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Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing Factors in the Skin

  • Author Footnotes
    11 These authors share first authorship
    Esther Hoste
    Footnotes
    11 These authors share first authorship
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Author Footnotes
    11 These authors share first authorship
    Patrick Kemperman
    Footnotes
    11 These authors share first authorship
    Affiliations
    Department of Dermatology and Venereology, Erasmus MC, Rotterdam, The Netherlands

    Department of Dermatology and Venereology, Waterlandziekenhuis, Purmerend, The Netherlands
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  • Author Footnotes
    11 These authors share first authorship
    Michael Devos
    Footnotes
    11 These authors share first authorship
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Geertrui Denecker
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Sanja Kezic
    Affiliations
    Coronel Institute of Occupational Health, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
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  • Nico Yau
    Affiliations
    Coronel Institute of Occupational Health, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
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  • Barbara Gilbert
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Saskia Lippens
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Philippe De Groote
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Ria Roelandt
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Petra Van Damme
    Affiliations
    Department of Medical Protein Research, VIB-Ghent University, Ghent, Belgium
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  • Kris Gevaert
    Affiliations
    Department of Medical Protein Research, VIB-Ghent University, Ghent, Belgium
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  • Richard B. Presland
    Affiliations
    Department of Oral Biology, University of Washington, Seattle, Washington, USA

    Department of Medicine (Dermatology), University of Washington, Seattle, Washington, USA
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  • Hidenari Takahara
    Affiliations
    Department of Applied Biological Resource Sciences, School of Agriculture, Ibaraki University, Ibaraki, Japan
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  • Gerwin Puppels
    Affiliations
    Department of Dermatology and Venereology, Erasmus MC, Rotterdam, The Netherlands

    River Diagnostics BV, Rotterdam, The Netherlands
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  • Peter Caspers
    Affiliations
    Department of Dermatology and Venereology, Erasmus MC, Rotterdam, The Netherlands

    River Diagnostics BV, Rotterdam, The Netherlands
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  • Peter Vandenabeele
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Wim Declercq
    Correspondence
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium.
    Affiliations
    Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Author Footnotes
    11 These authors share first authorship
      Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14-/- mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.

      Abbreviations

      FLG
      filaggrin
      NMF
      natural moisturizing factors
      PCA
      pyrrolidone carboxylic acid
      SC
      stratum corneum
      tUCA
      trans-urocanic acid

