TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function

Transcription

1 ORIGINAL ARTICLE TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function Dagmar Fehrenschild 1,2, Uwe Galli 1,2,8, Bernadette Breiden 3, Wilhelm Bloch 4, Peter Schettina 1,2, Susanne Brodesser 5,6, Christian Michels 1,6,7, Christian Günschmann 1,6,7, Konrad Sandhoff 3, Carien M. Niessen 1,6,7 and Catherin Niemann 1,2 Defects in the function of the skin barrier are associated with a wide variety of skin diseases, many of which are not well characterized at the molecular level. Using Lef1 (lymphoid enhancer-binding factor 1) dominantnegative mutant mice, we demonstrate here that altered epidermal TCF (T cell factor)/lef1 signaling results in severe impairment of the stratum corneum skin barrier and early postnatal death. Barrier defects were accompanied by major changes in lipid composition and ultrastructural abnormalities in assembly and extrusion of lipid lamellae of the interfollicular epidermis, as well as abnormal processing of profilaggrin. In contrast, tight-junction formation and stratified organization of the interfollicular epidermis was not obviously disturbed in Lef1 mutant mice. Molecular analysis revealed that TCF/Lef1 signaling regulates expression of lipidmodifying enzymes, such as Elovl3 and stearoyl coenzyme A desaturase 1 (SCD1), which are key regulators of barrier function. Promoter analysis and chromatin immunoprecipitation experiments indeed showed that SCD1 is a direct target of Lef1. Together, our data demonstrate that functional TCF/Lef1 signaling governs important aspects of epidermal differentiation and lipid metabolism, thereby regulating skin barrier function. Journal of Investigative Dermatology (212) 132, ; doi:1.138/jid ; published online 22 September 211 INTRODUCTION The skin provides a protective barrier against detrimental impact of the environment; consequently, multiple skin diseases are caused by a defective skin barrier function. During evolution, the skin has developed a number of structural features that facilitate adequate reactions to external signals, irritants, and injuries. This barrier is provided by the stratified epithelium of mammalian epidermis to prevent water loss to the terrestrial environment and damage of the organism by pathogenic and harmful agents (Segre, 26). Keratinocytes of the interfollicular epidermis (IFE) 1 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; 2 Institute of Pathology, University of Cologne, Cologne, Germany; 3 LIMES, c/o Kekulé-Institut, University of Bonn, Bonn, Germany; 4 Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne, Germany; 5 Institute for Medical Microbiology, Immunology, and Hygiene, University of Cologne, Cologne, Germany; 6 Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany and 7 Department of Dermatology, University of Cologne, Cologne, Germany 8 Current address: Department of Surgery and Cancer Cell Biology, University of Heidelberg, Heidelberg, Germany Correspondence: Catherin Niemann, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Strasse 21, D-5931 Cologne, Germany. cnieman1@uni-koeln.de Abbreviations: Cer, ceramide; FA, fatty acid; IFE, interfollicular epidermis; Lef1, lymphoid enhancer-binding factor 1; SCD1, stearoyl coenzyme A desaturase 1;, sebaceous gland; TCF, T cell factor; TEWL, transepidermal water loss;, wild type Received 23 August 21; revised 2 June 211; accepted 25 June 211; published online 22 September 211 undergo a sequence of coordinated differentiation steps to form the protective outermost layer, the stratum corneum. This layer consists of cornified cells known as corneocytes surrounded by intercellular lipid lamellae. The program of differentiation is initiated as cells of the basal layer withdraw from the cell cycle and detach from the underlying basement membrane to move upward through suprabasal layers of the IFE. Keratinocytes rearrange their keratin filaments to provide a durable cytoskeletal framework as prerequisite for mechanical strength of the tissue. Keratin bundles are assembled and proteins, including involucrin and loricrin, are crosslinked by transglutaminase to form an insoluble cornified envelope. Finally, lipid lamellae delivered to the extracellular environment fill the space between corneocytes, thereby sealing the tissue for definite protection of the organism (Nemes and Steinert, 1999). Work in recent years has begun to understand the basic elements governing the formation of a functional barrier of the epidermis (Tsuruta et al., 22; Segre, 26). In addition to a proper execution of the program of terminal differentiation and stratification of keratinocytes, it has been demonstrated that formation of intercellular tight junctions are required for acquisition of a protective barrier (Turksen and Troy, 22; Kirschner et al., 21). For instance, mice deficient in claudin-1, a transmembrane protein of the tightjunction complex, display marked defects of epidermal barrier function manifested by an increase in transepidermal water loss (TEWL) (Furuse et al., 22). The stratified epithelium of mammalian skin is derived from the embryonic ectoderm, and a variety of signals that & 212 The Society for Investigative Dermatology 337

