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Knockdown of Filaggrin Impairs Diffusion Barrier Function

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Knockdown of Filaggrin Impairs Diffusion Barrier Function ORIGINAL ARTICLE Knockdown of Filaggrin Impairs Diffusion Barrier Function and Increases UV Sensitivity in a Human Skin Model Michael Mildner1,5, Jiang Jin1,2,5, Leopold Eckhart1, Sanja Kezic3, Florian Gruber1, Caterina Barresi1, Caroline Stremnitzer1, Maria Buchberger1, Veronika Mlitz1, Claudia Ballaun1, Barbara Sterniczky1, Dagmar Fo¨dinger1 and Erwin Tschachler1,4 Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection. Journal of Investigative Dermatology (2010) 130, 2286–2294; doi:10.1038/jid.2010.115; published online 6 May 2010 INTRODUCTION highly phosphorylated precursor protein of around 500 kDa, Epidermal keratinocytes (KCs) undergo distinct morphologi- containing a calcium binding N terminus, followed by a cal and biochemical changes as they move from the basal hydrophilic domain, a truncated filaggrin repeat, 10–12 layer to the spinous and granular layers, eventually forming complete filaggrin repeats, another truncated repeat, and a the stratum corneum. Although keratins 5 and 14 are the tail peptide (Gan et al., 1990; Presland et al., 1992; Presland main keratins in the basal layer, suprabasal KCs express and Dale, 2000; McGrath and Uitto, 2008). During the last keratins 1, 2, and 10. In addition, several differentiation- steps of terminal KC differentiation, profilaggrin is depho- associated proteins uniquely found in the epidermis, such as sphorylated, and proteolytically processed into the involucrin, loricrin, and filaggrin, are expressed in the N-terminal domain and filaggrin monomers (Presland et al., terminally differentiated layers of the epidermis (Watt, 1997; Presland and Dale, 2000). The N terminus consists of 1983; Mehrel et al., 1990; Candi et al., 2005; Denecker an A domain that contains two Ca2 þ -binding S100-like et al., 2008). Human profilaggrin is synthesized as a large EF-hands, and a cationic B domain that contains a functional nuclear localization sequence. It was shown that the filaggrin N terminus is able to translocate into the nucleus and may 1Department of Dermatology, Medical University of Vienna, Vienna, Austria; 2 therefore have a crucial role in nuclear breakdown or other Department of Dermatology, Peking University People’s Hospital, Beijing, China; 3Academic Medical Center, Coronel Institute of Occupational Health, nuclear events of epidermal KC terminal differentiation Amsterdam, The Netherlands and 4Centre de Recherches et d’Investigations (Ishida-Yamamoto et al., 1998; Pearton et al., 2002). Epidermiques et Sensorielles, Neuilly, France However, the exact function of the filaggrin N terminus is 5 These authors contributed equally to this work. still unknown. Studies involving keratins and filaggrin Correspondence: Erwin Tschachler, Department of Dermatology, Medical purified from skin suggested that filaggrin can interact with University of Vienna, Wa¨hringer Gu¨rtel 18-20, Vienna A-1090, Austria. different types of intermediate filaments, thereby leading to E-mail: [email protected] their aggregation into macrofibrils (Dale et al., 1978). In Abbreviations: KC, keratinocyte; siRNA, small interfering RNA; UCA, urocanic acid addition, transient overexpression of human filaggrin in vitro Received 12 November 2009; revised 2 March 2010; accepted 17 March results in the collapse of the intermediate filament network 2010; published online 6 May 2010 (Dale et al., 1997; Presland et al., 2001). In the stratum 2286 Journal of Investigative Dermatology (2010), Volume 130 & 2010 The Society for Investigative Dermatology M Mildner et al. Filaggrin Knockdown in a Human Skin Model corneum, filaggrin is degraded into amino acids and other filaggrin in the granular layers of control organotypic components of the natural moisturizing factors and thereby skin samples and absence of filaggrin staining in cultures contributes to the retention of water within corneocytes, this containing KCs treated with filaggrin-specific siRNAs being essential for the osmolarity, flexibility, and barrier (Figure 1c). function of the stratum corneum (Scott et al., 1982; Scott and Histological investigation revealed a complete loss of Harding, 1986; McGrath and Uitto, 2008). keratohyalin granules in the filaggrin knockdown organotypic Clinically, loss-of-function mutations in the filaggrin gene cultures, whereas the morphology of the stratum corneum did are associated with ichthyosis vulgaris and atopic dermatitis. not appear to be compromised (Figure 2a and b). Ultra- R501X and 2282del4 are the two most common mutations structural examination confirmed that the number and size of resulting in a lack of functional filaggrin in populations of keratohyalin granules was strongly reduced (Figure 2c and e). European ancestry (Palmer et al., 2006; Smith et al., 2006; In addition, electron micrographs showed that the knock- McGrath and Uitto, 2008). Three other common mutations down of filaggrin did not affect the number but the and several rare or family-specific mutations are also reported morphology of lamellar bodies. As compared with control in European ancestral groups (Sandilands et al., 2007, 2009). samples, lamellar bodies in filaggrin-deficient skin models As filaggrin knockout mice are not available, recent loss-of- were smaller (Figure 2d and f) and lacked lamellae of the function filaggrin studies were performed using the flaky tail characteristic shape (Figure 2d, for higher resolution see (ft) mouse as a model system. This spontaneous recessive Supplementary Figure S2 online). mouse mutant arose in 1958 on the background of an existing recessive hair phenotype and is characterized by dry and Filaggrin knockdown in organotypic skin cultures affects neither flaky skin and neonatal abnormalities in the tail and paws KC differentiation and solubility nor lipid composition (Presland et al., 2000). It was shown that these mice express a A panel of genes differentially expressed in the various truncated profilaggrin that cannot be further processed into layers of the epidermis was investigated by quantitative real- filaggrin monomers (Presland et al., 2000). The epidermal time PCR (Supplementary Figure S3 online), western blot barrier function is impaired and percutaneous allergen (Figure 3a), and immunofluorescence analysis (Supplemen- priming is increased in the ft mouse, suggesting a similar tary Figure S4 online). By comparison with control samples, dysfunction as a cause of skin irritation in human atopic filaggrin-deficient organotypic skin cultures showed normal dermatitis (Fallon et al., 2009; Scharschmidt et al., 2009). mRNA expression levels of loricrin, matriptase-1, and However, considering the species-specific properties of keratins 1, 10, 5, and 14 (Supplementary Figure S3 online). filaggrin (Harding and Scott, 1983), caution should be taken Western blot analysis confirmed that protein expression of when interpreting and comparing the function of filaggrin in involucrin, loricrin, matriptase-1, transglutaminase-1, cas- animal models with that of human filaggrin. pase-14, histidase, and keratins 1, 2, and 10, as well as the Recently, we have reported the combination of RNA small proline-rich molecule SPRR3 was not influenced by interference technology and human organotypic skin cultures filaggrin knockdown in organotypic skin cultures (Figure 3a). as an efficient method to study gene functions in epidermal A slight increase in the expression of the small proline-rich development (Mildner et al., 2006). In this study, we knocked molecule SPRR2A was detected in the filaggrin-deficient down filaggrin in this organotypic skin model to address the organotypic skin. Immunofluorescence labeling of loricrin, questions: (1) whether and how the absence of filaggrin matriptase-1, transglutaminase-1, caspase-14, actin, and influences the maturation of the epidermal barrier; (2) keratins 1, 5, 10, and 14 did not reveal differences with whether filaggrin deficiency would influence the lipid regard to the intensity or pattern of labeling between the composition in this model; (3) whether filaggrin is necessary controls and filaggrin knockdown organotypic skin cultures for keratin aggregation in this model; (4)
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