      Introduction

      Keratinocytes, the main cell type in the epidermis, undergo a well-orchestrated differentiation program that leads to the formation of a functional skin barrier maintained by several components residing mainly in the stratum corneum (SC;
      • Proksch E.
      • Brandner J.M.
      • Jensen J.M.
      The skin: an indispensable barrier.
      ;
      • Schauber J.
      • Gallo R.L.
      Antimicrobial peptides and the skin immune defense system.
      ). In addition, tight junctions in the stratum granulosum are also key players in maintaining the water balance in skin (reviewed in
      • Morita K.
      • Miyachi Y.
      • Furuse M.
      Tight junctions in epidermis: from barrier to keratinization.
      ). The SC is the outermost epidermal layer and consists of corneocytes enshrouded by a cornified envelope embedded in a specialized lipid bilayer (
      • Elias P.M.
      Skin barrier function.
      ). This heterogeneous “brick and mortar” constitution of the outer epidermal layers provides a strong protective barrier. Water retention by the skin is governed by lipids as well as by the cornified envelope (
      • Imokawa G.
      • Akasaki S.
      • Minematsu Y.
      • et al.
      Importance of intercellular lipids in water-retention properties of the stratum corneum: induction and recovery study of surfactant dry skin.
      ). Cornified envelopes, which replace the plasma membrane in corneocytes, are insoluble protein structures that are tightly crosslinked by transglutaminases (
      • Candi E.
      • Schmidt R.
      • Melino G.
      The cornified envelope: a model of cell death in the skin.
      ). The so-called natural moisturizing factors (NMFs) are a mixture of hygroscopic molecules including amino acids and their derivatives, e.g., pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA), together with lactic acid, urea, citrate, and sugars (
      • Rawlings A.V.
      • Harding C.R.
      Moisturization and skin barrier function.
      ). These NMFs absorb water and maintain hydration in skin of terrestrial animals. In addition, the ionic interaction between NMF and keratins reduces the intermolecular binding between keratin filaments and thereby increases the elastic properties of the SC (
      • Jokura Y.
      • Ishikawa S.
      • Tokuda H.
      • et al.
      Molecular analysis of elastic properties of the stratum corneum by solid-state 13C-nuclear magnetic resonance spectroscopy.
      ). PCA and UCA are derived mainly from complete degradation of filaggrin (FLG, filament aggregating protein) in the top layers of the SC (
      • Scott I.R.
      • Harding C.R.
      • Barrett J.G.
      Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.
      ;
      • Scott I.R.
      • Harding C.R.
      Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.
      ;
      • Kezic S.
      • Kemperman P.M.
      • Koster E.S.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ,
      • Kezic S.
      • Kammeyer A.
      • Calkoen F.
      • et al.
      Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods.
      ).
      Filaggrin is thought to contribute to epidermal barrier function by promoting keratin filament aggregation into macrofibrils in the stratum granulosum and SC layers (
      • Dale B.A.
      • Resing K.A.
      • Lonsdale-Eccles J.D.
      Filaggrin: a keratin filament associated protein.
      ;
      • Sybert V.P.
      • Dale B.A.
      • Holbrook K.A.
      Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules.
      ). Loss-of-function mutations in the filaggrin gene (FLG) lead to the development of the dry skin disease, ichthyosis vulgaris in humans and to a similar neonatal skin disease in mice (
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Smith F.J.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • et al.
      Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
      ;
      • Fallon P.G.
      • Sasaki T.
      • Sandilands A.
      • et al.
      A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming.
      ). In addition, FLG gene mutations predispose individuals to atopic dermatitis (eczema), which is often associated with other allergies, including asthma (
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Schuttelaar M.L.
      • Kerkhof M.
      • Jonkman M.F.
      • et al.
      Filaggrin mutations in the onset of eczema, sensitization, asthma, hay fever and the interaction with cat exposure.
      ). Profilaggrin is synthesized as a large polyprotein precursor of >400kDa consisting of multiple FLG units flanked by distinct N- and C-terminal domains. The N-terminal domain contains a conserved S100 calcium-binding domain and a postulated B-domain. Profilaggrin comprises 10–12 FLG repeat units in humans and 12–20 in mice, depending on the strain (
      • Rothnagel J.A.
      • Steinert P.M.
      The structure of the gene for mouse filaggrin and a comparison of the repeating units.
      ;
      • Zhang D.
      • Karunaratne S.
      • Kessler M.
      • et al.
      Characterization of mouse profilaggrin: evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation.
      ;
      • Fallon P.G.
      • Sasaki T.
      • Sandilands A.
      • et al.
      A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • et al.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). In the stratum granulosum, profilaggrin is highly phosphorylated and retained in the keratohyalin granules. Profilaggrin is dephosphorylated in the transitional layer between stratum granulosum and SC before proteolytic cleavage into FLG units (reviewed in
      • Ovaere P.
      • Lippens S.
      • Vandenabeele P.
      • et al.
      The emerging roles of serine protease cascades in the epidermis.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • et al.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). Once FLG is released, it forms an aggregate with the keratin intermediate filaments (
      • Dale B.A.
      • Resing K.A.
      • Lonsdale-Eccles J.D.
      Filaggrin: a keratin filament associated protein.
      ). FLG is deiminated in the cornified layers by peptidylarginine deiminase (
      • Nachat R.
      • Mechin M.C.
      • Takahara H.
      • et al.
      Peptidylarginine deiminase isoforms 1-3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin.
      ), converting positively charged arginine residues into citrulline. Subsequently, the FLG–keratin interaction is broken and FLG is degraded further into free amino acids that contribute to the NMF (reviewed in
      • Rawlings A.V.
      • Harding C.R.
      Moisturization and skin barrier function.
      ). Consequently, patients carrying loss-of-function FLG mutations exhibit greatly reduced NMF levels (
      • Kezic S.
      • Kemperman P.M.
      • Koster E.S.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ,
      • Kezic S.
      • O’Regan G.M.
      • Yau N.
      • et al.
      Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.
      ,
      • O’Regan G.M.
      • Kemperman P.M.
      • Sandilands A.
      • et al.
      Raman profiles of the stratum corneum define 3 filaggrin genotype-determined atopic dermatitis endophenotypes.
      ).
      Although several proteases are involved in processing profilaggrin into FLG, little is known about the proteolytic events involved in the degradation of FLG into amino acids. We recently generated caspase-14-/- mice and showed that caspase-14 deficiency leads to the accumulation of fragments derived from FLG repeats (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ), suggesting that FLG degradation is defective in these mice. Calpain I and caspase-14 can cleave deiminated FLG repeats in vitro (
      • Kamata Y.
      • Taniguchi A.
      • Yamamoto M.
      • et al.
      Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.
      ), but the identity of the cleavage fragments was not reported. We compare FLG processing in wild-type and caspase-14-/- mice and show that FLG degradation is initiated in caspase-14-deficient mice but does not complete, resulting in the accumulation of FLG fragments. Consequently, we demonstrate lower NMF levels in caspase-14-/- mice relative to control mice. We also identified two caspase-14 cleavage sites in the FLG repeat unit. Taken together, our results indicate an important role for caspase-14 in FLG catabolism.

      Results

       Filaggrin degradation is defective in caspase-14-deficient skin

      As reported earlier, the proteolytic generation of the FLG repeat, which has an apparent molecular weight of ∼30kDa (
      • Presland R.B.
      • Boggess D.
      • Lewis S.P.
      • et al.
      Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.
      ), was not affected in caspase-14-/- skin, but FLG degradation products accumulated in the epidermis (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ,
      • Denecker G.
      • Ovaere P.
      • Vandenabeele P.
      • et al.
      Caspase-14 reveals its secrets.
      ). We show here that this accumulation is evident in adults but is even more pronounced in neonates ( ). Furthermore, minor FLG degradation products were also observed in caspase-14+/+ epidermis as detected by western blotting using an antibody directed against the FLG repeat. To validate the nature of these FLG fragments that accumulate in the absence of caspase-14, caspase-14+/+ and caspase-14-/- skin lysates were separated by gel electrophoresis and stained with Coomassie Blue (Figure 1b). The gel region in which FLG-derived fragments accumulated most prominently was subjected to protein elution and analyzed by mass spectrometry. All peptides but one that maps to the N-terminal B-domain of profilaggrin, map to the FLG repeat sequence (Figure 1c, Supplementary Figure S1 online). The number of spectra identified as FLG peptides was 6-fold higher in caspase-14 knockout skin than in wild-type skin, while the number of spectra derived from the subunits of the actin-related protein 2/3 complex was 5% in both samples (data not shown). This confirms that proteolytic fragments of the FLG repeat accumulate in caspase-14-deficient skin.
      Figure thumbnail gr1
      Figure 1Aberrant filaggrin processing in caspase-14-/- mice. (a) Filaggrin (FLG) immunoblotting of epidermal lysates from caspase-14+/+ and caspase-14-/- skin shows accumulation of FLG-derived peptides in caspase-14-deficient skin. Anti-actin blot is shown as control for equal loading. (b) Coomassie blue staining of P5.5 caspase-14+/+ and caspase-14-/- skin lysates. The region in which FLG fragments accumulated in caspase-14-/- skin was clearly visible on the Coomassie-stained gel and was cut out, as well as from the equivalent region of the caspase-14+/+ gel (indicated with a bar). Gel slices were analyzed by IonTrap ESI-MS/MS. (c) Table of identified FLG peptides, indicating sequence location within the mouse profilaggrin protein sequence (IPI00473837.2), and number of spectra by which the observed peptide was represented. Mw, molecular weight markers.
      It has been shown that degradation of FLG into NMF is blocked during the late fetal development, and that shortly after birth normal proteolysis of FLG proceeds in the outer part of the SC (
      • Scott I.R.
      • Harding C.R.
      Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.
      ). To investigate whether the timing of these events is affected in caspase-14-deficient mice, we analyzed skin lysates of caspase-14+/+ and caspase-14-/- mice at different developmental stages, ranging from E15.5 until P5.5 (Figure 2). FLG repeat formation, which coincides with SC formation and caspase-14 activation, was detectable from day E17.5 in caspase-14+/+ and caspase-14-/- mice, confirming previous reports (
      • Fischer H.
      • Rossiter H.
      • Ghannadan M.
      • et al.
      Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis.
      ). Our results indicate that FLG degradation is initiated from day E18.5 in both wild-type and caspase-14-/- mice. In wild-type mice, FLG degradation products were observed from day E18.5 to P0.5. From day P1.5 these fragments were hardly detectable in complete skin lysates from caspase-14+/+ mice, indicating that they are rapidly degraded after birth. In contrast, FLG fragments of 17–30kDa tended to accumulate in caspase-14-deficient skin from day P0.5. These data indicate that caspase-14 is crucial for FLG degradation in the SC.
      Figure thumbnail gr2
      Figure 2Accumulation of filaggrin-derived peptides in caspase-14-/- skin increases after birth. (a, b) Western blot analysis of filaggrin (FLG, a) and caspase-14 (b) was performed on total skin lysates from caspase-14+/+ and caspase-14-/- mice. Skin lysates were prepared from mice on embryonic days E15.5, E16.5, E17.5, and E18.5 and the first neonatal days (P0.5–P5.5). Complete skin was lysed in urea-Tris buffer. Positions of FLG repeat, procaspase-14, and the large (p20) and small (p10) catalytic subunits are indicated by arrows. Mw, molecular weight markers.

