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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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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.
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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.

Mentions: α3β1-dependence of laminin-γ2 processing was further assessed by immunoblotting of matrix deposited by a panel of mouse keratinocyte (MK) cell lines, including WT cells derived from a wildtype mouse, α3- cells derived from an α3- mouse, and α3+ cells in which α3β1 expression was restored in α3- cells through stable transfection with human α3 (DiPersio et al., 2000a; Iyer et al., 2005). Previously, we reported that these cells deposit only the unprocessed 155 kD form of laminin-γ2 when cultured in low-calcium medium (DiPersio et al., 2000a), as also reported in primary keratinocytes (deHart et al., 2003). Here, we cultured MK cells in high-calcium medium, which others have shown induces LN-332 processing in keratinocytes (Amano et al., 2000). Consistently, we detected the processed 105 kD fragment of laminin-γ2 in matrix fractions from WT cells cultured for 3-4 days under these conditions (Fig. 3g). We consistently observed reduced deposition of total laminin-γ2 in α3- cell cultures (see Fig. 5b). Importantly, we detected a significant reduction in the proportion of laminin-γ2 that is processed in the deposited matrix of α3- cells compared to α3 containing WT or α3+ cells (Fig. 3g,h), consistent with our in vivo data (Fig. 3a-f). While we occasionally detected enhanced processing in α3+ cells compared to WT, this difference was not statistically significant over several experiments (Fig. 3g,h).


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)

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.
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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.
Mentions: α3β1-dependence of laminin-γ2 processing was further assessed by immunoblotting of matrix deposited by a panel of mouse keratinocyte (MK) cell lines, including WT cells derived from a wildtype mouse, α3- cells derived from an α3- mouse, and α3+ cells in which α3β1 expression was restored in α3- cells through stable transfection with human α3 (DiPersio et al., 2000a; Iyer et al., 2005). Previously, we reported that these cells deposit only the unprocessed 155 kD form of laminin-γ2 when cultured in low-calcium medium (DiPersio et al., 2000a), as also reported in primary keratinocytes (deHart et al., 2003). Here, we cultured MK cells in high-calcium medium, which others have shown induces LN-332 processing in keratinocytes (Amano et al., 2000). Consistently, we detected the processed 105 kD fragment of laminin-γ2 in matrix fractions from WT cells cultured for 3-4 days under these conditions (Fig. 3g). We consistently observed reduced deposition of total laminin-γ2 in α3- cell cultures (see Fig. 5b). Importantly, we detected a significant reduction in the proportion of laminin-γ2 that is processed in the deposited matrix of α3- cells compared to α3 containing WT or α3+ cells (Fig. 3g,h), consistent with our in vivo data (Fig. 3a-f). While we occasionally detected enhanced processing in α3+ cells compared to WT, this difference was not statistically significant over several experiments (Fig. 3g,h).

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