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Reduced fibulin-2 contributes to loss of basement membrane integrity and skin blistering in mice lacking integrin α3β1 in the epidermis.

Longmate WM, Monichan R, Chu ML, Tsuda T, Mahoney MG, DiPersio CM - J. Invest. Dermatol. (2014)

Bottom Line: In the current study we identify a role for α3β1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332.Interestingly, α3- wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering.Our findings indicate a role for integrin α3β1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York, USA.

ABSTRACT
Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3β1, a major receptor in the epidermis for adhesion to laminin-332 (LN-332), has critical roles in basement membrane (BM) organization during skin development. In the current study we identify a role for α3β1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332. We demonstrate that mice lacking α3β1 in the epidermis display ruptured BM beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3- mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3β1 in BM organization is recapitulated during wound healing. Mice lacking epidermal α3β1 also have reduced fibulin-2 expression, and fibulin-2- mice display perinatal skin blisters similar to those in α3β1-deficient mice. Interestingly, α3- wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3β1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

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α3eKO neonatal mice develop blisters in paw skin but recover by adulthood. (a,b) Images of P2 paws from a control (a) or α3eKO (b) mouse. Bracket in panel b indicates a blood filled blister. (c-f) Cryosections of paw skin from control (c,e) or α3eKO (d,f) mice were stained by immunofluorescence with anti-LN-332 to visualize the basement membrane. Sections are from P0 pups (c,d) or adult mice (>6-weeks of age; e,f). Images are from representative mice of each genotype. Blisters were detected in P0 paws of 10/11 α3eKO mice, but not in P0 control mice (n=10) or in adult mice of either genotype (control, n=4; α3eKO, n=4). Scale bar, 100μm (applicable to panels c-f). e, epidermis; d, dermis; *, blister.
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Figure 1: α3eKO neonatal mice develop blisters in paw skin but recover by adulthood. (a,b) Images of P2 paws from a control (a) or α3eKO (b) mouse. Bracket in panel b indicates a blood filled blister. (c-f) Cryosections of paw skin from control (c,e) or α3eKO (d,f) mice were stained by immunofluorescence with anti-LN-332 to visualize the basement membrane. Sections are from P0 pups (c,d) or adult mice (>6-weeks of age; e,f). Images are from representative mice of each genotype. Blisters were detected in P0 paws of 10/11 α3eKO mice, but not in P0 control mice (n=10) or in adult mice of either genotype (control, n=4; α3eKO, n=4). Scale bar, 100μm (applicable to panels c-f). e, epidermis; d, dermis; *, blister.

Mentions: Global deletion of the integrin α3 subunit in mice results in disorganization of the epidermal BM during skin development, leading to formation of neonatal skin blisters caused by BM rupture (DiPersio et al., 1997). These blisters are restricted to the paws, likely due to increased friction in that area (DiPersio et al., 1997). It was recently shown that skin blisters form in human patients with mutations in the ITGA3 gene (which encodes the integrin α3 subunit) (Has et al., 2012). The onset of skin fragility was earlier in α3eKO mice than in human patients, possibly due to perinatal compensatory mechanisms that exist in humans, but not in mice. Nevertheless, human blisters bear remarkable similarities to those that form in α3- mice (Has et al., 2012). Like α3- mice, these patients developed skin blisters in which LN-332 localizes to both the epidermal and dermal sides, indicative of BM rupture. These similarities suggest that α3β1-deficient mice provide a useful genetic model for the blistering component of the human disorder. However, α3- mice die shortly after birth making it difficult to investigate post-developmental roles for α3β1. Therefore, we utilized α3eKO mice in which Cre recombinase is driven by the keratin 14 promoter to effect deletion of the floxed α3 subunit gene preferentially in basal epidermal keratinocytes (Mitchell et al., 2009). Epidermal deletion of α3 caused skin blisters in paws of α3eKO neonates (Fig. 1a-d), attributing this phenotype to loss of α3β1 within the epidermal compartment. Importantly, LN-332 localized to both sides of these blisters (Fig. 1d, asterisk), suggesting that blisters are generated via a similar mechanism as in global α3 knockout mice (DiPersio et al., 1997) or human patients with ITGA3 gene mutations (Has et al., 2012). Entactin/nidogen, a BM component that is contributed by mesenchymal cells (Thomas and Dziadek, 1993), also localized to both sides of α3eKO blisters, further indicating BM rupture (Fig. S1). Neonatal paw blisters could often be seen macroscopically, sometimes with subcutaneous hemorrhaging (Fig. 1b). Remarkably, we did not detect blisters in paws of adult α3eKO mice, which showed only minimal LN-332 disorganization at the BM (Fig. 1e,f). These observations are consistent with a previous report that only minor microblisters were detected in skin of adult α3eKO mice (Margadant et al., 2009), and they indicate that adult mice recover from the developmental blistering defect caused by absence of α3β1 (DiPersio et al., 1997).


