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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 of K14-CAP1/Prss8 transgenic mice.(a) Scheme of the K14-CAP1/Prss8 construct containing the mouse CAP1/Prss8 coding sequence (cDNA, g.i. 19111159), the human K14 promoter, the rabbit β-globin intron and the human growth hormone polyadenylation signal (polyA). Restriction sites used for isolation of the transgene (ClaI, NotI) and for Southern blot analysis (EcoRV) are indicated. For transgene-specific genotyping, primers 1 and 2, and for reverse transcriptase (RT)-PCR, primers 3 and 4 (arrows), were used. (b) Detection of K14-CAP1/Prss8 transgenic mice (line 1 and line 2) following Southern blot analysis (wild type, wt, 12 kb; transgenic, tg, 3.6 kb). (c) PCR genotyping using transgene-specific primers 1 and 2 (see a) and myogenin-specific primers (myo; endogenous control). (d) Immunofluorescence (green) shows transgenic CAP1/Prss8 expression in the basal layer of the epidermis in K14-CAP1/Prss8 transgenic mice. The white dotted line represents the basal membrane. Nuclei were counterstained with DAPI (blue). The white bar indicates 20 μm; (b–d) n≥4 mice/genotype. (e) CAP1/Prss8 gene expression in the skin was determined by quantitative RT–PCR analysis and normalized to β-actin. Line 1: n=3 animals/genotype. Line 2: wt, n=2 (data: 0.92 and 0.87) and tg, n=4 animals analysed. (f) CAP1/Prss8 protein expression was quantified by western blotting and loading was controlled by β-actin. Line 1: n=3 animals per genotype. Line 2: n=4 animals per genotype. All data are presented as mean±s.e.m.
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f1: Generation of K14-CAP1/Prss8 transgenic mice.(a) Scheme of the K14-CAP1/Prss8 construct containing the mouse CAP1/Prss8 coding sequence (cDNA, g.i. 19111159), the human K14 promoter, the rabbit β-globin intron and the human growth hormone polyadenylation signal (polyA). Restriction sites used for isolation of the transgene (ClaI, NotI) and for Southern blot analysis (EcoRV) are indicated. For transgene-specific genotyping, primers 1 and 2, and for reverse transcriptase (RT)-PCR, primers 3 and 4 (arrows), were used. (b) Detection of K14-CAP1/Prss8 transgenic mice (line 1 and line 2) following Southern blot analysis (wild type, wt, 12 kb; transgenic, tg, 3.6 kb). (c) PCR genotyping using transgene-specific primers 1 and 2 (see a) and myogenin-specific primers (myo; endogenous control). (d) Immunofluorescence (green) shows transgenic CAP1/Prss8 expression in the basal layer of the epidermis in K14-CAP1/Prss8 transgenic mice. The white dotted line represents the basal membrane. Nuclei were counterstained with DAPI (blue). The white bar indicates 20 μm; (b–d) n≥4 mice/genotype. (e) CAP1/Prss8 gene expression in the skin was determined by quantitative RT–PCR analysis and normalized to β-actin. Line 1: n=3 animals/genotype. Line 2: wt, n=2 (data: 0.92 and 0.87) and tg, n=4 animals analysed. (f) CAP1/Prss8 protein expression was quantified by western blotting and loading was controlled by β-actin. Line 1: n=3 animals per genotype. Line 2: n=4 animals per genotype. All data are presented as mean±s.e.m.

Mentions: To study the biological consequence of deregulated CAP1/Prss8 expression in skin, we generated transgenic mice expressing the full-length mouse CAP1/Prss8 coding sequence13 under the control of the human keratin-14 promoter25 (Fig. 1a). This promoter targets gene expression to keratinocytes of the basal layer of the epidermis and the outer root sheath of hair follicles25. Pronuclear injections of the K14-CAP1/Prss8 transgene construct yielded five founders, two of which were fertile and transmitted the transgene to produce two independent stable transgenic lines (termed lines 1 and 2; Fig. 1b,c). The transgene was transmitted according to Mendelian inheritance in both lines (line 1: 54% tg, n=95; line 2: 54% tg, n=72), suggesting that the level of CAP1/Prss8 expression obtained did not interfere with embryonic development. For line 1, only male mice (all of them) were transgenic, indicating integration of the transgene into the Y chromosome. Both transgenic lines expressed the transgene in skin as determined by immunohistochemistry (Fig. 1d; Supplementary Fig. S1a). Analysis of total (transgene-driven plus endogenous) expression of CAP1/Prss8 demonstrated a 1.1- and 4.7-fold increase in transcript levels and a 1.1- and 2.9-fold increase in protein levels in lines 1 and 2, respectively (Fig. 1e,f), indicating higher CAP1/Prss8 expression levels in line 2.


