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Loss of the desmosomal component perp impairs wound healing in vivo.

Beaudry VG, Ihrie RA, Jacobs SB, Nguyen B, Pathak N, Park E, Attardi LD - Dermatol Res Pract (2010)

Bottom Line: Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound.Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays.Thus, Perp's role in promoting cell adhesion is essential for wound closure.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound. Modulation of cell-cell adhesion junctions is thought to facilitate cellular proliferation and migration of keratinocytes across the wound. In particular, desmosomes, adhesion complexes critical for maintaining epithelial integrity, are downregulated at the wound edge. It is unclear, however, how compromised desmosomal adhesion would affect wound reepithelialization, given the need for a delicate balance between downmodulating adhesive strength to permit changes in cellular morphology and maintaining adhesion to allow coordinated migration of keratinocyte sheets. Here, we explore the contribution of desmosomal adhesion to wound healing using mice deficient for the desmosomal component Perp. We find that Perp conditional knockout mice display delayed wound healing relative to controls. Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays. Thus, Perp's role in promoting cell adhesion is essential for wound closure. Together, these studies suggest a role for desmosomal adhesion in efficient wound healing.

No MeSH data available.


Related in: MedlinePlus

Acute deletion of Perp leads to desmosome defects. (a) Perp immunohistochemistry on skin samples from control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. (b) H&E analysis of control (Perpfl/fl) and K14CreER;  Perpfl/fl  mice 4 weeks post tamoxifen injection, 200× and 400× magnification. (c) Graph displays the average percentage of PCNA positive cells in the basal cell layer in both control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. Graph represents the average of 3 separate fields from each of 4 mice +/− SEM. Statistical significance was determined using the Mann-Whitney test * = P < .03. (d) Representative image of the proliferation in the basal layer of the epidermis measured by PCNA immunostaining in control (Perpfl/fl) and K14CreER;  Perpfl/fl  mice 4 weeks post tamoxifen injection. (e) Immunofluorescence analysis of differentiation markers on control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. Keratin 14, Keratin 1, and Loricrin mark the basal, the spinous, and the granular layers, respectively. DAPI is used to mark nuclei. (f) Solubility/western blot analysis of Dsg1 and Pg in K14CreER;  Perpfl/fl or control (K14CreER; Perp+/+) mouse skin. Both Triton X-100-soluble and Urea fractions are presented. Gapdh and Keratin 14 serve as loading controls for the Triton X-100 and urea fractions, respectively. Scale bar for panels (a, b) (right column), (d), and (e) equals 20 μm. Scale bar for panel (b) (left column) equals  100 μm.
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fig1: Acute deletion of Perp leads to desmosome defects. (a) Perp immunohistochemistry on skin samples from control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. (b) H&E analysis of control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection, 200× and 400× magnification. (c) Graph displays the average percentage of PCNA positive cells in the basal cell layer in both control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. Graph represents the average of 3 separate fields from each of 4 mice +/− SEM. Statistical significance was determined using the Mann-Whitney test * = P < .03. (d) Representative image of the proliferation in the basal layer of the epidermis measured by PCNA immunostaining in control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. (e) Immunofluorescence analysis of differentiation markers on control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. Keratin 14, Keratin 1, and Loricrin mark the basal, the spinous, and the granular layers, respectively. DAPI is used to mark nuclei. (f) Solubility/western blot analysis of Dsg1 and Pg in K14CreER; Perpfl/fl or control (K14CreER; Perp+/+) mouse skin. Both Triton X-100-soluble and Urea fractions are presented. Gapdh and Keratin 14 serve as loading controls for the Triton X-100 and urea fractions, respectively. Scale bar for panels (a, b) (right column), (d), and (e) equals 20 μm. Scale bar for panel (b) (left column) equals  100 μm.