       Caspase-14 can directly cleave the FLG repeat

      Our biochemical analysis of skin lysates indicates that the FLG repeat needs to be cleaved by caspase-14 before it can be further degraded into amino acids (Figure 1). We previously reported that caspase-14 can directly cleave an N-terminal profilaggrin fragment spanning the A and B domains and a partial FLG repeat (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). To determine whether human caspase-14 can directly cleave the FLG repeat, we used recombinant human caspase-14 activated by incubation with an epidermal extract (because caspase-14 does not auto-activate by overexpression) to cleave a human FLG repeat (FLAG-FG) that was translated in vitro and labeled with [35S]methionine (
      • Dale B.A.
      • Presland R.B.
      • Lewis S.P.
      • et al.
      Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.
      ). The protein representing the FLG repeat was clearly degraded by caspase-14 (Figure 3a). However, no cleavage products were apparent on the autoradiogram, most likely because only one methionine residue (initiation AUG) is present in the radioactively labeled FLG repeat, which might be present in a small cleavage product that cannot be detected using 15% SDS–PAGE. Treatment of the FLG repeat with active caspase-14 in the presence of the pan-caspase inhibitor zVAD-fmk, or with a catalytic inactive mutant of caspase-14, did not result in FLG processing (Supplementary Figure S2 online). In addition, depletion of active caspase-14 from the incubation mixture abolished FLG repeat cleavage (Figure 3a). To identify the caspase-14 cleavage sites, we made use of a recombinant human FLG repeat, as used previously in other studies (
      • Kamata Y.
      • Taniguchi A.
      • Yamamoto M.
      • et al.
      Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.
      ). To avoid generating active caspase-14 by treatment of procaspase-14 with an undefined epidermal extract, we expressed the human caspase-14 p20 and p10 subunits separately in E. coli. During purification, these subunits were mixed to obtain enzymatically active p20/p10 caspase-14 heterodimers that mimic in vivo-activated caspase-14 (
      • Fischer H.
      • Stichenwirth M.
      • Dockal M.
      • et al.
      Stratum corneum-derived caspase-14 is catalytically active.
      ). This was verified by comparing the substrate specificity of p20/p10 caspase-14 to that of cleaved caspase-14 (Supplementary Figure S3 online). When the FLG repeat unit was incubated with the heterodimers, several FLG cleavage fragments were generated (Figure 3b). We analyzed these fragments by mass spectrometry and identified two caspase-14 cleavage sites (Figure 3c and d), namely, VSQD and HSED. These sites are situated near the middle of the FLG repeat and are conserved in most of the FLG repeats (Figure 3e). The FLG cleavage pattern indicates the presence of other caspase-14 cleavage sites, but the peptides corresponding to these cleavage sites might be difficult to detect. Taken together, our data show that the FLG repeat is a direct substrate of caspase-14.
      Figure thumbnail gr3
      Figure 3Direct cleavage of the human filaggrin repeat by caspase-14. (a) The construct harboring complementary DNA of a human filaggrin (FLG) repeat was transcribed and translated in vitro in the presence of 35S-methionine. Translated products were left untreated or incubated with the indicated concentrations of recombinant active cleaved human caspase-14 in buffer containing 1.1M kosmotropic salt. As a control, 20μM of the pan-caspase inhibitor zVADfmk was added or caspase-14 was depleted from the reaction. (b) Recombinantly expressed human FLG repeat (
      • Kanno T.
      • Kawada A.
      • Yamanouchi J.
      • et al.
      Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.
      ) was incubated with a 20:1 ratio of p20/p10 caspase-14 for 1hour in the absence or presence of 20μM zVADfmk. Numbered protein bands were cut out of the gel and analyzed by means of mass spectrometry to identify caspase cleavage sites. (c) Peptides identified during the mass spectrometry analysis that were generated as a result from cleavage by caspase-14 after an aspartate residue. These peptides were not identified in the non-cleaved FLG repeat band. We identified the HSED cleavage site also in cleaved protein band 1 (panel b), which is not in agreement with the apparent molecular weight of this band. Therefore we believe this is due to a minor contamination of band 1 with other cleaved FLG fragments, which can be detected by an ultrasensitive method such as mass spectrometry. (d) Amino-acid sequence of the recombinantly expressed human FLG repeat, with indication of the identified caspase-14 cleavage sites (underlined). The sequence of the partial linker sequence present in the construct is indicated in bold italics. C-terminal His-tag is indicated in bold. (e) Schematic representation of human profilaggrin with indication of VSQD and HSED positions throughout the molecule. In repeat 3 and in the C-terminal partial repeat the HSED site was replaced by HSDD or YPED, respectively. In repeat 7 the VSQD site was replaced by FSQD.

       NMF levels are strongly reduced in SC of caspase-14-/- mice

      NMF components derived from amino acids originate mainly from complete degradation of FLG (
      • Scott I.R.
      • Harding C.R.
      • Barrett J.G.
      Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.
      ). Because FLG degradation is defective in caspase-14-deficient epidermis, we used these mice to investigate the physiological role of caspase-14 in the formation of NMF. We analyzed the relative NMF levels of caspase-14+/+ and caspase-14-/- back skin by means of in situ Raman spectroscopy (
      • Caspers P.J.
      • Lucassen G.W.
      • Carter E.A.
      • et al.
      In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.
      ). The relative NMF levels in caspase-14-/- skin were three times lower than in caspase-14+/+ skin (Figure 4a). In keeping with the reduction in overall NMF level in caspase-14-/- skin, the levels of two NMF constituents, trans-urocanic acid (tUCA) and 2-pyrrolidone-5-carboxylic acid (PCA), were about 2-fold reduced in caspase-14-/- mice (Figure 4a). We wanted to exclude the possibility that decreased UCA and PCA levels are caused by inhibition of the conversion of histidine to UCA or glutamine to PCA, respectively. As the mouse FLG unit is composed of 15% glycine (Supplementary Figure S1 online), we analyzed the level of this amino acid in caspase-14+/+ and caspase-14-/- skin. As expected, glycine levels were drastically reduced in caspase-14-/- mice (Figure 4a), indicating that caspase-14 acts at the level of FLG degradation and not at the level of amino-acid conversion.
      Figure thumbnail gr4
      Figure 4NMF levels are significantly reduced in caspase-14-deficient mice. (a) Raman spectroscopic determination of the relative levels of natural moisturizing factor (NMF), pyrrolidone carboxylic acid (PCA), trans-urocanic acid (tUCA), and glycine in caspase-14+/+ (n=8) and caspase-14-/- (n=6) mice on day P5.5. All mice were measured at 10 different locations. For both genotypes the mean values of the measurements between 0 and 10μm below the skin surface were calculated per mouse, and compared by the Mann–Whitney U-test. (b) Biochemical determination of the average PCA and tUCA concentrations in caspase-14+/+ (n=6) and caspase-14-/- mice (n=6). TS1 (tape strip 1) represents the upper layer of the stratum corneum (SC), and TS10 is derived from the deeper layers of the SC. Concentrations of PCA and tUCA were normalized for the amount of protein determined on the tape strips and expressed as nmolg-1 protein. Error bars represent the standard deviation. All values from the two genotypes were compared using Mann–Whitney U-testing; NS, not significant; UCA, urocanic acid; P-values are indicated with * (<0.05), ** (0.05–0.001), or *** (<0.001).
      We also determined the amount of PCA and total UCA (trans and cis form) in caspase-14+/+ and caspase-14-/- SC by biochemically analyzing samples obtained from skin tape stripping experiments. The concentrations of PCA and UCA were normalized for the amount of total protein present in the tape strip samples. Consistent with the results obtained by Raman spectroscopy, this analysis indicated that the levels of both NMF constituents were significantly lower in caspase-14-deficient samples (Figure 4b).

      Discussion

      The adaptation of vertebrates to terrestrial life was accompanied by drastic changes in the integument to cope with the dry environment and protect against excessive water loss (
      • Alibardi L.
      Adaptation to the land: the skin of reptiles in comparison to that of amphibians and endotherm amniotes.
      ). The generation of a soft SC in mammals correlates with the occurrence of histidine-rich proteins such as profilaggrin. Patients or mice carrying FLG loss-of-function mutations suffer from dry and/or scaly skin (
      • Presland R.B.
      • Boggess D.
      • Lewis S.P.
      • et al.
      Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.
      ;
      • Nemoto-Hasebe I.
      • Akiyama M.
      • Nomura T.
      • et al.
      Clinical severity correlates with impaired barrier in filaggrin-related eczema.
      ). However, the pathways involved in FLG degradation to NMF are poorly characterized.
      We show that caspase-14 can directly cleave the FLG repeat, probably in preparation for complete breakdown by other, currently unidentified, proteases. The latter is supported by the finding that lysates from caspase-14-/- epidermis accumulate FLG fragments. In addition, immunohistochemical staining detects FLG only in the deeper layers of the wild-type SC, while FLG is present in all layers of the SC in caspase-14-deficient mice (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ,
      • Denecker G.
      • Ovaere P.
      • Vandenabeele P.
      • et al.
      Caspase-14 reveals its secrets.
      ). We identified two caspase-14 cleavage sites in the human FLG repeat: VSQD and HSED. The cleavage pattern suggests the presence of additional caspase-14 cleavage sites, but we could not identify them. The presence of valine and histidine at position P4 in the cleavage sites that we identified is in agreement with the observed preference of caspase-14 for hydrophobic or aromatic residues at P4 (
      • Mikolajczyk J.
      • Scott F.L.
      • Krajewski S.
      • et al.
      Activation and substrate specificity of caspase-14.
      ). These two caspase-14 cleavage sites were conserved in most of the human FLG repeats (Figure 3e). An additional HSED site was present in the C terminus of several repeats. These caspase-14 cleavage sites are not conserved in mouse FLG. However, several putative caspase-14 cleavage sites can be predicted in mouse filaggrin by using Siteprediction software (
      • Verspurten J.
      • Gevaert K.
      • Declercq W.
      • et al.
      SitePredicting the cleavage of proteinase substrates.
      ).
      In vivo Raman spectroscopy and biochemical analysis revealed that the total NMF levels and tUCA and PCA levels were ∼2.5 times lower in caspase-14-/- mice compared with wild-type mice, confirming the important role of caspase-14 in FLG degradation. This could explain the lower SC hydration levels in caspase-14-/- mice (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). tUCA represents ∼0.7% of the dry weight of the human skin and serves as an efficient UVB-absorbing chromophore and is thereby converted to cis-UCA, which has immunosuppressive activities (
      • de fine Olivarius F.
      • Wulf H.C.
      • Crosby J.
      • et al.
      The sunscreening effect of urocanic acid.
      ;
      • Walterscheid J.P.
      • Nghiem D.X.
      • Kazimi N.
      • et al.
      Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2A receptor.
      ). In view of this, one might expect that humans and mice deficient in FLG, caspase-14, or histidase are more prone to UVB-induced DNA damage and inflammatory stimuli. Indeed, caspase-14-/- and histidase-/- mice (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Barresi C.
      • Stremnitzer C.
      • Mlitz V.
      • et al.
      Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection.
      ), and in vitro human skin equivalents, in which FLG expression was suppressed (
      • Mildner M.
      • Jin J.
      • Eckhart L.
      • et al.
      Knockdown of Filaggrin impairs diffusion barrier Function and increases UV sensitivity in a human skin model.
      ), are more susceptible to UVB-induced DNA damage and subsequent apoptosis. Ft/ft mice, which do not have functional FLG, develop spontaneous skin inflammation and are more sensitive to cutaneous allergen priming (
      • Fallon P.G.
      • Sasaki T.
      • Sandilands A.
      • et al.
      A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming.
      ;
      • Oyoshi M.K.
      • Murphy G.F.
      • Geha R.S.
      Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen.
      ;
      • Scharschmidt T.C.
      • Man M.Q.
      • Hatano Y.
      • et al.
      Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.
      ). Whether this phenotype is caused by the absence of the FLG repeat or by lack of FLG-derived metabolites is not clear. A putative mechanism for impairment of epidermal barrier function in ft/ft mice is the abnormal lamellar body secretion and aberrant cornified envelope morphology observed in the skin of these mice (
      • Scharschmidt T.C.
      • Man M.Q.
      • Hatano Y.
      • et al.
      Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.
      ). Interestingly, mice deficient in the FLG degrading enzyme bleomycin hydrolase display a mild ichthyotic-like appearance with generalized scaling (
      • Schwartz D.R.
      • Homanics G.E.
      • Hoyt D.G.
      • et al.
      The neutral cysteine protease bleomycin hydrolase is essential for epidermal integrity and bleomycin resistance.
      ;
      • Kamata Y.
      • Taniguchi A.
      • Yamamoto M.
      • et al.
      Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.
