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

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-null 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-null mice display perinatal skin blisters similar to those in α3β1-deficient mice. Interestingly, α3-null 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.

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.

Figure 2

Wounds of adult α3eKO mice display junctional blisters and persistent basement membrane disorganization. (a-d) Cryosections of excisional wounds from control (a,c) or α3eKO (b,d) mice, isolated 5-days (a,b) or 20 days (c,d) post-wounding, were stained by immunofluorescence with anti-LN-332. (e,f) Paraffin sections from 5-day excisional wounds of a control (e) or α3eKO (f) mouse were stained with H&E. Blistering was detected in 5-day wounds of 10/12 α3eKO mice, but not in 5-day wounds of control mice (n=8). Blistering was not detected in 20-day wounds of either genotype (control, n=4; α3eKO, n=4). White scale bar, 100μm (in panels a-d); black scale bar, 200μm (in panels e,f). e, epidermis; wb, wound bed; s, eschar; b, blood; *, blister; arrow, migrating epidermis; arrowhead, BMZ.

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-null 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.

Figure 4

Absence of fibulin-2 causes skin blistering in neonatal mice. H&E staining of paraffin sections from paws of control (a) or fibulin-2-null (b) mice revealed blisters at the epidermal-dermal junction in the latter (n=6; representative images from P0 paws are shown). Cryosections of P2 neonatal paw skin from control (c), fibulin-2-null (d), or α3eKO (e,f) mice were stained by double-label immunofluorescence with anti-fibulin-2 (red) and anti-keratin 14 to mark basal keratinocytes (green). Representative images of an α3eKO paw show an unblistered region (e) and a blistered region (f). Fibulin-2 is reduced in non-blistered skin of neonatal α3eKO mice, but is upregulated in blistered regions. Reactivity to anti-fibulin-2 was not detected in fibulin-2-null mice (d; non-blistered region shown), demonstrating specificity. Scale bars, 100μm. e, epidermis; d, dermis; *, blister.

Figure 5

α3β1-dependent modulation in laminin-γ2 processing is independent of fibulin-2 levels. α3β1 expressing keratinocytes (WT) were transduced with lentivirus expressing non-targeting shRNA (ctrl) or two distinct shRNAs that target fibulin-2 (Fbln-2; numbered 1,2). As a control, α3β1-deficient keratinocytes (α3-) were tranduced with non-targeting shRNA (ctrl). (a) Cell lysates were assayed by immunoblot to verify fibulin-2 knockdown. Anti-α3 confirmed the presence or absence of α3 integrin, and anti-Erk served as normalization control. Quantification of relative fibulin-2, normalized to Erk, as a proportion of levels in control shRNA-treated WT cells, is shown below. Data are mean ± s.e.m.; n=3; 1-way ANOVA, P<0.001; Tukey’s multiple comparison, *P<0.05. (b) Matrix preparations or whole cell lysates were assayed by immunoblot with anti-laminin-γ2. As expected, both unprocessed (155 kD) and processed (105 kD) forms of laminin-γ2 were detected in matrix preparations, while only unprocessed laminin-γ2 was detected in whole cell lysates. Erk served as a loading control for whole cell lysates.

Figure 6

Healing of blisters in fibulin-2-null mice or α3eKO mice is evident from their displacement into the suprabasal layers of the epidermis. Cryosections of paws from P2 control pups (a,b), P2 fibulin-2-null pups (c,d), or P10 α3eKO pups (e,f) were stained by double-label immunofluorescence with anti-LN-332 (red; a,c,e) and anti-keratin 14 to mark basal keratinocytes (green; b,d,f). Note that staining for LN-332 or keratin 14 is seen to line or surround, respectively, the displaced blisters, indicating that these blisters derived from the basal layer of the epidermis. Scale bar, 100μm. e, epidermis; d, dermis; arrows, displaced blisters.