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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 wounds display persistent accumulation of unprocessed laminin-γ2 in the BMZ. Representative cryosections of adult unwounded skin (a,b), 5-day excisional wounds (c,d), or 10-day excisional wounds (e,f) were prepared from control (a,c,e) or α3eKO (b,d,f) mice and stained by immunofluorescence with anti-γ2L4m (specific for the L4 module of the laminin-γ2 chain). Scale bar, 100μm. e, epidermis; d, dermis; wb, wound bed; *, blister. (g) ECM fractions were collected from α3β1-expressing keratinocytes (WT), α3- keratinocytes (α3-), or α3- cells with restored α3 subunit expression (α3+), cultured in high calcium (see Materials and Methods) and assessed by immunoblot with anti-laminin-γ2. The unprocessed (155 kD) and processed (105 kD) forms of laminin-γ2 are indicated. (h) Quantification of processed laminin-γ2 as a proportion of total laminin-γ2, normalized to the daily mean to account for variability by day. Data are mean ± s.e.m.; n=4; 1-way ANOVA, P<0.01; Tukey’s multiple comparison.
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Figure 3: α3eKO wounds display persistent accumulation of unprocessed laminin-γ2 in the BMZ. Representative cryosections of adult unwounded skin (a,b), 5-day excisional wounds (c,d), or 10-day excisional wounds (e,f) were prepared from control (a,c,e) or α3eKO (b,d,f) mice and stained by immunofluorescence with anti-γ2L4m (specific for the L4 module of the laminin-γ2 chain). Scale bar, 100μm. e, epidermis; d, dermis; wb, wound bed; *, blister. (g) ECM fractions were collected from α3β1-expressing keratinocytes (WT), α3- keratinocytes (α3-), or α3- cells with restored α3 subunit expression (α3+), cultured in high calcium (see Materials and Methods) and assessed by immunoblot with anti-laminin-γ2. The unprocessed (155 kD) and processed (105 kD) forms of laminin-γ2 are indicated. (h) Quantification of processed laminin-γ2 as a proportion of total laminin-γ2, normalized to the daily mean to account for variability by day. Data are mean ± s.e.m.; n=4; 1-way ANOVA, P<0.01; Tukey’s multiple comparison.

Mentions: LN-332 is secreted by keratinocytes as a heterotrimer of three chains, α3 (190-200kDa), β3 (140 kDa) and γ2 (155 kDa), and differential processing of the α3 and γ2 chains has been linked to changes in stable adhesion and migration (Gianelli et al., 1997; Goldfinger et al., 1998). Since processing of the γ2 chain has been linked to stable incorporation of LN-332 into BM (Aumailley et al., 2003), we reasoned that epidermal α3β1 might modulate laminin-γ2 processing. To test this hypothesis, we utilized an antibody directed against the globular L4 module (anti-γ2L4m) of the laminin-γ2 chain (Sasaki et al., 2001). As this domain is cleaved off during γ2 chain processing, positive staining indicates the presence of the unprocessed, precursor form of LN-332. To assess differential laminin-γ2 processing, we performed immunofluorescence of wounds from adult control and α3eKO mice at various time-points post-wounding. Unwounded skin of both control and α3eKO mice showed weak anti-γ2L4m staining (Fig. 3a,b), compared to total LN-332 (see Fig. 1), indicative of LN-332 processing. Five-day wounds of both control and α3eKO mice displayed accumulation of unprocessed laminin-γ2 at the BMZ (Fig. 3c,d). Interestingly, accumulation was resolved in 10-day wounds of control animals (Fig. 3e) but persisted in 10-day wounds of α3eKO mice (Fig. 3f), suggesting a delay in LN-332 processing in the absence of α3β1.


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 wounds display persistent accumulation of unprocessed laminin-γ2 in the BMZ. Representative cryosections of adult unwounded skin (a,b), 5-day excisional wounds (c,d), or 10-day excisional wounds (e,f) were prepared from control (a,c,e) or α3eKO (b,d,f) mice and stained by immunofluorescence with anti-γ2L4m (specific for the L4 module of the laminin-γ2 chain). Scale bar, 100μm. e, epidermis; d, dermis; wb, wound bed; *, blister. (g) ECM fractions were collected from α3β1-expressing keratinocytes (WT), α3- keratinocytes (α3-), or α3- cells with restored α3 subunit expression (α3+), cultured in high calcium (see Materials and Methods) and assessed by immunoblot with anti-laminin-γ2. The unprocessed (155 kD) and processed (105 kD) forms of laminin-γ2 are indicated. (h) Quantification of processed laminin-γ2 as a proportion of total laminin-γ2, normalized to the daily mean to account for variability by day. Data are mean ± s.e.m.; n=4; 1-way ANOVA, P<0.01; Tukey’s multiple comparison.
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Figure 3: α3eKO wounds display persistent accumulation of unprocessed laminin-γ2 in the BMZ. Representative cryosections of adult unwounded skin (a,b), 5-day excisional wounds (c,d), or 10-day excisional wounds (e,f) were prepared from control (a,c,e) or α3eKO (b,d,f) mice and stained by immunofluorescence with anti-γ2L4m (specific for the L4 module of the laminin-γ2 chain). Scale bar, 100μm. e, epidermis; d, dermis; wb, wound bed; *, blister. (g) ECM fractions were collected from α3β1-expressing keratinocytes (WT), α3- keratinocytes (α3-), or α3- cells with restored α3 subunit expression (α3+), cultured in high calcium (see Materials and Methods) and assessed by immunoblot with anti-laminin-γ2. The unprocessed (155 kD) and processed (105 kD) forms of laminin-γ2 are indicated. (h) Quantification of processed laminin-γ2 as a proportion of total laminin-γ2, normalized to the daily mean to account for variability by day. Data are mean ± s.e.m.; n=4; 1-way ANOVA, P<0.01; Tukey’s multiple comparison.
Mentions: LN-332 is secreted by keratinocytes as a heterotrimer of three chains, α3 (190-200kDa), β3 (140 kDa) and γ2 (155 kDa), and differential processing of the α3 and γ2 chains has been linked to changes in stable adhesion and migration (Gianelli et al., 1997; Goldfinger et al., 1998). Since processing of the γ2 chain has been linked to stable incorporation of LN-332 into BM (Aumailley et al., 2003), we reasoned that epidermal α3β1 might modulate laminin-γ2 processing. To test this hypothesis, we utilized an antibody directed against the globular L4 module (anti-γ2L4m) of the laminin-γ2 chain (Sasaki et al., 2001). As this domain is cleaved off during γ2 chain processing, positive staining indicates the presence of the unprocessed, precursor form of LN-332. To assess differential laminin-γ2 processing, we performed immunofluorescence of wounds from adult control and α3eKO mice at various time-points post-wounding. Unwounded skin of both control and α3eKO mice showed weak anti-γ2L4m staining (Fig. 3a,b), compared to total LN-332 (see Fig. 1), indicative of LN-332 processing. Five-day wounds of both control and α3eKO mice displayed accumulation of unprocessed laminin-γ2 at the BMZ (Fig. 3c,d). Interestingly, accumulation was resolved in 10-day wounds of control animals (Fig. 3e) but persisted in 10-day wounds of α3eKO mice (Fig. 3f), suggesting a delay in LN-332 processing in the absence of α3β1.

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