2 are crucial for development and homeostasis have been identified previously. Several signaling molecules, including Delta/Notch, Hedgehog, and BMP family members, are implicated in regulation of keratinocyte differentiation during development (Blanpain and Fuchs, 29). In addition, an important role emerged for canonical Wnt/b-catenin signaling in controlling a defined differentiation program and specifying cell fate in mouse epidermis (Niemann, 26). The differentiation of hair follicle progenitor cells is proposed to be driven by b-catenin/lef1 (lymphoid enhancerbinding factor 1)-dependent transcription (DasGupta and Fuchs, 1999; Lowry et al., 25). Accordingly, hair formation is blocked, whereas differentiation of stratifying keratinocytes and sebocytes is stimulated in the epidermis in which b-catenin signaling is blocked, either through epidermal inactivation of b-catenin or by transgenic expression of a mutant Lef1 unable to associate with b-catenin (DNLef1; Huelsken et al., 21; Merrill et al., 21; Niemann et al., 22). The impact of an abnormal function of canonical Wnt/ b-catenin or TCF (T cell factor)/lef1 signaling in skin barrier function has not been investigated yet. To address this issue, we set out to investigate the consequences of altering TCF/Lef1 signaling for proper barrier function of mammalian skin by expressing a mutant DNLef1 in the epidermis. RESULTS Defective epidermal barrier function in Lef1 mutant mice To block functional TCF/Lef1 signaling in mouse epidermis, we previously generated a transgenic mouse model expressing an N-terminally deleted dominant-negative mutant of the transcription factor Lef1 under the control of the keratin14 promoter (Niemann et al., 22). This Lef1 mutant cannot bind b-catenin, and thus prevents formation of the transcriptional complex required to activate the expression of Wnt/ b-catenin target genes. The mutant Lef1 also inhibits signaling by other TCF proteins (Niemann et al., 22). Genotyping of newborn mice revealed a Mendelian inheritance ratio of mutant mice (31 wild-type () mice and 35 mutant mice). In contrast, at weaning age, a reduction of B5% of mutant mice was observed (29 mice and 18 mutant mice; Figure 1a). ant mice surviving the first 5 days appeared smaller in size and developed a severe scaling skin phenotype (Figure 1b). To examine whether impaired barrier function explains the early postnatal death of mutant mice, we measured TEWL. Compared with control littermates, Lef1 mutant mice showed a significant increase in the rate of TEWL (Figure 1c), indicating impaired skin barrier function. Thus, expression of mutant Lef1 in mouse epidermis has a profound effect on early postnatal survival likely due to a strongly reduced skin barrier function. Next, we wanted to find out whether the epidermal barrier defect is initiated before birth. Therefore, expression of mutant Lef1 was analyzed during epidermal morphogenesis. As expected, mutant Lef1 was strongly expressed from E15.5 onward in basal cells of the IFE, but was also seen in suprabasal keratinocytes, because of the expansion of keratin 14-positive cell compartment of the IFE. At E18.5, mutant Lef1 was also detected in the outer root sheath of the hair follicle (Figure 1d, Supplementary Figure S1a online). Endogenous Lef1 protein was found in hair bulbs of developing hair follicles at E18.5 in control skin (Figure 1d). To analyze whether functional barrier is already disturbed during late embryonic development, we investigated its competence to exclude percutaneous dye penetration. Functional barrier formation starts at the dorsal surface around E16.5, as demonstrated by toluidine blue dye % Of mice b -b -w -w E15.5 E18.5 E18.5 E17.5 TEWL (g m 2 h 1 ) Figure 1. Disrupted epidermal barrier function in Lef1 (lymphoid enhancer-binding factor 1) mutant mice. (a) Postnatal lethality in mutant mice. Wild-type () and mutant animals expressed as percentage of living mice at birth (-b and -b) and at weaning age (-w and -w). (b) Image of mice at 5 days of age displaying reduced body size and scaling of the skin of mutant animals. (c) Transepidermal water loss (TEWL) measured on the back skin of postnatal mice (n ¼ 4). (d) Immunostaining for Lef1 (green) detects mutant Lef1 protein in the skin of mutant embryos (arrows, right panels). Endogenous Lef1 production is seen in the bulbs of developing hair follicles at E18.5 (arrowhead). Nuclear counterstaining with propidium iodide is shown in red. Bar ¼ 8 mm. (e) Toluidine blue dye exclusion assay of and Lef1 mutant embryos. 338 Journal of Investigative Dermatology (212), Volume 132

3 exclusion, and spreads ventrally, resulting in complete dye impermeability of the skin around E18.5 in mice (Hardman et al., 1998). Similar to control mice, Lef1 mutants were able to exclude dye before birth (Figure 1e), indicating that the outside-in barrier was formed relatively normal in these mice. This was confirmed by Lucifer yellow penetration assays on newborn mice (not shown), which also showed that Lef1 mutant mice did not exhibit a defective outside-in barrier at birth that could account for the increased water loss and subsequent lethality (Figure 1a c). These data reveal that despite strong mutant Lef1 expression, the formation of the epidermal barrier during embryogenesis is not affected. Instead, the barrier defect becomes evident after birth. Previous experiments have demonstrated that TCF/Lef1- dependent reporter gene activity is blocked in primary keratinocytes isolated from Lef1 mutant mice (Niemann et al., 22). We next asked which members of the TCF/ Lef1 transcription factor family are expressed within the IFE of newborn mice and thus might be responsible for the barrier defect. Low level of RNA expression of TCF3, TCF4, and Lef1 was detected in IFE using quantitative real time-polymerase chain reaction (RT-PCR). Therefore TCF3, TCF4, or Lef1 could potentially regulate barrier function. ant Lef1 expression did not alter TCF3 or TCF4 expression levels (Supplementary Figure S1b online). Tight-junction barrier is not impaired by mutant Lef1 It was previously reported that the outside-in barrier function was not obviously disturbed in mice with impaired tight junctions that suffered from water loss (Furuse et al., 22; Tungal et al., 25). We therefore investigated whether tight junctions were defective in Lef1 mutant mice. First, we analyzed the expression pattern of tight-junction components. Claudin-1 and claudin-4 were normally distributed and detected at the membrane in 6-day-old and adult and mutant mice (Supplementary Figure S2a online). To investigate tight-junction formation and function in primary keratinocytes upon induction of differentiation, a switch in calcium ion concentration was applied (Mertens et al., 25). We detected an increase in claudin-1 and claudin-4 protein production upon differentiation in primary keratinocyte cultures of both and mutant mice (Supplementary Figure S2b online). Transepithelial resistance measurements showed that keratinocytes of both genotypes were able to build up comparable epithelial resistance, indicating that the expression of mutant Lef1 (Supplementary Figure S3 online) does not interfere with the formation of the ionic tightjunction barrier (Supplementary Figure S2c online). In addition, mutant keratinocytes were similarly protected against the penetration of a neutral tracer (Supplementary Figure S2d online), demonstrating that the tight-junction size barrier is also properly formed. These results show that the observed barrier defect in Lef1 mutant epidermis cannot be explained by impaired function of the tight junctions. Defective maturation of corneocytes IFE of newborn and adult Lef1 mutant mice is distinguished by an increase in the number of epidermal layers and a thickened stratum corneum (Supplementary Figure S4 online). Ultrastructural analysis of back skin of mice revealed that corneocytes are flat, of regular shape, and densely packed to form a protective stratum corneum (Figure 2a). kd Filaggrin 15 SC K1 Involucrin Actin SC Circumference (µm 2 ) >3, 2,1 3, 1,91 2, 1,81 1,9 1,71 1,8 1,61 1,7 1,51 1,6 1,41 1,5 1,31 1,4 1,21 1,3 1,11 1,2 1,1 1,1 <1, Number of corneocytes Figure 2. Maturation of corneocytes and profilaggrin processing are defective in Lef1 (lymphoid enhancer-binding factor 1) mutant skin. (a, b) Morphological analysis of back skin by transmission electron microscopy of mutant (b) and control littermates (a). SC, stratum corneum;, stratum granulosum. (c) Morphology of corneocytes isolated from wild-type () and Lef1 mutant mice. (d) Surface areas of corneocytes of control (n ¼ 73, light gray bars) and mutant mice (n ¼ 884, dark gray bars) were defined using Analysis computer software. (e) Western blot analysis of keratin 1 (K1), involucrin, and profilaggrin processing in epidermal lysates of 6-day-old and Lef1 mutant mice. Bar ¼ 7 nm (a, b), 1 mm (c)

4 In contrast, keratinized cells of stratum corneum of Lef1 mutant skin displayed abnormal shape and size with the majority of corneocytes being enlarged, thickened, and distorted (Figure 2b). Many corneosomes were still present in the initial layers of the stratum corneum maturing from the stratum granulosum in the epidermis of Lef1 mutant mice when compared with controls (Figure 2b, arrows). The surface of the stratum granulosum was even and smooth in skin (Figure 2a), whereas the junction between upper granular layer and corneocytes was rough and of irregular shape in mutant skin samples (Figure 2b). Corneocytes were isolated to assess their size and structure in detail. Close examination revealed a different form of corneocytes isolated from mutant mice when compared with corneocytes (Figure 2c). In addition, the average size of corneocytes from mutant mice was larger (1,683.3 mm 2 ) compared with that of corneocytes from control animals (1,318.6 mm 2 ) (Figure 2d). Next, we investigated whether an altered program of keratinocyte differentiation could potentially contribute to defects seen in the IFE. Immunofluorescence analysis of the hyperproliferative keratin 6 did not detect an increase in 6-day-old Lef1 mutant. Instead, a slight increase in expression of keratin 1, involucrin, and loricrin was detected in Lef1 mutant mice (Supplementary Figure S4 online). However, western blot analysis revealed that overall levels of keratin 1 and involucrin were not altered in Lef1 mutant versus control mice (Figure 2e). Surprisingly, a reduction in distribution of filaggrin, an important component of cornified envelopes, was detected by immunofluorescence in the skin of mutant animals (Supplementary Figure S4 online). During terminal differentiation of keratinocytes, profilaggrin is processed into mature filaggrin that is subsequently crosslinked and fragmented in mature cells (Scott and Harding, 1986; Smith et al., 26). Western blots showed that the monomer band of filaggrin was strongly reduced in the epidermis of 5-day-old Lef1 mutant mice compared with control tissue. In addition, filaggrin fragments of 15 25K were also decreased in Lef1 mutant epidermis (Figure 2e), indicating disturbed filaggrin production in Lef1 mutant skin. Taken together, these results indicate that impairment of both differentiation of keratinocytes and maturation of the stratum corneum are most likely to contribute to the barrier defect observed in Lef1 mutant mice. Severe defects in lipid matrix composition To test whether the lipid barrier is disturbed in the epidermis of Lef1 mutant mice, freely extractable lipids and those covalently bound to corneocytes were analyzed from the skin of mutant and mice. First, the content of cholesterol, free fatty acids, and ceramides of isolated stratum corneum was determined. The level of total ceramides (cer) was slightly elevated in Lef1 mutant mice, but levels of cholesterol and free fatty acids were not significantly altered (Figure 3a). Quantification of extractable ceramides of stratum corneum revealed an overall gain in ceramides containing longer fatty acid (FA) chains in Lef1 mutant mice. Specifically, we observed an increase in the amount of Cer(EOS) and Cer (26-AS) and a decrease in Cer(C16-AH/AP) and Cer(AS-16) (Figure 3c and d). In metabolic studies of epidermis with [ 14 C] serine, we also found a higher level of Cer(EOS) (data not shown). Sphingomyelin is the ceramide precursor for Cer(NS) and Cer(AS) (Uchida et al., 2) and glucosylceramide for all ceramide species, especially ceramides with long- and very long-chain fatty acids (Hamanaka et al., 22). Densitometric quantification of the probarrier lipids showed a decrease of glucosylceramide and an increase of sphingomyelin (Figure 3b). Covalently linked lipids are of major importance for the lipid organization of lamellar structures, and therefore constitute an essential part of the lipid barrier. Analysis of covalently bound lipids revealed no alteration in the amount of o-oh-fa in Lef1 mutant mice (.71 ng mg 1 dry stratum corneum (SC) in vs..68 ng mg 1 dry SC in mut). However, a significant reduction in Cer(OS) was seen in mutant skin (2.27±.98 ng mg 1 dry SC) compared with control skin (14.75±1.3 ng mg 1 dry SC; Figure 3e). Therefore, the increase in Cer(EOS) in mutant Lef1 skin could be caused by a block of the processing of free Cer(EOS) to proteinbound ceramide (OS). Given the strong increase in sebaceous gland () differentiation in Lef1 mutant mice (Niemann et al., 22; Braun et al., 23), we analyzed the composition of sebum lipids, such as triglycerides, diglycerides, and esters, in more detail. Skin samples of mutant mice show a significant decrease in triglycerides when compared with control samples (Figure 3f). To a lesser extent, diglyceride fraction was also reduced, whereas wax esters and cholesterol esters were not significantly altered within s of mutant mice (Figure 3f). The decline in triglyceride point to a specific regulation of sebum lipid synthesis by mutant Lef1 and are not a pure consequence of increase in sebocyte differentiation evident in Lef1 mutant mice. Regulation of lipid barrier is an early phenotype as similar major changes in the lipid composition were detected in 5-day-old mutant mice (Figure 3, Supplementary Figure S5 online). Importantly, lipid defects are a cell-autonomous consequence of altered TCF/Lef signaling as no differences in lipid composition were observed in the liver tissue (Supplementary Figure S6 online). Impairment of lamellar lipid assembly To investigate whether aberrant lipid composition affects assembly of lamellar lipid layers and structure of lamellar bodies, transmission electron microscopy was performed. Ultrastructural analysis of Lef1 mutant mouse skin showed drastic morphological changes of intercellular lipid lamellae. ant keratinocytes in the granular layer contained disorganized lipid lamellae and vesicular structures (Figure 4b and c). These abnormal lipid vesicles were prominent at the intersection from the upper granular layer to the stratum corneum and persisted in corneocytes of the lower stratum corneum (Figure 4b and c and data not shown). In contrast, lipid lamellae were tightly packed and secretion occurred normally in the skin of mice (Figure 4a). Wild-type lipid lamellae were equally spaced in both the lamellar body structures and intercellular spaces of the stratum corneum (6.4±1.16 nm). In contrast, in Lef1 mutant mice, lamellar 34 Journal of Investigative Dermatology (212), Volume 132

5 μg mg 1 dry SC * μg mg 1 dry SC Cer Chol FA SM CSO 4 GlcCer * ** Chol FA Cer(EOS) Cer(NS) Cer(NP) Cer(AS) Cer(AP)/Cer(AH) Start FA ω-oh-fa 5 Months 3 Weeks Cer(EOS) Cer(C26-NS) Cer(C16-NS) Cer(NP) Cer(C16-AS) Cer(C26-AS) Cer(C16-AH/AP) Cer/C26-AH/AP) **** *** * *** *** μg mg 1 dry SC CE WE TAG Cer(OS) DAG Start 5 Months 3 Weeks Chol FA Start Figure 3. Abnormal lipid composition in Lef1 (lymphoid enhancer-binding factor 1) mutant mice. (a) Free stratum corneum (SC) lipids of Lef1 mutant mice and control littermates. Cer, total ceramides; Chol, cholesterol; FA, free fatty acids;, wild type. (b) Quantitative analysis of probarrier lipids sphingomyelin (SM), cholesterol sulfate (CSO 4 ), and glucosyl ceramide (GlcCer). (c, d) Free extractable ceramides from stratum corneum. (c) Representative thin layer chromatography (TLC) analysis at 5 months and 3 weeks of age. (d) Individual lipid levels of 3-week-old mice quantified by densitometric analysis. (e) Representative TLC of covalently bound lipids analyzed at 5 months and 3 weeks of age. (f) Decrease in sebum lipids in Lef1 mutant mice demonstrated by TLC. CE, cholesterol ester; DAG, diglyceride; TAG, triglyceride; WE, wax ester. *Po.5; **Po.1; ***Po.1; ****Po.5. SC SC Figure 4. Defective lamellar bodies in Lef1 (lymphoid enhancer-binding factor 1) mutant mice. (a c) Transmission electron microscopy reveals abnormal lamellar body structures within the granular layer of mutant mice (b, c, arrows). Normal lamellar bodies with typical regular lipid lamellae are seen in skin samples from wild-type mice (a, arrow). SC, stratum corneum;, stratum granulosum. Bars ¼ 1 nm, inset in a ¼ 3 nm

6 spacing was consistently increased (8.7±1.19 nm). No lipid lamellae were seen in lipid vesicles in mutant mice. These data reveal that specific defects in the lipid content result in abnormal lipid organization, thereby affecting the selective barrier in Lef1 mutant mice. Regulation in lipid-modifying enzymes in Lef1 mutant mice To unravel the molecular mechanism underlying the lipid barrier defect induced by aberrant Lef1 signaling, we screened in an unbiased manner for differentially expressed genes in mutant mice by subtractive hybridization experiments (data not shown). This revealed that Elovl3 and SCD1 (stearoyl coenzyme A desaturase 1), both important enzymes of the lipid metabolism, were abnormally expressed in Lef1 mutant mice. Quantitative RT-PCR confirmed that expressions of both Elovl3 and SCD1 were increased in newborn and adult Lef1 mutant mice (Figure 5a and b and Supplementary Figure S9a online). Protein levels of SCD1 were also elevated in mutant mice (Figure 5c). Importantly, a strong reduction in C18: saturated fatty acids (and to a lower extent C16:), together with an increase in unsaturated fatty acids (C18:1 and C16:1), in the epidermis from Lef1 mutant mice when compared with controls indicates an increase in SCD1 enzyme activity in mutant skin samples (Supplementary Figure S7 online). Expression of both enzymes SCD1 and Elovl3 has been detected within the previously. To investigate whether SCD1 and Elovl3 are also expressed within the IFE, quantitative RT-PCR analysis was performed on the epidermal tissue that had been separated from hair follicle and underlying dermis. Interestingly, we detect mrna for both enzymes within the IFE. However, the level of SCD1 and Elovl3 expression is not increased in IFE of mutant Lef1 epidermis, suggesting that SCD1 and Elovl3 made by Fold regulation ATG Prom3 LUC Prom2 LUC Prom1 LUC -2d -2d -2m -2m 1 15 Fold regulation Relative light units Pd Pd mut Pd2 Pd2 mut Pd5 Pd5 mut Pd9 Pd9 mut pgl Prom1 Prom2 Prom3 ΔNLef Lef Input Tag Ig kda 37 - Wt SCD1 co M bp BS prom α-tubulin bp BS prom3 TCF/Lef1 Elovl TCF/Lef1 Free ceramides Prot-bound ceramides VLCFA Ceramide composition Lipid lamellae TCF/Lef1 Profilaggrin processing BARRIER Figure 5. SCD1 (stearoyl coenzyme A desaturase 1) is a direct target of mutant Lef1 (lymphoid enhancer-binding factor 1). (a) Increase in ELOVL3 RNA in skin from 2-day-old (2d) and 2-month-old (2m) Lef1 mutant mice. (b) Analysis of SCD1 RNA in Lef1 mutant mice at birth (Pd) up to Pd9. (c) Western blot for SCD1 of wild-type and Lef1 mutant skin lysates. (d) Mouse SCD1 promoter sequence containing putative TCF (T cell factor)/lef1-binding sites (dark boxes) and deletion constructs for reporter assays (Prom1, Prom2, and Prom3). (e) Promoter assays detect opposite regulation by mutant Lef1 (DNLef1) and full-length Lef1 (Lef1). (f) Chromatin immunoprecipitation (ChIP) experiments demonstrate binding of mutant Lef1 (mut) by applying myc tag antibody to Prom2 and Prom3 fragment of the SCD1 regulatory sequence. Chromatin precipitates were analyzed by PCR. (g) Model of mutant Lef1 function in barrier acquisition. 342 Journal of Investigative Dermatology (212), Volume 132

7 keratinocytes of the IFE are most likely not responsible for the defects of the epidermal lipid barrier in Lef1 mutant mice (Supplementary Figure S9b online). Next, we performed immunofluorescence analysis of SCD1 on epidermal whole mounts of tail skin. As expected, SCD1 protein was detected in of control mice. In contrast, SCD1 protein localized to and ectopic sebocytes along the deformed hair follicle structures in the skin of Lef1 mutant mice (Supplementary Figure S8 online). To investigate whether SCD1 constitutes a potential direct target of Lef1, the mouse SCD1 upstream promoter sequence was screened for potential TCF/Lef1-binding sites. Seven putative TCF/Lef1 consensus-binding sites were identified within a 2.5-kb region upstream of the ATG translation start (Figure 5d). Co-transfection of a reporter construct containing a 2,221-bp upstream region with mutant Lef1 into HaCat or SW48 cells induced luciferase expression and activity (Figure 5e), indicating that these sites respond to Lef1 signaling. In contrast, full-length Lef1 cdna transfection decreased reporter gene activity significantly. To identify which of the potential Lef1/TCF consensus sites are important for the responsiveness, we generated serial deletion mutants (Figure 5d). Co-transfection of these fragments together with DNLef1 revealed that the promoter fragment containing four potential Lef1/TCF sites (Prom2) was slightly more induced than the shortest SCD1 promoter fragment harboring only one site (Prom1). The longest construct spanning all seven binding sites induced the most potent response. These data suggest that several Lef1/TCF sites of the SCD1 promoter are involved in the regulation of SCD1 expression by Lef1 (Figure 5d and e). To determine whether mutant Lef1 can directly bind to the endogenous SCD1 promoter, we performed chromatin immunoprecipitation experiments. An antibody against the myc tag, which is fused to mutant Lef1, immunoprecipitated the SCD1 promoter target sequence in keratinocytes isolated from the mutant but not from the control mice (Figure 5f). Specific binding of mutant Lef1 protein was seen for TCF/Lef1 consensus-binding sites in Prom2 and Prom3 sequences. These data reveal that SCD1 is indeed a direct target of mutant Lef1 in vivo. DISCUSSION Our results demonstrate a previously unreported and important function for TCF/Lef1 DNA-binding proteins in the formation and maintenance of epidermal barrier. Interference with TCF/Lef1 signaling in mouse skin induces drastic changes in the lipid composition and lipid lamellae formation. As a consequence, a disturbed lipid matrix causes profound defects in the epidermal lipid barrier. The results presented here demonstrate that functional TCF/Lef1 signaling governs important aspects of metabolic lipid processing in mammalian tissues. In addition to directing crucial aspects of the lipid metabolism, TCF/Lef1 proteins affect proliferation and differentiation of keratinocytes of the IFE. In Lef1 mutant mice, squamous differentiation and formation of corneocytes was disturbed, and less profilaggrin and filaggrin monomers were detected. Abnormal differentiation is often seen as a consequence of a skin barrier defect. However, expansion of the keratin 14-positive compartment of the IFE was already evident before the barrier defect at E18 in mutant mice. This strongly suggests that mutant Lef1 affects differentiation and maturation of epidermal keratinocytes directly. How mutant Lef1 modulates the squamous differentiation program of keratinocytes is at present unclear. Interestingly, Lef1, TCF3, and TCF4 are expressed within the IFE of newborn mice, and an important function of TCF3/TCF4 factors for maintenance of long-term homeostasis of the IFE has been reported previously (Nguyen et al., 29). Thus, the results implicate that Lef1, TCF3, and TCF4 can potentially regulate the barrier function of mammalian epidermis. Here we identified SCD1 and Elovl3 as primary targets of mutant Lef1. Both molecules are key enzymes of mammalian lipid metabolism and have an important role for skin barrier function. SCD1, a direct target of mutant Lef1, is a key enzyme that converts saturated fatty acids into monounsaturated fatty acids. Palmitoyl- and stearoyl-coenzyme A are preferred substrates used by SCD1 to synthesize palmitoleic (D9-16:1) and oleic acid (D9-18:1), respectively (Enoch and Strittmatter, 1978). Global loss of SCD1 and mutations within the SCD1 locus seen in the asebia mouse model as 2J disrupt the epidermal lipid barrier and increase TEWL (Sundberg et al., 2; Binczek et al., 27). In addition, lipid matrix and lipid lamellae are disturbed in SCD1-deficient mice due to the loss of o-hydroxylated very long-chain fatty acids and ceramides normally substituted with o-hydroxylated very long chain fatty acids (Binczek et al., 27). In contrast, the fraction of total ceramides, including o-oh ceramides (EOS), is increased in Lef1 mutant mice and composition of free extractable ceramides from stratum corneum is altered. Thus, either lack of SCD1 (SCD1-deficient mice) or increased SCD1 enzyme activity (Lef1 mutant mice) results in disturbances of the lipid matrix and severe skin barrier defects. ant Lef1 also upregulates transcription of Elovl3, a member of a highly conserved family of microsomal enzymes (Tvrdiket al., 2) that mediate formation of very long-chain fatty acids. This is especially interesting in the light of our findings that mutant Lef1 is increasing the level of ceramides containing longer FA, which is associated with a decrease in ceramides with shorter FA. It has been shown previously that targeted mutation of the elovl3 gene results in severe defects of the epidermal barrier and abnormal lipid composition (Westerberg et al., 24). The characterization of the barrier defect of Lef1 mutant mice complements a list of mouse mutants carrying defects in the skin barrier function that are accompanied by abnormal sebaceous lipid composition and changes in sebaceous gland physiology (see, e.g., Binczek et al., 27). However, the exact mechanism of how disturbed sebaceous gland functions contribute to the defective lipid and skin barrier is not understood yet. Covalently linked lipids are unique to the epidermis (Werzt and Downing, 1987) and are key components of the extracellular lamellae and corneocyte lipid envelope, which is the covalently attached bilayer of the cornified envelope 343

8 (Marekov and Steinert, 1998; Bouwstra et al., 23). We demonstrate a significant decrease in the Cer(OS) proteinbound fraction of ceramides in skin of Lef1 mutant mice. This is due to a block in processing of free ceramides (e.g., Cer(EOS)), a lipid fraction that was accumulating in mutant skin. As a result, the lamellar lipid matrix and consequently lamellar bodies in stratum granulosum are highly disturbed. This became evident by lipid lamellae that are not well organized and often displaced by vesicular lipid aggregates. Furthermore, our results suggest that variations in ceramide and lipid composition can profoundly affect the spacing of lamellar structures (Figure 5g). Many human sebaceous tumors harbor mutations within the Lef1 gene. These mutations lie within the b-cateninbinding domain and abolish interaction of Lef1 with b-catenin, resulting in a block of transcriptional activation (Takeda et al., 26). Lef1 mutant mice also develop spontaneous sebaceous tumors (Niemann et al., 22). Until now, it is not known whether a defective epidermal barrier could also be contributing to the formation of skin cancer. Furthermore, abnormal constitution of the lipid matrix could profoundly affect tumor groh. In the future, it will be exciting to explore whether mutations within the components of TCF/Lef1 signaling are also underlying pathological defects in human barrier diseases. MATERIALS AND METHODS Mice Lef1 mutant mice (K14DNLef1) have been described previously (Niemann et al., 22) and have been backcrossed into a pure C57Bl/6 background. Postnatal lethality was monitored from a total of 1 litters. Experiments were performed according to institutional guidelines and animal license given by the State Office North Rhine- Westphalia, Germany. Epidermal protein extraction and western blot analysis Protein extracts were isolated from skin samples and processed for western blot analysis following standard protocols (Supplementary Information online). Isolation of corneocytes Mouse ear tips were incubated with 25 mm dithiothreitol and 2% SDS for 15 minutes at 1 1C and centrifuged afterward. Corneocytes were resuspended in 1 mm Tris, 1 mm EDTA buffer (ph 8.) and images of cornified envelopes were taken using an Olympus CK 4 microscope and analyzed with an Analysis computer program (Tokyo, Japan). Ultrastructural analysis Back skin from and K14DNLef1 mutant mice was fixed in 2% formaldehyde, 2% glutaraldehyde in 1 mm cacodylate buffer (ph 7.4) and postfixed with either.2% ruthenium tetroxide or 1% osmium tetroxide. Samples were dehydrated and then embedded in Araldite (Serva, Heidelberg, Germany). Semithin (5 nm) and ultrathin sections (3 6 nm) for light and electron microscopic observation were processed on an ultramicrotome (Fa. Reichert, Vienna, Austria). Semithin sections were stained using methylene blue. Transmission electron microscopy was performed using a 92A electron microscope from Zeiss (Oberkochen, Germany). Quantitative RT-PCR Quantitative RT-PCR experiments were conducted according to standard methods (Supplementary Information online). Chromatin immunoprecipitation Chromatin immunoprecipitation assays and reporter assays were performed as described in Supplementary Information online. Lipid analysis Stratum corneum preparation and lipid analyses were performed as described previously (Reichelt et al., 24, Supplementary Information online). Functional tests of the epidermal barrier The rate of TEWL was determined with a Tewameter (Courage and Khazaka Electronic GmbH, Cologne, Germany) as described by Clarys and Barel (1995). Dye exclusion assays were performed as described before (Hardman et al., 1998). CONFLICT OF INTEREST The authors state no conflict of interest. ACKNOWLEDGMENTS We are very grateful to Andreas Kraus, Gilles Séquaris, Daniela Bobermien, Monika Petersson, Karen Reuter, and Mojgan Ghilav for excellent technical assistance, and Thomas Krieg (Dermatology, University of Cologne) and Johanna Brandner (Dermatology, University of Hamburg) for critical reading of the manuscript and for helpful comments. The work was supported by SFB 829 (to CN and CMN), SFB 645 (to KS), SFB 832 (to CMN), German Cancer Aid (to CN and CMN), and by the seventh framework program of the EUfunded LipidomicNet, proposal number (to KS). SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at REFERENCES Binczek E, Jenke B, Holz B et al. (27) Obesity resistance of the stearoyl-coa desaturase-deficient (scd1 / ) mouse results from disruption of the epidermal lipid barrier and adaptive thermoregulation. Biol Bioch 388:45 18 Blanpain C, Fuchs E (29) Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev Mol Cell Biol 1:27 17 Bouwstra JA, Honeywell-Nguyen PL, Gooris GS et al. (23) Structure of the skin barrier and its modulation by vesicular formulations. Prog Lipid Res 42:1 36 Braun KM, Niemann C, Jensen UB et al. (23) Manipulation of stem cell proliferation and lineage commitment: visualization of label-retaining cells in wholemounts of mouse epidermis. Development 13: Clarys P, Barel A (1995) Quantitative evaluation of skin surface lipids. Clin Dermatol 13:37 21 DasGupta R, Fuchs E (1999) Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126: Enoch HG, Strittmatter P (1978) Role of tyrosyl and arginyl residues in rat liver microsomal stearylcoenzyme A desaturase. Biochemistry 17: Furuse M, Hata M, Furuse K et al. (22) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1- deficient mice. J Cell Biol 156: Hamanaka S, Hara M, Nishio H et al. (22) Human epidermal glucosylceramides are major precursors of stratum corneum ceramides. J Invest Dermatol 119: Journal of Investigative Dermatology (212), Volume 132

9 Hardman MJ, Sisi P, Banbury DN et al. (1998) Patterned acquisition of skin barrier function during development. Development 125: Huelsken J, Vogel R, Erdmann B et al. (21) b-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 15: Kirschner N, Bohner C, Rachow S et al. (21) Tight junctions: is there a role in dermatology? Arch Dermatol Res 32: Lowry WE, Blanpain C, Nowak JA et al. (25) Defining the impact of betacatenin/tcf transactivation on epithelial stem cells. Genes Dev 19: Marekov LN, Steinert PM (1998) Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope. J Biol Chem 273: Merrill BJ, Gat U, DasGupta R et al. (21) Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev 15: Mertens AEE, Rygiel TP, Olivio C et al. (25) The rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol 17: Nemes Z, Steinert P (1999) Bricks and motar of the epidermal barrier. Exp Mol Med 31:5 19 Nguyen H, Merrill B, Polak L et al. (29) Tcf3 and Tcf4 are essential for longterm homeostasis of skin epithelia. Nat Genet 41: Niemann C (26) Controlling the stem cell niche: right time, right place, right strength. BioEssays 28:1 5 Niemann C, Owens DM, Hülsken J et al. (22) Expression of DNLef1inmouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 129:95 19 Reichelt J, Breiden B, Sandhoff K et al. (24) Loss of keratin 1 is accompanied by increased sebocyte proliferation and differentiation. Eur J Cell Biol 83: Scott IR, Harding CR (1986) Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment. Dev Biol 115:84 92 Segre JA (26) Epidermal barrier formation and recovery in skin disorders. J Clin Invest 116:115 8 Smith FJ, Irvine AD, Terron-Kwiatkowski A et al. (26) Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat Genet 38: Sundberg JP, Boggess D, Sundberg BA et al. (2) Asebia-2J [Scd1(ab2J)]: a new allele and a model for scarring alopecia. Am J Pathol 156: Takeda H, Lyle S, Lazar AJF et al. (26) Human sebaceous tumors harbor inactivating mutations in LEF1. Nat Med 12:395 7 Tsuruta D, Green KJ, Getsios S et al. (22) The barrier function of skin: how to keep a lid on water loss. Trends Cell Biol 12:355 7 Tungal JA, Helfrich I, Schmitz A et al. (25) E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J 24: Turksen K, Troy TC (22) Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129: Tvrdik P, Westerberg R, Silve S et al. (2) Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids. J Cell Biol 149:77 18 Uchida Y, Hara M, Nishio H et al. (2) Epidermal sphingomyelins are precursors for selected stratum corneum ceramides. J Lipid Res 41: Werzt PW, Downing DT (1987) Covalently bound omegahydroxyacylsphingosine in stratum corneum. Biochim Biophys Acta 917:18 11 Westerberg R, Tvrdik P, Unden AB et al. (24) Role of ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem 279: