alpha3beta1 Integrin is required for normal development of the epidermal basement membrane - PubMed (original) (raw)
alpha3beta1 Integrin is required for normal development of the epidermal basement membrane
C M DiPersio et al. J Cell Biol. 1997.
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.
Figures
Figure 1
α3β1 integrin is absent from the skin of α3-null mice. Frozen skin sections were prepared from the limbs of wild-type (A–D) or α3-null (E–H) mice and stained by immunofluorescence with an antiserum against the cytoplasmic domain of α3 (B and F), or with the preimmune serum (D and H). The corresponding phase contrast (A, C, E, and G) is shown to the left of each immunofluorescence panel. e, epidermis; d, dermis. Arrowheads point to basal keratinocytes of the epidermis. Bar, 50 μm.
Figure 2
α3-null mice form skin blisters. (A and B) Frozen skin sections from wildtype (A) or α3-null (B) mice were stained with hematoxylin and eosin and the epidermal-dermal junctions were compared. Arrowheads point to basal keratinocytes of the epidermis. (C and D) A frozen skin section from an α3null mouse showing a blister viewed by phase contrast (C) or stained by immunofluorescence with an antiserum against laminin-5 (D). Arrowheads and arrows point to areas of laminin-5 staining at the dermal and epidermal sides of the blister, respectively. e, epidermis; d, dermis. Bars, 50 μm.
Figure 3
The basement membrane in α3-null skin is disorganized. (A–D) Frozen skin sections from wild-type (A and C) or α3-null (B and D) mice were viewed by phase contrast (A and B) or stained by immunofluorescence with an antiserum against laminin-5 (C and D). Arrowheads point to areas of laminin-5 staining at the basement membrane in the wild-type skin (A and C) or at regions of disorganized basement membrane in α3-null skin (B and D). (E and F) Electron micrographs comparing ultrastructure of the basement membrane zone in wild-type (E) and α3-null (F) skin. (G and H) Schematic illustration of relevant structures seen in E and F. BK, basal keratinocyte; LL, lamina lucida; LD, lamina densa; arrowheads point to hemidesmosomes along the basal aspect of the plasma membranes in the basal keratinocytes. Bars: (D) 50 μm; (F) 200 nm.
Figure 4
Entactin and type VII collagen codistribute with laminin-5 over regions of disorganized basement membrane and to both dermal and epidermal sides of blisters in α3-null skin. Frozen sections from neonatal skin were stained by double-label immunofluorescence with GoH3 monoclonal antibody against the α6 integrin subunit and antiserum against laminin-5 (A, E, I, and B, F, J, respectively), or by immunofluorescence with antisera against type VII collagen (C, G, and K) or entactin (D, H, and L). (A–D) Representative fields from wild-type skin. (E–H) Adjacent sections through a region of disorganized basement membrane in α3-null skin; arrows point to areas in the dermis, outside the basement membrane zone as defined by α6-staining (E) where basement membrane proteins are detected (F–H). (I–L) Adjacent sections through a blister in α3-null skin; arrowheads and arrows point to the dermal and epidermal sides of the blister, respectively. Bar, 50 μm.
Figure 5
Fibronectin distributes to both epidermal and dermal sides of blisters in α3null skin. Frozen sections from wild-type (A, B, D, and E) or α3-null (C and F) skin were stained with an antiserum against fibronectin (B and C) or the preimmune serum (A), or with antiserum specific for the EIIIB segment of fibronectin (D–F). Recognition of fibronectin by anti-EIIIB requires treatment with N-glycanase (E and F), as described previously (Peters and Hynes, 1996); as a control, the section in D was not treated with N-glycanase. (A, B, D, and E) Arrowheads point to the dermal-epidermal junction. (C and F) Arrowheads and arrows point to the dermal and epidermal sides of blisters, respectively. Bar, 50 μm.
Figure 6
Distributions of entactin and type VII collagen in the developing skin. Frozen sections from mouse embryonic skin at days E11.5 (A and B), E15.5 (C and D), or E17.5 (E and F) of development were stained with antisera against entactin (A, C, and E) or type VII collagen (B, D, and F). Staining patterns for entactin or type VII collagen were identical in wild-type (A, C, and D) and heterozygous (E and F) embryos; type VII collagen staining in B is from an α3-null, E11.5 embryo, but was identical to that of a wild-type embryo at this stage. e, epidermis; d, dermis. Bar, 50 μm.
Figure 7
Distributions of α6β4 and laminin-5 in the developing skin of normal and α3-null 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-null 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-null 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.
Figure 8
Surface expression of integrins in primary keratinocytes isolated from wild-type (lanes 1–3) or α3-null (lanes 4–6), neonatal mice. Detergent lysates from 125I surface-labeled cells were immunoprecipitated with antisera against the cytoplasmic domains of the β1, α3, or α6 integrin subunits, as indicated at the top of each lane. Molecular weight markers are shown to the right of the autoradiograph. Migratory positions of certain integrin subunits are indicated to the left, including proteolytic fragments of β4, a, b, and c, described previously by Hemler et al. (1989). The β1-associated band in α3-null cells that comigrates with α3 (lane 4) represents other integrin α subunits that dimerize with β1 in keratinocytes, since α3 was not detected in α3-null cells (lane 5). ?, an unidentified band that may represent a proteolytic fragment of β4 (see text).
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 α3null 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-null (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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