Reduced fibulin-2 contributes to loss of basement membrane integrity and skin blistering in mice lacking integrin α3β1 in the epidermis.

Longmate WM, Monichan R, Chu ML, Tsuda T, Mahoney MG, DiPersio CM - J. Invest. Dermatol. (2014)

α3eKO neonatal mice develop blisters in paw skin but recover by adulthood. (a,b) Images of P2 paws from a control (a) or α3eKO (b) mouse. Bracket in panel b indicates a blood filled blister. (c-f) Cryosections of paw skin from control (c,e) or α3eKO (d,f) mice were stained by immunofluorescence with anti-LN-332 to visualize the basement membrane. Sections are from P0 pups (c,d) or adult mice (>6-weeks of age; e,f). Images are from representative mice of each genotype. Blisters were detected in P0 paws of 10/11 α3eKO mice, but not in P0 control mice (n=10) or in adult mice of either genotype (control, n=4; α3eKO, n=4). Scale bar, 100μm (applicable to panels c-f). e, epidermis; d, dermis; *, blister.
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Figure 1: α3eKO neonatal mice develop blisters in paw skin but recover by adulthood. (a,b) Images of P2 paws from a control (a) or α3eKO (b) mouse. Bracket in panel b indicates a blood filled blister. (c-f) Cryosections of paw skin from control (c,e) or α3eKO (d,f) mice were stained by immunofluorescence with anti-LN-332 to visualize the basement membrane. Sections are from P0 pups (c,d) or adult mice (>6-weeks of age; e,f). Images are from representative mice of each genotype. Blisters were detected in P0 paws of 10/11 α3eKO mice, but not in P0 control mice (n=10) or in adult mice of either genotype (control, n=4; α3eKO, n=4). Scale bar, 100μm (applicable to panels c-f). e, epidermis; d, dermis; *, blister.
Mentions: Global deletion of the integrin α3 subunit in mice results in disorganization of the epidermal BM during skin development, leading to formation of neonatal skin blisters caused by BM rupture (DiPersio et al., 1997). These blisters are restricted to the paws, likely due to increased friction in that area (DiPersio et al., 1997). It was recently shown that skin blisters form in human patients with mutations in the ITGA3 gene (which encodes the integrin α3 subunit) (Has et al., 2012). The onset of skin fragility was earlier in α3eKO mice than in human patients, possibly due to perinatal compensatory mechanisms that exist in humans, but not in mice. Nevertheless, human blisters bear remarkable similarities to those that form in α3- mice (Has et al., 2012). Like α3- mice, these patients developed skin blisters in which LN-332 localizes to both the epidermal and dermal sides, indicative of BM rupture. These similarities suggest that α3β1-deficient mice provide a useful genetic model for the blistering component of the human disorder. However, α3- mice die shortly after birth making it difficult to investigate post-developmental roles for α3β1. Therefore, we utilized α3eKO mice in which Cre recombinase is driven by the keratin 14 promoter to effect deletion of the floxed α3 subunit gene preferentially in basal epidermal keratinocytes (Mitchell et al., 2009). Epidermal deletion of α3 caused skin blisters in paws of α3eKO neonates (Fig. 1a-d), attributing this phenotype to loss of α3β1 within the epidermal compartment. Importantly, LN-332 localized to both sides of these blisters (Fig. 1d, asterisk), suggesting that blisters are generated via a similar mechanism as in global α3 knockout mice (DiPersio et al., 1997) or human patients with ITGA3 gene mutations (Has et al., 2012). Entactin/nidogen, a BM component that is contributed by mesenchymal cells (Thomas and Dziadek, 1993), also localized to both sides of α3eKO blisters, further indicating BM rupture (Fig. S1). Neonatal paw blisters could often be seen macroscopically, sometimes with subcutaneous hemorrhaging (Fig. 1b). Remarkably, we did not detect blisters in paws of adult α3eKO mice, which showed only minimal LN-332 disorganization at the BM (Fig. 1e,f). These observations are consistent with a previous report that only minor microblisters were detected in skin of adult α3eKO mice (Margadant et al., 2009), and they indicate that adult mice recover from the developmental blistering defect caused by absence of α3β1 (DiPersio et al., 1997).

Bottom Line: In the current study we identify a role for α3β1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332.Interestingly, α3- wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering.Our findings indicate a role for integrin α3β1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York, USA.

ABSTRACT
Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3β1, a major receptor in the epidermis for adhesion to laminin-332 (LN-332), has critical roles in basement membrane (BM) organization during skin development. In the current study we identify a role for α3β1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332. We demonstrate that mice lacking α3β1 in the epidermis display ruptured BM beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3- mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3β1 in BM organization is recapitulated during wound healing. Mice lacking epidermal α3β1 also have reduced fibulin-2 expression, and fibulin-2- mice display perinatal skin blisters similar to those in α3β1-deficient mice. Interestingly, α3- wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3β1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

Show MeSH
Related in: MedlinePlus