Mentions: To define the role of Perp and desmosomes in wound healing, we analyzed wound reepithelialization in vivo using Perp-deficient mice. We generated cohorts of 6-week-old control and conditional Perp knockout mice (Perpfl/fl; fl = floxed) expressing a K14CreER transgene, which allows deletion of the Perp locus in the epidermis upon introduction of tamoxifen. Immunohistochemistry confirmed that Perp expression was successfully abolished in the majority of these mice 4 weeks after tamoxifen injection (Figure 1(a)). Specifically, ~70% of these mice exhibited highly efficient Perp ablation (>90%) throughout the epidermis, while the other ~30% displayed somewhat less efficient Perp loss in the epidermis (>50%). Analysis of the skin from these mice revealed specific alterations in the epidermis of K14CreER; Perpfl/fl mice compared to controls (Figures 1(b)–1(e)). For example, we noted an increase in the percentage of proliferating basal cells in the K14CreER; Perpfl/fl mice compared to controls (Figures 1(c), 1(d)), similar to that observed in constitutive Perp knockout mice. Furthermore, this enhanced proliferation led to the expansion of differentiation marker staining in the skin (Figure 1(e)), although there were no apparent aberrations in the differentiation patterns of the skin in the K14CreER; Perpfl/fl mice compared to controls. Occasional blisters were also observed in K14CreER; Perpfl/fl mice, and accordingly, we found that desmosomes were functionally compromised in the skin of K14CreER; Perpfl/fl mice using a solubility assay (Figure 1(f)). This assay relies on the fact that stably formed desmosomal complexes can only be solubilized by chaotropic agents, whereas improperly assembled desmosomal components can be solubilized by the nonionic detergent Triton X-100. We found that Desmoglein 1 and Plakoglobin display enhanced Triton X-100 solubility in skin from K14CreER; Perpfl/fl mice compared to skin from control mice, confirming that acute deletion of Perp leads to impaired desmosome formation similar to that observed in constitutive Perp−/− mice [13].


Loss of the desmosomal component perp impairs wound healing in vivo.

Beaudry VG, Ihrie RA, Jacobs SB, Nguyen B, Pathak N, Park E, Attardi LD - Dermatol Res Pract (2010)

Acute deletion of Perp leads to desmosome defects. (a) Perp immunohistochemistry on skin samples from control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. (b) H&E analysis of control (Perpfl/fl) and K14CreER;  Perpfl/fl  mice 4 weeks post tamoxifen injection, 200× and 400× magnification. (c) Graph displays the average percentage of PCNA positive cells in the basal cell layer in both control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. Graph represents the average of 3 separate fields from each of 4 mice +/− SEM. Statistical significance was determined using the Mann-Whitney test * = P < .03. (d) Representative image of the proliferation in the basal layer of the epidermis measured by PCNA immunostaining in control (Perpfl/fl) and K14CreER;  Perpfl/fl  mice 4 weeks post tamoxifen injection. (e) Immunofluorescence analysis of differentiation markers on control (Perpfl/fl) and K14CreER;  Perpfl/fl mice 4 weeks post tamoxifen injection. Keratin 14, Keratin 1, and Loricrin mark the basal, the spinous, and the granular layers, respectively. DAPI is used to mark nuclei. (f) Solubility/western blot analysis of Dsg1 and Pg in K14CreER;  Perpfl/fl or control (K14CreER; Perp+/+) mouse skin. Both Triton X-100-soluble and Urea fractions are presented. Gapdh and Keratin 14 serve as loading controls for the Triton X-100 and urea fractions, respectively. Scale bar for panels (a, b) (right column), (d), and (e) equals 20 μm. Scale bar for panel (b) (left column) equals  100 μm.
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fig1: Acute deletion of Perp leads to desmosome defects. (a) Perp immunohistochemistry on skin samples from control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. (b) H&E analysis of control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection, 200× and 400× magnification. (c) Graph displays the average percentage of PCNA positive cells in the basal cell layer in both control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. Graph represents the average of 3 separate fields from each of 4 mice +/− SEM. Statistical significance was determined using the Mann-Whitney test * = P < .03. (d) Representative image of the proliferation in the basal layer of the epidermis measured by PCNA immunostaining in control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. (e) Immunofluorescence analysis of differentiation markers on control (Perpfl/fl) and K14CreER; Perpfl/fl mice 4 weeks post tamoxifen injection. Keratin 14, Keratin 1, and Loricrin mark the basal, the spinous, and the granular layers, respectively. DAPI is used to mark nuclei. (f) Solubility/western blot analysis of Dsg1 and Pg in K14CreER; Perpfl/fl or control (K14CreER; Perp+/+) mouse skin. Both Triton X-100-soluble and Urea fractions are presented. Gapdh and Keratin 14 serve as loading controls for the Triton X-100 and urea fractions, respectively. Scale bar for panels (a, b) (right column), (d), and (e) equals 20 μm. Scale bar for panel (b) (left column) equals  100 μm.
Mentions: To define the role of Perp and desmosomes in wound healing, we analyzed wound reepithelialization in vivo using Perp-deficient mice. We generated cohorts of 6-week-old control and conditional Perp knockout mice (Perpfl/fl; fl = floxed) expressing a K14CreER transgene, which allows deletion of the Perp locus in the epidermis upon introduction of tamoxifen. Immunohistochemistry confirmed that Perp expression was successfully abolished in the majority of these mice 4 weeks after tamoxifen injection (Figure 1(a)). Specifically, ~70% of these mice exhibited highly efficient Perp ablation (>90%) throughout the epidermis, while the other ~30% displayed somewhat less efficient Perp loss in the epidermis (>50%). Analysis of the skin from these mice revealed specific alterations in the epidermis of K14CreER; Perpfl/fl mice compared to controls (Figures 1(b)–1(e)). For example, we noted an increase in the percentage of proliferating basal cells in the K14CreER; Perpfl/fl mice compared to controls (Figures 1(c), 1(d)), similar to that observed in constitutive Perp knockout mice. Furthermore, this enhanced proliferation led to the expansion of differentiation marker staining in the skin (Figure 1(e)), although there were no apparent aberrations in the differentiation patterns of the skin in the K14CreER; Perpfl/fl mice compared to controls. Occasional blisters were also observed in K14CreER; Perpfl/fl mice, and accordingly, we found that desmosomes were functionally compromised in the skin of K14CreER; Perpfl/fl mice using a solubility assay (Figure 1(f)). This assay relies on the fact that stably formed desmosomal complexes can only be solubilized by chaotropic agents, whereas improperly assembled desmosomal components can be solubilized by the nonionic detergent Triton X-100. We found that Desmoglein 1 and Plakoglobin display enhanced Triton X-100 solubility in skin from K14CreER; Perpfl/fl mice compared to skin from control mice, confirming that acute deletion of Perp leads to impaired desmosome formation similar to that observed in constitutive Perp−/− mice [13].

Bottom Line: Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound.Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays.Thus, Perp's role in promoting cell adhesion is essential for wound closure.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound. Modulation of cell-cell adhesion junctions is thought to facilitate cellular proliferation and migration of keratinocytes across the wound. In particular, desmosomes, adhesion complexes critical for maintaining epithelial integrity, are downregulated at the wound edge. It is unclear, however, how compromised desmosomal adhesion would affect wound reepithelialization, given the need for a delicate balance between downmodulating adhesive strength to permit changes in cellular morphology and maintaining adhesion to allow coordinated migration of keratinocyte sheets. Here, we explore the contribution of desmosomal adhesion to wound healing using mice deficient for the desmosomal component Perp. We find that Perp conditional knockout mice display delayed wound healing relative to controls. Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays. Thus, Perp's role in promoting cell adhesion is essential for wound closure. Together, these studies suggest a role for desmosomal adhesion in efficient wound healing.

No MeSH data available.


Related in: MedlinePlus