      ). The “spontaneous” skin phenotype observed in ft/ft mice and bleomycin hydrolase-deficient mice is more severe than that seen in caspase-14-/- mice. Apparently, homeostatic formation of FLG repeats is crucial for skin barrier function, whereas defects in degradation of FLG lead to milder phenotypes, such as increased transepidermal water loss, lower levels of NMFs, or mild scaling. In keeping with the above hypothesis, decreased NMF levels in nonlesional skin are a recurring feature in atopic dermatitis (
      • Kezic S.
      • O’Regan G.M.
      • Yau N.
      • et al.
      Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.
      ). This could be explained by FLG deficiency or lower levels of active caspase-14 in such patients (
      • Yamamoto M.
      • Kamata Y.
      • Iida T.
      • et al.
      Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.
      ). However, we cannot exclude that other defects, such as changes in pH or lipid structure, may also contribute to the caspase-14-/- phenotype.
      The decreased water content in the upper epidermal layers, which is caused by the low humidity of the terrestrial environment, is required for induction of FLG degradation (
      • Warner R.R.
      • Myers M.C.
      • Taylor D.A.
      Electron probe analysis of human skin: determination of the water concentration profile.
      ;
      • Caspers P.J.
      • Lucassen G.W.
      • Carter E.A.
      • et al.
      In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.
      ). Consequently, experiments using in vitro skin equivalents indicated that PCA levels are dependent on the environment's relative humidity (
      • Bouwstra J.A.
      • Groenink H.W.
      • Kempenaar J.A.
      • et al.
      Water distribution and natural moisturizer factor content in human skin equivalents are regulated by environmental relative humidity.
      ). This is in accordance with the reduced level of NMF in humans who live in areas with high relative humidity (
      • Declercq L.
      • Muizzuddi N.
      • Hellemans L.
      • et al.
      Adaptation response in human skin barrier to a hot and dry environment.
      ). The requirement for high concentrations of kosmotropic salt to enhance caspase-14 activity in vitro might mimic the low hydration status of the top layers of the SC (
      • Mikolajczyk J.
      • Scott F.L.
      • Krajewski S.
      • et al.
      Activation and substrate specificity of caspase-14.
      ). Kosmotropic salts induce both dimerization and ordering of active site loops by partial desolvation of the protein.
      It should be noted that residual NMF, tUCA, and PCA levels were detected in caspase-14-deficient skin. This could imply that a pool of FLG repeats was still being broken down or that FLG was not the only source of amino acids contributing to NMF synthesis. The latter is consistent with the presence of some NMF in SC of patients who carry homozygous loss-of-function FLG mutations (
      • Kezic S.
      • Kemperman P.M.
      • Koster E.S.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ,
      • Kezic S.
      • Kammeyer A.
      • Calkoen F.
      • et al.
      Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods.
      ,
      • O’Regan G.M.
      • Kemperman P.M.
      • Sandilands A.
      • et al.
      Raman profiles of the stratum corneum define 3 filaggrin genotype-determined atopic dermatitis endophenotypes.
      ; ). It also confirms quantitative studies showing that between 70 and 100% of the total SC free amino acids are derived from FLG (). It is tempting to consider the other members of the S100-fused gene family as the main contributors of these residual NMF. It would be interesting to analyze NMF levels in mice that lack multiple fused S100 proteins, especially the two more closely related members hornerin and ifapsoriasin (also referred to as FLG-2). Further research is needed to determine the in vivo role of these fused S100 family members.
      To conclude, we provide in vivo and in vitro evidence that identifies caspase-14 as a crucial protease in the generation of NMF (Figure 5). FLG repeat degradation is initiated in the deeper cornified layers, eventually leading to the generation of free amino acids, the main source of NMF (
      • Scott I.R.
      • Harding C.R.
      Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.
      ;
      • Jokura Y.
      • Ishikawa S.
      • Tokuda H.
      • et al.
      Molecular analysis of elastic properties of the stratum corneum by solid-state 13C-nuclear magnetic resonance spectroscopy.
      ). In caspase-14-/- mice, FLG peptides accumulate in these layers and NMF formation is defective. These findings indicate that caspase-14 is a crucial protease in FLG catabolism.
      Figure thumbnail gr5
      Figure 5Hypothetical overview of the pathway involved in filaggrin degradation. Insoluble profilaggrin is stored in the stratum granulosum (SG) in keratohyalin granules. Upon dephosphorylation, profilaggrin is released in the cytosol, where it is proteolysed into filaggrin (FLG) repeats. FLG binds and strengthens keratin intermediate filaments (KIFs), thereby promoting cell flattening at the SG-SC transitional layer. Deimination of FLG within the SC, probably by PAD 1 and PAD 3, has been suggested to render FLG susceptible to further proteolysis. FLG repeats also become crosslinked by transglutaminases to strengthen the cornified envelopes. FLG degradation starts in the deeper SC layers by partial proteolysis by one or more unknown proteases. Next, caspase-14-mediated cleavage prepares these fragments for efficient degradation into free amino acids, which contribute to the natural moisturizing factors (NMFs). Calpain 1 and bleomycin hydrolase also participate in this process. However, the order of events is not clear. NMFs provide SC hydration and possibly also UVB protection and immune suppression. PAD, peptidylarginine deiminase.

      Materials and Methods

       Animals

      Caspase-14 knockout mice were generated as described (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). Skin was prepared from embryos at E15.5, E16.5, E17.5, and E18.5, and on postpartum days P0.5, P1.5, P2.5, P3.5, P4.5, and P5.5. We also used adult female mice at the age of 8–12 weeks. The animals were housed under specific pathogen free conditions, and all experiments were approved by the local ethics committee of Ghent University.

       Immunoblotting

      Mice were killed and full skin or epidermal samples were obtained and frozen in NP-40 buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2 and 1% (w/v) NP-40) and freeze-thawed 10 times in liquid nitrogen. Skin samples from timed mating experiments were frozen in urea-Tris buffer (8M urea, 10mM EDTA, 50mM Tris-HCl, pH 8, 0.3mM orthophenanthroline and 20μgml-1 phenylmethylsulfonyl fluoride) and homogenized by sonication. Homogenates were centrifuged at 0°C for 30minutes at 14,000 × g. Epidermal lysates were obtained by separating the epidermis from dermis after incubating the skin for 10minutes at 56°C in phosphate-buffered saline with 10mM EDTA. We separated 20μg of protein in 15% SDS–PAGE and transferred them to a nitrocellulose membrane. The membrane was incubated with primary antibodies and appropriate horseradish peroxidase-labeled secondary antibodies (GE Healthcare, Diegem, Belgium). The primary antibodies were 1:2,000 rabbit polyclonal caspase-14 (
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ), 1:1,000 rabbit polyclonal anti-FLG (Covance, Princeton, NJ), and 1:20,000 mouse monoclonal anti-actin (MP Biomedicals, Illkirch, France). Detection was performed with the Western Lightning chemiluminescent reagent plus kit (PerkinElmer, Waltham, MA).

       Caspase-14 cleavage assay

      The sequence of a human FLG repeat flanked by an N-terminal Flag-tag was cloned in the pcDNA3 vector (
      • Dale B.A.
      • Presland R.B.
      • Lewis S.P.
      • et al.
      Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.
      ). Using this vector [35S]Methionine-labeled human FLG protein was prepared with the coupled transcription-translation assay TNT kit (Promega, Leiden, The Netherlands). Recombinant bacterially expressed human FLG repeat was produced and purified according to the method reported by
      • Kanno T.
      • Kawada A.
      • Yamanouchi J.
      • et al.
      Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.
      . Recombinant, processed, enzymatically active human caspase-14 (C14 WT) was obtained by incubating purified human recombinant procaspase-14 either with an epidermal extract containing a caspase-14-activating enzyme (i.e., cleaved caspase-14, see Supplementary Material and Methods online) or with enzymatically active p20/p10 human caspase-14 (p20/p10 caspase-14) produced by expressing the p20 and p10 subunits from the pLT32 bacterial expression vector (
      • Mertens N.
      • Remaut E.
      • Fiers W.
      Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines.
      ). The p20 and p10 subunits were purified from inclusion bodies by denaturation–renaturation and Q-source chromatography. To check whether they have the same substrate specificity as the native recombinant human caspase-14 obtained by cleavage of procaspase-14 between the p20 and p10 subunits, we compared their activities on fluorescent caspase substrates (Supplementary Figure S3 online). The enzymatic activity profiles of these two preparations of active human caspase-14 were identical to those described by others (
      • Fischer H.
      • Stichenwirth M.
      • Dockal M.
      • et al.
      Stratum corneum-derived caspase-14 is catalytically active.
      ;
      • Mikolajczyk J.
      • Scott F.L.
      • Krajewski S.
      • et al.
      Activation and substrate specificity of caspase-14.
      ). Radioactive-labeled FLG repeat translation products were incubated with caspase-14 for 3hours at 37°C as described (
      • Mikolajczyk J.
      • Scott F.L.
      • Krajewski S.
      • et al.
      Activation and substrate specificity of caspase-14.
      ). We added 1μM of the pan-caspase inhibitor zVAD-fmk (Bachem, Bubendorf, Switzerland) to inactivate caspase-14, or else we used a recombinant, processed, catalytic inactive human caspase-14 mutant (C14 C/A), which has a cysteine to alanine mutation at the catalytic site C132. Depletion of His-tagged recombinant caspase-14 was achieved by incubation with Ni2+ Sepharose beads (Amersham Biosciences, Diegem, Belgium) for 2hours at 4°C on a rotating platform. Reaction samples, containing 4μl of translation mix (6 microCurie), were precipitated with trichloroacetic acid overnight at -20°C. After centrifugation, pellets were washed twice with acetone and dissolved in loading buffer. Proteins were separated by 15% SDS–PAGE. Gels were incubated for 30minutes in fixation buffer (10% acetic acid, 25% isopropanol and 0.5% (w/v) L-methionine) and afterwards incubated in amplifier solution (Amersham Amplify Fluorographic Reagent, Amersham Biosciences). Gels were vacuum-dried and cleavage events were examined after autoradiography. Bacterially expressed FLG monomer was incubated with p20/p10 caspase-14 (20:1 ratio) for 1hour at 37°C in the presence or absence of 1μM zVAD-fmk. Samples were analyzed using SDS–PAGE and Coomassie Blue staining.

       Protein identification by liquid chromatography–mass spectrometry/mass spectrometry analysis

      Proteins were in-gel digested overnight at 37°C with 0.1μg of sequencing-grade-modified trypsin (Promega, Madison, WI). The resulting peptide mixture was applied for nano-liquid chromatography–mass spectrometry/mass spectrometry analysis on an Ultimate (Dionex, Amsterdam, The Netherlands) in-line connected to an Esquire HCT mass spectrometer (Bruker, Bremen, Germany). The fragmentation spectra were converted to Mascot generic files (mgf files) using the Automation Engine software (version 3.2, Bruker) and were searched using the MASCOT database search engine against the IPI database (taxonomy Mus Musculus) extracted from the EBI website (v3.16—http://www.ebi.ac.uk/IPI). MASCOT's parameter settings were as follows: enzyme: trypsin/P and variable modifications: acetyl (N-term), propionamide (C), oxidation(M), pyro-glu (N-term Q), peptide mass and fragment mass tolerances: ±0.5kDa, maximum number of missed cleavages: 1 and instrument type: ESI-TRAP. Only spectra exceeding Mascot's identity threshold score (set at the 95% confidence level) and that were ranked first were retained.

       Raman spectroscopy

      Raman measurements were performed using model 3510 Skin Composition Analyzer (River Diagnostics, Rotterdam, The Netherlands). The instrument is optimized for rapid in vivo measurements on the skin and offers an axial spatial resolution of <5μm. It comprises a dispersive spectrometer and a continuous-wave solid-state laser with an emission wavelength of 785nm. Raman spectra are recorded in the 400–1800cm-1 spectral range with a spectral resolution of 5cm-1. The spectrometer is connected to a confocal measurement stage, which is essentially an inverted microscope. The skin is placed on a fused silica window in the measurement stage. Focusing at different depths in the skin at 0.1μm resolution is controlled by the instrument and the axial displacements are stored with the collected Raman spectra. For lateral positioning, the fused silica window can be moved in one dimension in steps of 50μm. A built-in video camera enables visual inspection of the skin surface to avoid wrinkles and hairs during data collection. For ex vivo Raman measurements of NMF concentration profiles, a piece of 1 × 1cm2 full-thickness skin from the back of newborn (P5.5) caspase-14 wild-type and knockout mice was placed on the measurement stage. The Raman spectra were recorded from the skin surface down to a depth of 30μm in 2-μm steps. In this way, detailed Raman profiles were acquired across the SC and the upper part of the viable epidermis. Because of the heterogeneity of the skin at the microscopic scale, all measurements were repeated 10 times at different locations that were 0.1–5mm apart. Raman spectra were recorded from eight caspase-14+/+ mice and six caspase-14-/- mice. For each mouse, we calculated the average NMF, PCA, and tUCA content of measurements between 0 and 10μm from the skin surface. Both groups were compared by the Mann–Whitney U-test. A P-value of 0.05 was used as a cut-off for statistical significance. All tests were two-sided; analyses were performed using SPSS 15.0 (SPSS, IBM, Chicago, IL).

       Sequential SC tape stripping and measurement of UCA and PCA by high-pressure liquid chromatography

      The levels of PCA and tUCA on lysates from tape-strips obtained from caspase-14+/+ and caspase-14-/- mice were determined by using a method based on high-pressure liquid chromatography quantification (
      • Kezic S.
      • Kammeyer A.
      • Calkoen F.
      • et al.
      Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods.
      ,
      • Kezic S.
      • O’Regan G.M.
      • Yau N.
      • et al.
      Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.
      ). For technical details see online section. Data from the groups were compared by Mann–Whitney U-testing.

      ACKNOWLEDGMENTS

      We thank A. Bredan for editing the manuscript. This research was supported by the Flanders Institute for Biotechnology (VIB), Ghent University, and by several grants. European grants: FP6 Integrated Project Epistem LSHB-CT-2005-019067 and COST action SKINBAD BM0903. Belgian grants: Interuniversity Attraction Poles, IAP 6/18. Flemish grants: Fonds Wetenschappelijke Onderzoek Vlaanderen, 3G.0218.06, G.0226.09, and 1.5.169.08. Ghent University grants: BOF-GOA—12.0505.02. E. Hoste held a predoctoral research grant of the IWT-Vlaanderen. PV holds a Methusalem grant from the Flemish Government.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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