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Impaired Cutaneous Permeability Barrier Function, Skin Hydration, and Sphingomyelinase Activity in Keratin 10 Deficient Mice1
Impaired Cutaneous Permeability Barrier Function, Skin Hydration, and Sphingomyelinase Activity in Keratin 10 De®cient Mice1 Jens-Michael Jensen,*2 Stefan SchuÈtze,² Claudia Neumann,* and Ehrhardt Proksch* *Department of Dermatology and ²Institute of Immunology, University of Kiel, Germany
Point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) in humans have been shown to be the cause of the congenital ichthyosis epidermolytic hyperkeratosis. Recently, a K10 de®cient mouse model was established serving as a model for epidermolytic hyperkeratosis. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes developed hyperkeratosis with age. To see whether phenotypic abnormalities in the mouse model were associated with changes in skin barrier function and skin water content we studied basal transepidermal water loss and capacity for barrier repair after experimental barrier disruption as well as stratum corneum hydration. Also, we determined the activities of acid and neutral sphingomyelinase key enzymes of the tumor necrosis factor and interleukin-1 signal transduction pathways generating the ceramides most important for epidermal permeability barrier homeostasis. Neonatal homozygotes showed an 8-fold increase in basal transepidermal
water loss compared with wild type controls. Adult heterozygotes exhibited delayed barrier repair after experimental barrier disruption. Stratum corneum hydration was reduced in homozygous and heterozygous mice. Acid sphingomyelinase activity, which is localized in the epidermal lamellar bodies and generates ceramides for extracellular lipid lamellae in the stratum corneum permeability barrier, was reduced in homozygous as well as heterozygous animals. Neutral sphingomyelinase activity, which has a different location and generates ceramides involved in cell signaling, was increased. The reduction in acid sphingomyelinase activity may explain the recently described decreased ratio of ceramides to total lipids in K10 de®cient mice. In summary, our results demonstrate the crucial role of the keratin ®lament for permeability barrier function and stratum corneum hydration. Key words: ceramide/epidermolytic hyperkeratosis/skin barrier function/skin hydration/sphingomyelinase. J Invest Dermatol 115:708±713, 2000
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Gene targeting at the mouse K10 locus resulted in mice with different genotypes in the homozygotes and heterozygotes, both of which exhibit similarities to speci®c clinical signs of EHK. Homozygotes revealed very fragile skin with small blisters and large areas of erosions and died within several hours after birth. Heterozygotes showed no clinical phenotype at birth, but developed hyperkeratosis and ¯aking in adults. Morphologically in both genotypes, alterations in the programme of epidermal differentiation, abnormal aggregation of cytokeratin intermediate ®laments, and changes in cytokeratin expression were observed. In addition, increased expression of K16 and K17 in the de®cient mice has been described (Porter et al, 1996, 1998). A reduced skin barrier function has been described in EHK and several other types of ichthyosis (Frost et al, 1968; Lavrijsen et al, 1993; 1995). The permeability barrier of the skin is localized in the stratum corneum, consists of corneocytes and lipid-enriched intercellular domains, and develops during epidermal differentiation. The intercellular domains mainly contain cholesterol, free fatty acids, and, most important, ceramides. Several studies by Elias, Feingold and coworkers have shown that an increase in the synthesis of these lipids in the nucleated epidermis occurs after experimental barrier disruption (reviewed by Elias, 1983; Feingold, 1991). These lipids or the respective precursors, glucosylceramides and phospholipids including sphingomyelin, are stored in the epidermal lamellar bodies, extruded, and converted to ceramides
pidermolytic hyperkeratosis (EHK, also known as bullous congenital ichthyosiform erythroderma Brocq) is caused by point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992; Yang et al, 1997; Suga et al, 1998). Clinically EHK includes localized or extensive blistering and erosions in severe cases. Adults show hystrix-like hyperkeratosis. The disease is complicated by erythroderma and secondary infections. Widespread disease can be very debilitating and dis®guring. Histologic features are cytokeratin ®lament aggregation, vacuolization of spinous and granular layer keratinocytes, cytolysis, and alteration of nuclear shape (Traube, 1989; DiGiovanna and Bale, 1994). Manuscript received November 25, 1999; revised June 7, 2000; accepted for publication July 7, 2000. Reprint requests to: Dr. Ehrhardt Proksch, Department of Dermatology, University of Kiel, Schittenhelmstr. 7, 24105 Kiel, Germany. Email: [email protected] 1Part of this study was presented at the 66th Annual Meeting of the Society for Investigative Dermatology (SID), April 23±27, 1997, Washington, DC. 2Present address: The Harvard Skin Disease Research Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Abbreviation: TEWL, transcutaneous/transepidermal water loss. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 708
