Limits...
PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin.

Frateschi S, Camerer E, Crisante G, Rieser S, Membrez M, Charles RP, Beermann F, Stehle JC, Breiden B, Sandhoff K, Rotman S, Haftek M, Wilson A, Ryser S, Steinhoff M, Coughlin SR, Hummler E - Nat Commun (2011)

Bottom Line: K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations.Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2- background, establishing PAR2 as a pivotal mediator of pathogenesis.Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne 1005, Switzerland.

ABSTRACT
Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2- background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

No MeSH data available.


Related in: MedlinePlus

Generation and characterization of Grhl3PAR2/+ transgenic mice.(a) Scheme of the Grhl3PAR2/+ knock-in targeting vector, part of the targeted Grhl3 gene locus and the resulting targeted allele. Exons (black boxes), transgenic sequences (from the left, white boxes: mouse PAR2 coding sequence [PAR2 cDNA], internal ribosome entry site [IRES], lacZ coding sequence [lacZ], poly A signal [pA], neomycin cassette [neo]) and Frt sites (open triangles) are shown. Primers 1, 2 and 3 used for PCR genotyping are represented (arrows). Primers 1 and 2 amplify the mutant Grhl3PAR2 allele, and primers 2 and 3 amplify the endogenous Grhl3 allele. (b) PCR-based genotyping of Grhl3PAR2/+ transgenic mice. Amplified fragments using specific primers reveal the mutant (460 bp) and endogenous (702 bp) Grhl3 alleles. (c) β-Galactosidase staining (light blue) on skin autopsy samples and on skin sections from Grhl3PAR2/+ mice counterstained with neutral red (pink) of the respective genotypes. Bar represents 10 μm. (d) Phenotypic appearance and haematoxylin- and eosin-stained skin of Grhl3PAR2/+ mice versus control littermates at 2 weeks of age (top panel) and in adulthood (lower panel). Scale bars represent 20 μm. (e) Dot plot indicating the percentage of monitoring time spent scratching or grooming. Bars indicate the average of the time spent scratching. Each dot represents a single animal (P<0.05). (f) Skin injuries (white arrows) become evident in adult Grhl3PAR2/+ transgenic mice with occasional swelling of regional lymph nodes (dotted circle). (c, d, f) n≥3 mice per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105307&req=5

f5: Generation and characterization of Grhl3PAR2/+ transgenic mice.(a) Scheme of the Grhl3PAR2/+ knock-in targeting vector, part of the targeted Grhl3 gene locus and the resulting targeted allele. Exons (black boxes), transgenic sequences (from the left, white boxes: mouse PAR2 coding sequence [PAR2 cDNA], internal ribosome entry site [IRES], lacZ coding sequence [lacZ], poly A signal [pA], neomycin cassette [neo]) and Frt sites (open triangles) are shown. Primers 1, 2 and 3 used for PCR genotyping are represented (arrows). Primers 1 and 2 amplify the mutant Grhl3PAR2 allele, and primers 2 and 3 amplify the endogenous Grhl3 allele. (b) PCR-based genotyping of Grhl3PAR2/+ transgenic mice. Amplified fragments using specific primers reveal the mutant (460 bp) and endogenous (702 bp) Grhl3 alleles. (c) β-Galactosidase staining (light blue) on skin autopsy samples and on skin sections from Grhl3PAR2/+ mice counterstained with neutral red (pink) of the respective genotypes. Bar represents 10 μm. (d) Phenotypic appearance and haematoxylin- and eosin-stained skin of Grhl3PAR2/+ mice versus control littermates at 2 weeks of age (top panel) and in adulthood (lower panel). Scale bars represent 20 μm. (e) Dot plot indicating the percentage of monitoring time spent scratching or grooming. Bars indicate the average of the time spent scratching. Each dot represents a single animal (P<0.05). (f) Skin injuries (white arrows) become evident in adult Grhl3PAR2/+ transgenic mice with occasional swelling of regional lymph nodes (dotted circle). (c, d, f) n≥3 mice per group.

Mentions: To probe the contribution of PAR2 signalling to skin disease, we addressed whether increased expression of PAR2 in mouse skin might induce a pathological transformation similar to what had been observed with altered CAP1/Prss8 activity. We inserted a cassette consisting of the mouse PAR2 coding sequence followed by an internal ribosomal entry site and the lacZ reporter gene at the start codon of the grainyhead-like-3 (Grhl3) gene (Grhl3PAR2/+) by homologous recombination (Fig. 5a,b). The Grhl3 locus drives the expression of PAR2 throughout the epithelial ectoderm from mid-gestation onwards163435. Consistent with simultaneous interruption of the Grhl3 gene, mice with PAR2 inserted in both alleles (Grhl3PAR2/PAR2) died perinatally with spina bifida34. Therefore, Grhl3PAR2/+ mice were used throughout this study for transgenic PAR2 overexpression. Importantly, mice carrying one functional allele of the Grhl3 gene (Grhl3+/−) do not exhibit phenotypic differences compared to wt controls34, and knock-in of the Cre recombinase into the Grhl3 gene locus also affects Grhl3 gene expression, but does not trigger skin pathology16.


PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin.

Frateschi S, Camerer E, Crisante G, Rieser S, Membrez M, Charles RP, Beermann F, Stehle JC, Breiden B, Sandhoff K, Rotman S, Haftek M, Wilson A, Ryser S, Steinhoff M, Coughlin SR, Hummler E - Nat Commun (2011)

Generation and characterization of Grhl3PAR2/+ transgenic mice.(a) Scheme of the Grhl3PAR2/+ knock-in targeting vector, part of the targeted Grhl3 gene locus and the resulting targeted allele. Exons (black boxes), transgenic sequences (from the left, white boxes: mouse PAR2 coding sequence [PAR2 cDNA], internal ribosome entry site [IRES], lacZ coding sequence [lacZ], poly A signal [pA], neomycin cassette [neo]) and Frt sites (open triangles) are shown. Primers 1, 2 and 3 used for PCR genotyping are represented (arrows). Primers 1 and 2 amplify the mutant Grhl3PAR2 allele, and primers 2 and 3 amplify the endogenous Grhl3 allele. (b) PCR-based genotyping of Grhl3PAR2/+ transgenic mice. Amplified fragments using specific primers reveal the mutant (460 bp) and endogenous (702 bp) Grhl3 alleles. (c) β-Galactosidase staining (light blue) on skin autopsy samples and on skin sections from Grhl3PAR2/+ mice counterstained with neutral red (pink) of the respective genotypes. Bar represents 10 μm. (d) Phenotypic appearance and haematoxylin- and eosin-stained skin of Grhl3PAR2/+ mice versus control littermates at 2 weeks of age (top panel) and in adulthood (lower panel). Scale bars represent 20 μm. (e) Dot plot indicating the percentage of monitoring time spent scratching or grooming. Bars indicate the average of the time spent scratching. Each dot represents a single animal (P<0.05). (f) Skin injuries (white arrows) become evident in adult Grhl3PAR2/+ transgenic mice with occasional swelling of regional lymph nodes (dotted circle). (c, d, f) n≥3 mice per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105307&req=5

f5: Generation and characterization of Grhl3PAR2/+ transgenic mice.(a) Scheme of the Grhl3PAR2/+ knock-in targeting vector, part of the targeted Grhl3 gene locus and the resulting targeted allele. Exons (black boxes), transgenic sequences (from the left, white boxes: mouse PAR2 coding sequence [PAR2 cDNA], internal ribosome entry site [IRES], lacZ coding sequence [lacZ], poly A signal [pA], neomycin cassette [neo]) and Frt sites (open triangles) are shown. Primers 1, 2 and 3 used for PCR genotyping are represented (arrows). Primers 1 and 2 amplify the mutant Grhl3PAR2 allele, and primers 2 and 3 amplify the endogenous Grhl3 allele. (b) PCR-based genotyping of Grhl3PAR2/+ transgenic mice. Amplified fragments using specific primers reveal the mutant (460 bp) and endogenous (702 bp) Grhl3 alleles. (c) β-Galactosidase staining (light blue) on skin autopsy samples and on skin sections from Grhl3PAR2/+ mice counterstained with neutral red (pink) of the respective genotypes. Bar represents 10 μm. (d) Phenotypic appearance and haematoxylin- and eosin-stained skin of Grhl3PAR2/+ mice versus control littermates at 2 weeks of age (top panel) and in adulthood (lower panel). Scale bars represent 20 μm. (e) Dot plot indicating the percentage of monitoring time spent scratching or grooming. Bars indicate the average of the time spent scratching. Each dot represents a single animal (P<0.05). (f) Skin injuries (white arrows) become evident in adult Grhl3PAR2/+ transgenic mice with occasional swelling of regional lymph nodes (dotted circle). (c, d, f) n≥3 mice per group.
Mentions: To probe the contribution of PAR2 signalling to skin disease, we addressed whether increased expression of PAR2 in mouse skin might induce a pathological transformation similar to what had been observed with altered CAP1/Prss8 activity. We inserted a cassette consisting of the mouse PAR2 coding sequence followed by an internal ribosomal entry site and the lacZ reporter gene at the start codon of the grainyhead-like-3 (Grhl3) gene (Grhl3PAR2/+) by homologous recombination (Fig. 5a,b). The Grhl3 locus drives the expression of PAR2 throughout the epithelial ectoderm from mid-gestation onwards163435. Consistent with simultaneous interruption of the Grhl3 gene, mice with PAR2 inserted in both alleles (Grhl3PAR2/PAR2) died perinatally with spina bifida34. Therefore, Grhl3PAR2/+ mice were used throughout this study for transgenic PAR2 overexpression. Importantly, mice carrying one functional allele of the Grhl3 gene (Grhl3+/−) do not exhibit phenotypic differences compared to wt controls34, and knock-in of the Cre recombinase into the Grhl3 gene locus also affects Grhl3 gene expression, but does not trigger skin pathology16.

Bottom Line: K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations.Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2- background, establishing PAR2 as a pivotal mediator of pathogenesis.Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne 1005, Switzerland.

ABSTRACT
Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2- background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

No MeSH data available.


Related in: MedlinePlus