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 of K14-CAP1/Prss8 transgenic mice.(a) Scheme of the K14-CAP1/Prss8 construct containing the mouse CAP1/Prss8 coding sequence (cDNA, g.i. 19111159), the human K14 promoter, the rabbit β-globin intron and the human growth hormone polyadenylation signal (polyA). Restriction sites used for isolation of the transgene (ClaI, NotI) and for Southern blot analysis (EcoRV) are indicated. For transgene-specific genotyping, primers 1 and 2, and for reverse transcriptase (RT)-PCR, primers 3 and 4 (arrows), were used. (b) Detection of K14-CAP1/Prss8 transgenic mice (line 1 and line 2) following Southern blot analysis (wild type, wt, 12 kb; transgenic, tg, 3.6 kb). (c) PCR genotyping using transgene-specific primers 1 and 2 (see a) and myogenin-specific primers (myo; endogenous control). (d) Immunofluorescence (green) shows transgenic CAP1/Prss8 expression in the basal layer of the epidermis in K14-CAP1/Prss8 transgenic mice. The white dotted line represents the basal membrane. Nuclei were counterstained with DAPI (blue). The white bar indicates 20 μm; (b–d) n≥4 mice/genotype. (e) CAP1/Prss8 gene expression in the skin was determined by quantitative RT–PCR analysis and normalized to β-actin. Line 1: n=3 animals/genotype. Line 2: wt, n=2 (data: 0.92 and 0.87) and tg, n=4 animals analysed. (f) CAP1/Prss8 protein expression was quantified by western blotting and loading was controlled by β-actin. Line 1: n=3 animals per genotype. Line 2: n=4 animals per genotype. All data are presented as mean±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Generation of K14-CAP1/Prss8 transgenic mice.(a) Scheme of the K14-CAP1/Prss8 construct containing the mouse CAP1/Prss8 coding sequence (cDNA, g.i. 19111159), the human K14 promoter, the rabbit β-globin intron and the human growth hormone polyadenylation signal (polyA). Restriction sites used for isolation of the transgene (ClaI, NotI) and for Southern blot analysis (EcoRV) are indicated. For transgene-specific genotyping, primers 1 and 2, and for reverse transcriptase (RT)-PCR, primers 3 and 4 (arrows), were used. (b) Detection of K14-CAP1/Prss8 transgenic mice (line 1 and line 2) following Southern blot analysis (wild type, wt, 12 kb; transgenic, tg, 3.6 kb). (c) PCR genotyping using transgene-specific primers 1 and 2 (see a) and myogenin-specific primers (myo; endogenous control). (d) Immunofluorescence (green) shows transgenic CAP1/Prss8 expression in the basal layer of the epidermis in K14-CAP1/Prss8 transgenic mice. The white dotted line represents the basal membrane. Nuclei were counterstained with DAPI (blue). The white bar indicates 20 μm; (b–d) n≥4 mice/genotype. (e) CAP1/Prss8 gene expression in the skin was determined by quantitative RT–PCR analysis and normalized to β-actin. Line 1: n=3 animals/genotype. Line 2: wt, n=2 (data: 0.92 and 0.87) and tg, n=4 animals analysed. (f) CAP1/Prss8 protein expression was quantified by western blotting and loading was controlled by β-actin. Line 1: n=3 animals per genotype. Line 2: n=4 animals per genotype. All data are presented as mean±s.e.m.
Mentions: To study the biological consequence of deregulated CAP1/Prss8 expression in skin, we generated transgenic mice expressing the full-length mouse CAP1/Prss8 coding sequence13 under the control of the human keratin-14 promoter25 (Fig. 1a). This promoter targets gene expression to keratinocytes of the basal layer of the epidermis and the outer root sheath of hair follicles25. Pronuclear injections of the K14-CAP1/Prss8 transgene construct yielded five founders, two of which were fertile and transmitted the transgene to produce two independent stable transgenic lines (termed lines 1 and 2; Fig. 1b,c). The transgene was transmitted according to Mendelian inheritance in both lines (line 1: 54% tg, n=95; line 2: 54% tg, n=72), suggesting that the level of CAP1/Prss8 expression obtained did not interfere with embryonic development. For line 1, only male mice (all of them) were transgenic, indicating integration of the transgene into the Y chromosome. Both transgenic lines expressed the transgene in skin as determined by immunohistochemistry (Fig. 1d; Supplementary Fig. S1a). Analysis of total (transgene-driven plus endogenous) expression of CAP1/Prss8 demonstrated a 1.1- and 4.7-fold increase in transcript levels and a 1.1- and 2.9-fold increase in protein levels in lines 1 and 2, respectively (Fig. 1e,f), indicating higher CAP1/Prss8 expression levels in line 2.

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