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Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing

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Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector See related commentary on pg 2173 ORIGINAL ARTICLE Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing Factors in the Skin Esther Hoste1,2,11, Patrick Kemperman3,4,11, Michael Devos1,2,11, Geertrui Denecker1,2, Sanja Kezic5, Nico Yau5, Barbara Gilbert1,2, Saskia Lippens1,2, Philippe De Groote1,2, Ria Roelandt1,2, Petra Van Damme6, Kris Gevaert6, Richard B. Presland7,8, Hidenari Takahara9, Gerwin Puppels3,10, Peter Caspers3,10, Peter Vandenabeele1,2 and Wim Declercq1,2 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-14À/À 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. Journal of Investigative Dermatology (2011) 131, 2233–2241; doi:10.1038/jid.2011.153; published online 9 June 2011 INTRODUCTION consists of corneocytes enshrouded by a cornified envelope Keratinocytes, the main cell type in the epidermis, undergo a embedded in a specialized lipid bilayer (Elias, 2008). This well-orchestrated differentiation program that leads to the heterogeneous ‘‘brick and mortar’’ constitution of the outer formation of a functional skin barrier maintained by several epidermal layers provides a strong protective barrier. Water components residing mainly in the stratum corneum (SC; retention by the skin is governed by lipids as well as by the Proksch et al., 2008; Schauber and Gallo, 2009). In addition, cornified envelope (Imokawa et al., 1989). Cornified envel- tight junctions in the stratum granulosum are also key players opes, which replace the plasma membrane in corneocytes, in maintaining the water balance in skin (reviewed in Morita are insoluble protein structures that are tightly crosslinked by et al., 2011). The SC is the outermost epidermal layer and transglutaminases (Candi et al., 2005). 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), 1Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium; 2Department of Biomedical together with lactic acid, urea, citrate, and sugars (Rawlings Molecular Biology, Ghent University, Ghent, Belgium; 3Department of and Harding, 2004). These NMFs absorb water and maintain Dermatology and Venereology, Erasmus MC, Rotterdam, The Netherlands; hydration in skin of terrestrial animals. In addition, the ionic 4 Department of Dermatology and Venereology, Waterlandziekenhuis, interaction between NMF and keratins reduces the inter- Purmerend, The Netherlands; 5Coronel Institute of Occupational Health, Academic Medical Centre, University of Amsterdam, Amsterdam, The molecular binding between keratin filaments and thereby Netherlands; 6Department of Medical Protein Research, VIB-Ghent increases the elastic properties of the SC (Jokura et al., 1995). University, Ghent, Belgium; 7Department of Oral Biology, University of PCA and UCA are derived mainly from complete degradation Washington, Seattle, Washington, USA; 8Department of Medicine (Dermatology), University of Washington, Seattle, Washington, USA; of filaggrin (FLG, filament aggregating protein) in the top 9Department of Applied Biological Resource Sciences, School of Agriculture, layers of the SC (Scott et al., 1982; Scott and Harding, 1986; Ibaraki University, Ibaraki, Japan and 10River Diagnostics BV, Rotterdam, Kezic et al., 2008, 2009). The Netherlands Filaggrin is thought to contribute to epidermal barrier 11 These authors share first authorship function by promoting keratin filament aggregation into Correspondence: Wim Declercq, Molecular Signaling and Cell Death Unit, macrofibrils in the stratum granulosum and SC layers (Dale Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium. E-mail: [email protected] et al., 1985; Sybert et al., 1985). Loss-of-function mutations in the filaggrin gene (FLG) lead to the development of the dry Abbreviations: FLG, filaggrin; NMF, natural moisturizing factors; PCA, pyrrolidone carboxylic acid; SC, stratum corneum; tUCA, trans-urocanic acid skin disease, ichthyosis vulgaris in humans and to a similar Received 8 March 2011; revised 8 April 2011; accepted 12 April 2011; neonatal skin disease in mice (Palmer et al., 2006; Smith published online 9 June 2011 et al., 2006; Fallon et al., 2009). In addition, FLG gene & 2011 The Society for Investigative Dermatology www.jidonline.org 2233 E Hoste et al. Caspase-14 Required for NMF Generation mutations predispose individuals to atopic dermatitis (ecze- fragments was not reported. We compare FLG processing in ma), which is often associated with other allergies, including wild-type and caspase-14À/À mice and show that FLG asthma (Palmer et al., 2006; Schuttelaar et al., 2009). degradation is initiated in caspase-14-deficient mice but Profilaggrin is synthesized as a large polyprotein precursor does not complete, resulting in the accumulation of FLG of 4400 kDa consisting of multiple FLG units flanked by fragments. Consequently, we demonstrate lower NMF levels distinct N- and C-terminal domains. The N-terminal domain in caspase-14À/À mice relative to control mice. We also contains a conserved S100 calcium-binding domain and a identified two caspase-14 cleavage sites in the FLG repeat postulated B-domain. Profilaggrin comprises 10–12 FLG unit. Taken together, our results indicate an important role for repeat units in humans and 12–20 in mice, depending on caspase-14 in FLG catabolism. the strain (Rothnagel and Steinert, 1990; Zhang et al., 2002; Fallon et al., 2009; Sandilands et al., 2009). In the stratum RESULTS granulosum, profilaggrin is highly phosphorylated and Filaggrin degradation is defective in caspase-14-deficient skin retained in the keratohyalin granules. Profilaggrin is dephos- As reported earlier, the proteolytic generation of the FLG phorylated in the transitional layer between stratum granu- repeat, which has an apparent molecular weight of B30 kDa losum and SC before proteolytic cleavage into FLG units (Presland et al., 2000), was not affected in caspase-14À/À (reviewed in Ovaere et al., 2009; Sandilands et al., 2009). skin, but FLG degradation products accumulated in the Once FLG is released, it forms an aggregate with the keratin epidermis (Denecker et al., 2007, 2008). We show here that intermediate filaments (Dale et al., 1985). FLG is deiminated this accumulation is evident in adults but is even more in the cornified layers by peptidylarginine deiminase (Nachat pronounced in neonates (Figure 1a). Furthermore, minor FLG et al., 2005), converting positively charged arginine residues degradation products were also observed in caspase-14 þ / þ into citrulline. Subsequently, the FLG–keratin interaction is epidermis as detected by western blotting using an antibody broken and FLG is degraded further into free amino acids that directed against the FLG repeat. To validate the nature of contribute to the NMF (reviewed in Rawlings and Harding, these FLG fragments that accumulate in the absence of 2004). Consequently, patients carrying loss-of-function FLG caspase-14, caspase-14 þ / þ and caspase-14À/À skin lysates mutations exhibit greatly reduced NMF levels (Kezic et al., were separated by gel electrophoresis and stained with 2008, 2011; O’Regan et al., 2010). Coomassie Blue (Figure 1b). The gel region in which FLG- Although several proteases are involved in processing derived fragments accumulated most prominently was sub- profilaggrin into FLG, little is known about the proteolytic jected to protein elution and analyzed by mass spectrometry. events involved in the degradation of FLG into amino acids. All peptides but one that maps to the N-terminal B-domain of We recently generated caspase-14À/À mice and showed that profilaggrin, map to the FLG repeat sequence (Figure 1c, caspase-14 deficiency leads to the accumulation of fragments Supplementary Figure S1 online). The number of spectra derived from FLG repeats (Denecker et al., 2007), suggesting identified as FLG peptides was 6-fold higher in caspase-14 that FLG degradation is defective in these mice. Calpain I and knockout skin than in wild-type skin, while the number of caspase-14 can cleave deiminated FLG repeats in vitro spectra derived from the subunits of the actin-related protein (Kamata et al., 2009), but the identity of the cleavage 2/3 complex was 5% in both samples (data not shown). acMw P5.5
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