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and fatty acids during the transition from the stratum granulosum to the stratum corneum. We recently showed that ceramides for permeability barrier function are generated not only using a synthetic pathway but also by the hydrolytic enzyme acid sphingomyelinase (A-SMase), localized in the endosomal lamellar body compartment (Jensen et al, 1999). In response to acute and chronic permeability barrier disruption A-SMase hydrolyzes sphingomyelin to ceramides. Ceramides are not only structural components for the physical permeability barrier, but they are also important second messengers in cell signaling. We showed recently that the neutral sphingomyelinase (N-SMase), which is localized in the cell membranes, is involved in cell signaling for permeability barrier repair (Jensen et al, 1999; Kreder et al, 1999). A- and NSMase are activated by tumor necrosis factor (TNF) and interleukin-1 (Il-1) during distinctive signal transduction pathways (Kolesnik and Golde, 1994; Wiegmann et al, 1994). We described previously that experimental barrier disruption in hairless mouse skin induces an increase in epidermal proliferation and changes in epidermal differentiation. We found an increase in the expression of the hyperproliferation- and in¯ammationassociated keratins K6, K16, and K17, but also in the suprabasal keratin K10, and a premature expression of involucrin after experimental barrier disruption in hairless mice (Proksch et al, 1991; Ekanayake-Mudiyanselage et al, 1998). Therefore, we and colleagues suggested that keratins and corni®ed envelope proteins are important for permeability barrier function and repair (Bickenbach et al, 1995; Roop, 1995). Reduced hydration of the stratum corneum and aggregated desquamating corneocytes appearing as white scales are the hallmarks of dry skin. Dry skin is common in otherwise healthy persons but is often observed in atopic dermatitis and in different types of ichthyosis (Grif®ths, 1982; Fartasch et al, 1989; Mukherjee and Gupta, 1994; Rabinowitz and Esterly, 1994; Johansen et al, 1995). The cause of dry skin is only known in part: using a commercially available corneometer or hygrometer, a reduced stratum corneum water content was detected in dry skin. Several studies on dry skin and ichthyosis showed changes in lipid content and lipid distribution, especially in ceramides (Paige et al, 1994; Lavrijsen et al, 1995). Also ®laggrin, ®laggrin degradation to natural moisturizing factors, and amino acid content are important for water binding in the stratum corneum (reviewed by Rawlings et al, 1994). In addition, in dry skin of healthy persons we previously found changes in epidermal differentiation including a decreased expression in K1 and K10 (Engelke et al, 1997). In this report we asked if the genetically determined cytokeratin intermediate ®lament abnormalities in the K10 de®cient mouse model result in changes in epidermal permeability barrier homeostasis and repair as well as in skin hydration and A- and N-SMase activity. MATERIALS AND METHODS Biochemicals and radiochemicals Biochemicals were purchased from Sigma-Aldrich Chemie (Munich, Germany). 14C-sphingomyelin (CFA566, 47.0 mCi per mmol) was purchased from Amersham Pharmacia Biotech Europe (Braunschweig, Germany). Experimental protocols Transepidermal water loss (TEWL) and stratum corneum hydration in homozygous and heterozygous mice TEWL and stratum corneum hydration in homozygous, heterozygous, and wild type K10 de®cient mice bred on a hairless background (Porter et al, 1996) (kindly provided by T.M. Magin and J. Reichelt, Bonn, Germany) were determined on ¯ank skin with the Tewameter water analyzer and the Corneometer (both by Courage & Khazaka, Cologne, Germany). Stratum corneum hydration was recorded as units (U) (Hashimoto-Kumasaka et al, 1993). Wild type animals served as control. Measurements in newborn mice were performed 2-6 h after birth because homozygous K10 de®cient mice died within 24 h (Porter et al, 1996). TEWL was studied in skin with normal appearance but also in sites of erosions in homozygous animals. In addition, TEWL and stratum corneum hydration were determined in adult (6±12 wk of age) hetero-
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zygous and wild type mice. Ambient room temperature was 22°C 6 2°C, and humidity was 45% 6 2%. Integrity of the stratum corneum and capacity in skin barrier repair in heterozygous mice Experimental disruption of the permeability barrier was induced in heterozygous hairless K10 de®cient mice (age 6±12 wk) by tape stripping (cellophane tape, Scotch type) until a 5±6-fold increase in TEWL was achieved (Tewameter) (Zettersten et al, 1998). The number of tape strips to disrupt the permeability barrier was counted and barrier recovery was monitored by TEWL at 1, 3, 5, 7, 24, and 48 h after treatment. In vitro assay of A- and N-SMase activity Flank skin samples (about 4 cm2) were excised. The epidermis isolated by incubation of the skin in 10 mM ethylenediamine tetraacetic acid (EDTA) at 37°C for 30 min was homogenized in a glass homogenizator on ice with a Potter S (Braun, Melsungen, Germany) at 500 rpm in 400 ml. For measuring A-SMase, buffer A containing 0.2% Triton-X 100 was used, and for N-SMase, buffer B containing 20 mM HEPES, 10 mM NaF, 2 mM EDTA, 10 mM MgCl2, 30 mM p-nitrophenyl phosphate, 100 mM sodium vanadate, 10 mM bglycerophosphate, 1 mM phenylmethylsulfonyl ¯uoride, 1 mM pepstatin A, leupeptin, antipain (PLA), 750 mM adenosine-5¢-triphosphate, and 0.2% Nonidet P-40 was used. The cell debris and nuclei were removed by lowspeed centrifugation at 1500 3 g for 10 min. The supernatants were used for the in vitro assay as described by Wiegmann et al (1994): 50 mg protein from the supernatants were incubated for 2 h at 37°C in reaction buffer A or B (50 mg ®nal volume), for A- or N-SMase, containing 2.25 ml of [Nmethyl-14C]-sphingomyelin (0.2 mCi per ml, speci®c activity 56.6 mCi per Mol, Amersham) as substrate. The reactions were stopped by addition of 800 ml chloroform:methanol (2:1, vol/vol) and 250 ml of H2O. [14C]phosphorylcholine, produced from [14C]-SM, was extracted from the aqueous phase, identi®ed by thin-layer chromatography, and routinely determined by liquid scintillation counting. Protein determination The protein content in epidermal homogenates was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as the standard (BCA-protein assay). Statistics Statistical signi®cance was determined using the two-tailed Student t test.
RESULTS Impaired basal permeability barrier homeostasis in newborn homozygous and heterozygous K10 de®cient mice As homozygous K10 de®cient mice exhibit severe skin fragility with small blisters and erosions we determined TEWL as a marker of barrier function in skin with normal appearance and in focally denuded skin at 2-6 h after birth. In skin with normal appearance TEWL was increased 8-fold compared with wild type animals (p < 0.001, n = 6) (Fig 1). In focally denuded skin areas there was a totally disrupted permeability barrier: TEWL was extremely high and out of range for the Tewameter instrument (not shown). In newborn heterozygous mice TEWL was slightly increased compared with the wild type (+21.1%, p < 0.025, n = 5, Fig 1). In parallel with the development of severe scales in adult heterozygous mice basal TEWL was slightly lower than in wild type mice (heterozygotes, 6.88 6 0.74, n = 14; wild type, 8.57 6 0.36, n = 34; ±20%, p < 0.05). By comparing newborn and adult mice we noted that TEWL was slightly increased in adult heterozygous and wild type mice. These results show an impaired basal skin permeability barrier function in newborn homozygous and heterozygous K10 de®cient mice. Delayed permeability barrier repair after experimental barrier disruption, but no changes in stratum corneum integrity (tape stripping) in adult heterozygous K10 de®cient mice Because in heterozygous mice we found a small increase in basal TEWL in the newborns only, we performed functional studies and determined permeability barrier repair after experimental disruption by tape stripping (Zettersten et al, 1998). These studies could only be performed in adult mice because of the small size of newborn mice. The physical forces necessary to disrupt the permeability barrier in adult heterozygous K10 de®cient mice did not differ from those in wild type controls. In both types of animals six to eight tape strips were necessary to obtain a 5±6-fold
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Figure 1. TEWL is signi®cantly increased in homozygous K10 de®cient mice. TEWL was determined in newborn homozygous (skin with normal appearance) and heterozygous K10 de®cient mice as well as in adult heterozygotes compared with wild type controls of the some age [*p < 0.05; newborn, n = 6 (homozygotes), n = 5 (heterozygotes and wild type); adult, n = 14 (heterozygotes), n = 34 (wild type)].
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Figure 3. Signi®cant reduced stratum corneum hydration in newborn homozygous and adult heterozygous mice. Hydration of the stratum corneum on ¯ank skin was determined using the Corneometerq in newborn homozygous mice (in skin with normal appearance) and heterozygous mice and also in adult heterozygous animals [*p < 0.025; newborn, n = 5 (homozygotes), n = 34 (heterozygotes), n = 18 (wild type); adult, n = 42 (heterozygotes) and n = 51 (wild type)].
newborn heterozygous mice hydration was only slightly reduced (54.3 6 1.7 U, n = 34, ±3%, NS). In adult mice, however, in parallel with the progress in scaliness a signi®cant reduction in stratum corneum hydration occurred [adult heterozygotes, 78.3 6 0.8 U; adult wild type, 84.0 6 0.6 U; ±7%, p < 0.005, n = 42 (heterozygotes), n = 51 (wild type)] (Fig 3). Remarkably, hydration in adult animals was higher than in young animals. The results exhibit a reduced skin hydration in newborn homozygous and adult heterozygous K10 de®cient mice compared with wild type mice of the same age.
Figure 2. Reduced capacity in permeability barrier repair in heterozygous K10 de®cient mice. In adult heterozygous K10 de®cient mice and in wild type controls the permeability barrier was experimentally disrupted by tape stripping and barrier repair was monitored at different time points after treatment [*p < 0.0001, n = 5 (heterozygotes and wild type)].
increase in TEWL. Permeability barrier repair in de®cient mice compared with wild type mice, however, was signi®cantly delayed at all time points after treatment (1 h, ±58%; 3 h, ±44%; 5 h, ±38%; 7 h, ±27%; and until 24 h, ±12%; p < 0.05, n = 5 at each time point). The time delay to achieve the same degree in barrier repair (same TEWL) in the de®cient mice compared with wild type was about 3±4 h (Fig 2). At 48 h after treatment there was still a small delay in barrier repair in the K10 de®cient mice (NS, data not shown). These studies reveal a signi®cantly reduced capacity for permeability barrier repair in adult heterozygous K10 de®cient mice. Reduced hydration of the stratum corneum in homozygous and heterozygous K10 de®cient mice Dry skin is a wellknown clinical feature of ichthyosis in humans (Williams, 1983; Traube, 1989). Therefore, we examined stratum corneum hydration in our K10 de®cient mouse model. In newborn mice at the age of 2 h we found a signi®cant reduced hydration of the ¯ank skin in homozygous compared with wild type mice [newborn homozygotes, 42.6 6 5.8 U; newborn wild type, 55.8 6 1.5 U; ±24%, p < 0.025, n = 5 (homozygotes), n = 18 (wild type)]. In
Reduced A-SMase in homozygous and heterozygous K10 de®cient mice We recently showed that the hydrolytic enzyme A-SMase generates ceramides from sphingomyelin with structural functions in the lipid intercellular layers of the stratum corneum permeability barrier (Jensen et al, 1999). In this study we examined epidermal A-SMase activity in K10 de®cient mice. A-SMase activity in homozygous de®cient mice compared with wild type was decreased by 35% [p < 0.005, n = 6 (homozygous), n = 18 (wild type)]. Also, in age-matched heterozygous animals a decrease in the activity of epidermal A-SMase was noted (±32%, p < 0.005, n = 18) (Fig 4a). These ®ndings show a reduced activity of the enzyme ASMase in K10 de®cient mice. Increased N-SMase activity in homozygous and heterozygous K10 de®cient mice Several cell culture studies have revealed the importance of N-SMase in cell signaling (Kolesnik and Golde, 1994; Wiegmann et al, 1994). In addition, we have shown that N-SMase activity is involved in cell signaling for permeability barrier repair in vivo (Kreder et al, 1999). Here we found that the absolute activity for N-SMase in each model was much lower compared with A-SMase (about a thirtieth). N-SMase activity was signi®cantly increased in age-matched homozygous and heterozygous K10 de®cient mice compared with wild type animals (homozygotes, +273%, p < 0.05, n = 6; adult heterozygotes, +232%, p < 0.01, n = 6) (Fig 4b). The results reveal increased N-SMase activity in K10 de®cient mice. DISCUSSION We found that K10 de®ciency in hairless mice leads to profound changes in permeability barrier function. Baseline TEWL in skin with normal appearance of newborn homozygous K10 de®cient mice was increased 8-fold compared with wild type controls. In newborn heterozygous animals baseline TEWL was slightly increased and in adult heterozygotes TEWL was slightly reduced in parallel with the increase in hyperkeratosis (Porter et al, 1996). In functional studies after experi-
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Figure 4. Reduced A-SMase and increased N-SMase in K10 de®cient mice. Epidermal samples from newborn homozygous and heterozygous K10 de®cient mice as well as wild type mice were isolated and A- and N-SMase activity was determined by speci®c enzyme assays: (a) A-SMase (*p < 0.05), n = 6 (homozygotes), n = 18 (heterozygotes and wild type); (b) N-SMase, n = 6, n = 12 (heterozygotes and wild type).
mental barrier disruption by tape stripping, the capacity for barrier repair was reduced in adult heterozygous mice. Barrier repair was signi®cantly delayed at 1, 3, 5, 7, and 24 h after treatment. These results show a relationship between keratin ®lament abnormalities (K10 de®ciency) and permeability barrier function. The recently described mild abnormalities in keratin ®laments in the heterozygous mice lead to a diminished repair capacity only, whereas profound changes in keratin ®laments in homozygous mice (Porter et al, 1996) lead to a signi®cant reduction in baseline barrier function.3 Our results are in accordance with studies of the human disease EHK, where a reduced barrier function is known. These patients show a 2±5fold increase in TEWL (Frost et al, 1968). Also in another type of congenital ichthyosis, lamellar ichthyosis (a nonbullous 3Jensen J-M, Proksch E, Reichelt J, Magin T, Swensson O: Disturbed barrier function and delayed corni®cation in keratin 10 knockout mice. J Invest Dermatol 108: 593, 1997 (abstr.)
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congenital ichthyosis), which involves defects in structural proteins caused by mutations in transglutaminase (Huber et al, 1995), a reduced skin barrier function and abnormal stratum corneum lipid organization have been described (Lavrijsen et al, 1993; 1995; Paige et al, 1994). The reduced permeability barrier function in the K10 de®cient mouse model may be explained by changes in epidermal differentiation and in lipids. It has been described that K10 is tightly bound to the corni®ed envelope (Ming et al, 1994). Reduced expression of K10 leads to changes in the expression of the corni®ed envelope protein involucrin in the de®cient mouse model, as has been shown by Reichelt et al (1999). The corni®ed envelope proteins involucrin, envoplakin, and periplakin are covalently linked to w- hydroxyceramides (Marekov and Steinert, 1998; Nemes et al, 1999), which serve as a scaffold for the attachment of further lipids forming multilamellar intercellular layers crucial for permeability barrier function (Swartzendruber et al, 1989; Downing, 1992). A decreased ratio of ceramides, especially ceramides 1, 3, 4, and 5, to total stratum corneum lipids in the K10 de®cient mouse model has been described recently (Reichelt et al, 1999). Therefore we suggest that the disturbed epidermal differentiation and corni®cation, including changes in keratins, corni®ed envelope proteins, and lipids, is functionally related to impaired permeability barrier homeostasis in K10 de®cient mice. Recently, Elias et al described a normal barrier repair in K10 de®cient heterozygous mice. In contrast to our studies, they used hairy mice that were clipped and shaved prior to barrier disruption (Elias et al, 2000). In our experience these pretreatments lead to irritation of the skin and therefore in¯uence permeability barrier repair. Therefore, we used hairless de®cient mice only. Hydration of the stratum corneum was signi®cantly reduced in newborn homozygous mice at the age of 2-6 h compared with wild type mice. In adult heterozygous mice a signi®cant reduction in hydration compared with wild type animals occurred in parallel with the appearance of an ichthyosis-like scaliness. The K10 de®cient mouse model therefore exhibits dry skin, which is well known by clinical signs and by measurements of stratum corneum hydration in different types of ichthyosis (Johansen et al, 1995; Wang et al, 1997; Ganemo et al, 1999). Our results suggest a relationship between K10 ®lament abnormalities and dry skin conditions. This relationship has also been described in our previous studies in healthy humans with dry and aged dry skin conditions where we found a reduced expression of the differentiation-related keratins K1 and K10 (Engelke et al, 1997). Also, a reduced skin hydration (and a reduced barrier function) accompanied by decreased expression of K1 and K10 has been reported in psoriasis lesional skin (Hagemann and Proksch, 1996). K10 de®ciency is therefore associated with dry skin conditions from different causes. Reduced water content in the stratum corneum of K10 de®cient mice may be explained by pertubations in keratins and lipids. Leveque et al (1987) described a direct role of keratins in water binding: water molecules are bound to the keratin chain up to a water content of 12%. Stratum corneum hydration could also be in¯uenced indirectly by changes in the lipid content shown in the K10 de®cient model. Stratum corneum lipids serve a water-holding function through the formation of lamellar structures within the stratum corneum (Imokawa et al, 1991; Rawlings et al, 1994). Lipids protect water-soluble substances and therefore in¯uence stratum corneum hydration (Middleton, 1968). Also, the water content in¯uences the ordering of stratum corneum lipids (Bouwstra et al, 1991; Pechthold et al, 1998; Schreiner et al, 2000). The reduced stratum corneum hydration in our K10 de®cient mice could therefore be a direct effect related to changes in keratins or an indirect effect related to changes in lipids.
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The role of ®laggrin and ®laggrin degradation to natural moisturizing factors for stratum corneum hydration has been described in several studies (reviewed by Rawlings et al, 1994). Increased staining of ®laggrin has been found for K10 de®cient mice (Fuchs et al, 1992) and for EHK (Ishida-Yamamoto et al, 1994), though hydration is reduced in both cases. Electron microscopy revealed a pertubation of keratin±®laggrin interactions. Instead of normal K1-K10 association, predominantly K6/K16± ®laggrin interactions were observed (Magin, 1998). Besides keratins and ceramides, these altered keratin±®laggrin interactions might be the cause of the reduced skin hydration, to which ®laggrin contributes. Sphingomyelinase, generating ceramide from sphingomyelin, is involved in Il-1a and TNF cell signaling (Kolesnik and Golde, 1994; Wiegmann et al, 1994). The activated keratinocytes in response to epidermal injury, e.g., wound healing and barrier disruption, release Il-1 and TNF (Wood et al, 1992; Nickoloff and Naidu, 1994; Komine et al, 1995; Geilen et al, 1997). We very recently showed the importance of the TNF signaling pathway and SMases generating ceramides for skin barrier repair in normal hairless mice (Jensen et al, 1999). ASMase is colocalized with lipids including sphingomyelin and other hydrolytic enzymes in the lamellar bodies of the upper stratum spinosum and the stratum granulosum. Activation of ASMase is involved in early barrier repair, generating ceramide with structural function (Jensen et al, 1999). In the homozygous and heterozygous K10 de®cient mice in this report we found a decreased A-SMase activity. These results are in accordance with the decreased ratio of ceramides to total lipids in K10 de®cient mice recently described by Reichelt et al (1999). Very recently Uchida et al showed that stratum corneum ceramide 2 and most important ceramide 5 in mouse and human skin are derived by sphingomyelin hydrolysis.4 Our results suggest that the reduced ratio of ceramides to the total amount of lipids is related to a decreased activity in the enzyme A-SMase. The cell-membrane-associated N-SMase activity was signi®cantly increased in homozygous and heterozygous K10 de®cient mice, in contrast to A-SMase activity. But the activity of NSMase was a thirtieth of that of A-SMase. This activity is too low to generate an essential part of the ceramides for structural function in the stratum corneum lipid bilayers. Therefore we recently proposed during our studies on barrier function in normal hairless mice that N-SMase is most probably involved in cell signaling for skin barrier repair (Jensen et al, 1999). The relationship between N-SMase and epidermal proliferation was very recently shown in the FAN-de®cient mouse model, too. De®ciency in the FAN-adapter protein of the TNF receptor p55/N-SMase pathway leads to a decreased epidermal proliferation after stimulation (Kreder et al, 1999). Increase in N-SMase activity as described in this report is most probably related to the known increase in epidermal proliferation in the disease EHK and in K10 de®cient animals (Fuchs et al, 1992; Kanitakis et al, 1993; Bickenbach et al, 1996; Porter et al, 1998). Because of ongoing keratin ®lament abnormalities in the K10 de®cient mice the increase in N-SMase activity and in epidermal proliferation does not lead to normal barrier function or barrier repair. In summary, we found a disrupted permeability barrier in homozygous K10 de®cient mice and a delayed permeability barrier repair in heterozygous K10 de®cient mice. Also, a reduced stratum corneum hydration was noted in both types of de®ciency. The defects are related in part to changes in A- and N-SMase activities generating the ceramides most important for structural and signaling functions in the skin. Our results show 4Uchida Y, Hara M, Nishio H, Inoue S, Ohtuka F, Elias PM, Holleran WM, Hamanaka S: Epidermal sphingomyelins are precursors for speci®c ceramides in the stratum corneum. J Invest Dermatol 114: 878, 2000 (abstr.)
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the crucial role of keratin ®lament expression and organization for permeability barrier function and skin hydration. We would like to thank T. Magin and J. Reichelt, Institute of Molecular Genetics, University of Bonn, Germany, for providing the K10 de®cient mice and for helpful discussions. J.-M. Jensen is a recipient of a grant from the European Academy of Dermatology (EADV and Yamanouchi Europe), and the Deutsche Forschungsgemeinschaft (DFG). This work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 415) given to E. Proksch and S. SchuÈtze.
REFERENCES Bickenbach JR, Greer JM, Bundman DS, Rothnagel JA, Roop DR: Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development. J Invest Dermatol 104:405±410, 1995 Bickenbach JR, Longley MA, Bundman DS, Dominey AM, Bowden PE, Rothnagel JA, Roop DR: A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis. Differentiation 61:129±139, 1996 Bouwstra JA, Gooris GS, van der Spek JA, Bras W: Structural investigations of human stratum corneum by small-angle X-ray scattering. J Invest Dermatol 97:1005±1012, 1991 Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 72:248±254, 1976 Cheng J, Syder AJ, Yu QC, Letai A, Paller AS, Fuchs E: The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-speci®c epidermal keratin genes. Cell 70:811±819, 1992 Chipev CC, Korge BP, Markova N, Bale SJ, DiGiovanna JJ, Compton JG, Steinert PM: A leucine-proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 70:821±828, 1992 DiGiovanna JJ, Bale SJ: Clinical heterogeneity in epidermolytic hyperkeratosis. Arch Dermatol 130:1026±1035, 1994 Downing DT: Lipid and protein structures in the permeability barrier of mammalian epidermis. J Lipid Res 33:301±313, 1992 Ekanayake-Mudiyanselage S, Aschauer H, Schmook FP, Jensen JM, Meingassner JG, Proksch E: Expression of epidermal keratins and the corni®ed envelope protein involucrin is in¯uenced by permeability barrier disruption. J Invest Dermatol 111:517±523, 1998 Elias PM: Epidermal lipids, barrier function, and desquamation. J Invest Dermatol 80:44±49, 1983 Elias PM, Man M-Q, Williams ML, Feingold KR, Magin T: Barrier function in K10 heterozygote knockout mice. J Invest Dermatol 114:396±397, 2000 Engelke M, Jensen JM, Ekanayake-Mudiyanselage S, Proksch E: Effects of xerosis and ageing on epidermal proliferation and differentiation. Br J Dermatol 137:219±225, 1997 Fartasch M, Diepgen TL, Hornstein OP: Are hyperlinear palms and dry skin signs of a concomitant autosomal ichthyosis vulgaris in atopic dermatitis? Acta Derm Venereol (Suppl.)(Stockh) 144:143±145, 1989 Feingold KR: The regulation and role of epidermal lipid synthesis. Adv Lipid Res 24:57±82, 1991 Frost P, Weinstein GD, Bothwell JW, Wildnauer R: Ichthyosiform dermatoses. Arch Derm 98:230±233, 1968 Fuchs E, Esteves RA, Coulombe PA: Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis. Proc Natl Acad Sci (USA) 89:6906±6910, 1992 Ganemo A, Kirtanen M, Vahlquist A: Improved topical treatment of lamellar ichthyosis: a double blind study of four different cream formulations. Br J Dermatol 141:1027±1032, 1999 Geilen CC, Wieder T, Orfanos CF: Ceramide signalling regulatory role in cell proliferation, differentiation and apoptosis in human epidermis. Arch Dermatol Res 289:559±566, 1997 Grif®ths WAAD: Ichthyosis and dry skin. Clin Exp Dermatol 7:551±556, 1982 Hagemann I, Proksch E: Topical treatment by urea reduces epidermal hyperproliferation and induces differentiation in psoriasis. Acta Derm Vernereol (Stockh) 76:353±356, 1996 Hashimoto-Kumasaka K, Takahashi K, Tagami H: Electrical measurement of the water content of the stratum corneum in vivo and in vitro under various conditions: comparison between skin surface hygrometer and corneometer in evaluation of the skin surface hydration state. Acta Derm Venereol (Stockh) 73:335±339, 1993 Huber M, Rettler I, Bernasconi K, et al: Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science 267:525±528, 1995 Imokawa G, Kuno H, Kawai M: Stratum corneum lipids serve as a bound-water modulator. J Invest Dermatol 96:845±851, 1991 Ishida-Yamamoto A, Eady RA, Underwood RA, Dale BA, Holbrook KA: Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis). Br J Dermatol 131:767±779, 1994 Jensen JM, SchuÈtze S, FoÈrl M, KroÈnke M, Proksch E: Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier repair. J Clin Invest 104:1761±1770, 1999 Johansen JD, Ramsing D, Vejlsgaard G, Agner T: Skin barrier properties in patients
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with recessive X-linked ichthyosis. Acta Derm Venereol (Stockh) 75:202±204, 1995 Kanitakis J, Hoyo E, Chouvet B, Thivolet J, Faure M, Claudy A: Keratinocyte proliferation in epidermal keratinocyte disorders evaluated through PCNA/ Cyclin immunolabelling and AgNOR counting. Acta Derm Venereol (Stockh) 73:370±375, 1993 Kolesnik R, Golde DW: The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77:328, 1994 Komine M, Freedberg IM, Blumenberg M: The activated keratinocytes. Acta Derm Venerol (APA) 4:169±173, 1995 Kreder D, Krut O, Adam-Klages S, et al: Impaired neutral sphingomyelinase activation and cutaneous barrier repair in FAN-de®cient mice. EMBO J 18:2472±2479, 1999 Lavrijsen APM, Oestmann E, Hermans J, Bodde HE, Vermeer BJ, Ponec M: Barrier function parameters in various keratinization disorders: transepidermal water loss and vascular response to hexyl nicotinate. Br J Dermatol 129:547±554, 1993 Lavrijsen APM, Bouwstra JA, Gooris GS, Weerheim A, Bodde HE, Ponce M: Reduced skin barrier function parallels abnormal stratum corneum lipid organization in patients with lamellar ichthyosis. J Invest Dermatol 105:619±624, 1995 Leveque JL, Escoubez M, Rasseneur L: Water±keratin interaction in human stratum corneum. Bioeng Skin J 3:227±242, 1987 Magin TM: Lessons from keratin transgenic and knockout mice. Subcell Biochem 31:141±172, 1998 Marekov LN, Steinert PM: Ceramides are bound to structural proteins of the human foreskin epidermal corni®ed cell envelope. J Biol Chem 273:17763±17770, 1998 Middleton JD: The mechanism of water binding in stratum corneum. Br J Derm 80:437±449, 1968 Ming ME, Daryanani HA, Roberts LP, Baden HP, Kvedar JC: Binding of keratin intermediate ®laments (K10) to the corni®ed envelope in mouse epidermis: implications for barrier function. J Invest Dermatol 103:780±784, 1994 Mukherjee S, Gupta AB: A statistical study on in vivo sorption and desorption of water in ichthyosis vulgaris. J Dermatol 21:78±83, 1994 Nemes Z, Marekov LN, Fsus L, Steinert PM: A novel function for transglutaminase 1: attachment of long-chain omega-hydroxyceramides to involucrin by ester bond formation. Proc Natl Acad Sci USA 96:8402±8407, 1999 Nickoloff BJ, Naidu Y: Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. J Am Acad Dermatol 30:535±546, 1994 Paige DG, Morse-Fisher N, Harper JI: Quanti®cation of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyosis. Br J Dermatol 131:23± 27, 1994 Pechthold LA, Abraham W, Potts RO: Characterization of the stratum corneum lipid matrix using ¯uorescence spectroscopy. J Invest Dermatol Symp Proc The 3:105±109, 1998
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Porter RM, Leitgeb S, Melton DW, Swensson O, Eady RAJ, Magin TM: Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis. J Cell Biol 132:925±936, 1996 Porter RM, Reichelt J, Lunny DP, Magin TM, Lane EB: The relationship between hyperproliferation and epidermal thickening in a mouse model for BCIE. J Invest Dermatol 110:951±957, 1998 Proksch E, Feingold KR, Elias PM: Barrier function regulates epidermal DNAsynthesis. J Clin Invest 87:1668±1673, 1991 Rabinowitz LD, Esterly NB: Atopic dermatitis and ichthyosis vulgaris. Pediatr Rev 15:220±226, 1994 Rawlings AV, Scott IR, Harding CR, Bowser PA: Stratum corneum moisturization at the molecular level. J Invest Dermatol 103:731±741, 1994 Reichelt J, DoÈring T, