratified regions it was concentrated at the basal surfaces of the basal keratinocytes, adjacent to laminin-5 in the basement membrane (compare Fig. 7, C and D). Therefore, recruitment of α6β4 to the basal aspects of basal keratinocytes appeared to coincide with incorporation of laminin-5 into the basement membrane, and may reflect the establishment of cell adhesion to laminin-5 mediated by α6β4. In E17.5 epidermis, α6β4 was always concentrated in the basal surfaces of basal keratinocytes adjacent to the laminin-5-rich basement membrane (Fig. 7, G and H), as in newborn skin (Fig. 4, A and B).


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)

Distributions of α6β4 and laminin-5 in the developing skin of normal and α3- embryos. Frozen sections from mouse embryonic skin at days E15.5 (A–F) or E17.5 (G–L) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit (A, C, and G) or GoH3 monoclonal antibody against the α6 integrin subunit (E,  I, and K), and anti–laminin-5 serum (B, D, H, and F, J, L, respectively). Control sections were from wild-type embryos (A–D) or heterozygous embryos (G and H). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more  stratified regions (C and D), but not in less stratified regions (A and B); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3- embryos at E15.5 (E and F) and E17.5 (I and J), arrowheads point to areas of laminin-5 staining in areas of  disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. (K and L)  Higher magnification of α3- skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive  basement membrane, marked by arrowheads. e, epidermis; d, dermis. Bars: (shown in J for A–J) 50 μm; and (in L for K and L) 50 μm.
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Figure 7: Distributions of α6β4 and laminin-5 in the developing skin of normal and α3- embryos. Frozen sections from mouse embryonic skin at days E15.5 (A–F) or E17.5 (G–L) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit (A, C, and G) or GoH3 monoclonal antibody against the α6 integrin subunit (E, I, and K), and anti–laminin-5 serum (B, D, H, and F, J, L, respectively). Control sections were from wild-type embryos (A–D) or heterozygous embryos (G and H). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more stratified regions (C and D), but not in less stratified regions (A and B); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3- embryos at E15.5 (E and F) and E17.5 (I and J), arrowheads point to areas of laminin-5 staining in areas of disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. (K and L) Higher magnification of α3- skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive basement membrane, marked by arrowheads. e, epidermis; d, dermis. Bars: (shown in J for A–J) 50 μm; and (in L for K and L) 50 μm.
Mentions: To compare developmental expression of laminin-5 and its receptors α3β1 and α6β4, we first examined expression of these proteins in wild-type or heterozygous mice. α3β1 was expressed in the embryonic epidermis as early as E9.5 and remained restricted to basal keratinocytes, in a basolateral distribution, throughout development (Fig. 1 B and data not shown). The relative distributions of α6β4 and laminin-5 were determined by double-label immunofluorescence using a monoclonal antibody against the β4 subunit (346-11A; Kennel et al., 1989) and anti–laminin-5 serum (Fig. 7, A–D, G, and H). Laminin-5 was not detected in the unstratified skin of E11.5 embryos (data not shown). At E15.5, the degree of stratification varied throughout the embryo, and the appearance of laminin-5 in the basement membrane zone coincided roughly with increased epidermal stratification; anti–laminin-5 did not stain the basement membrane in less stratified regions (Fig. 7 B), but clearly stained the basement membrane in more stratified regions (Fig. 7 D). The distribution of β4 at E15.5 was similar; in less stratified regions it was present in a basolateral distribution within the basal keratinocyte layer (Fig. 7 A), similar to the distribution of α3β1 (not shown), and in more stratified regions it was concentrated at the basal surfaces of the basal keratinocytes, adjacent to laminin-5 in the basement membrane (compare Fig. 7, C and D). Therefore, recruitment of α6β4 to the basal aspects of basal keratinocytes appeared to coincide with incorporation of laminin-5 into the basement membrane, and may reflect the establishment of cell adhesion to laminin-5 mediated by α6β4. In E17.5 epidermis, α6β4 was always concentrated in the basal surfaces of basal keratinocytes adjacent to the laminin-5-rich basement membrane (Fig. 7, G and H), as in newborn skin (Fig. 4, A and B).

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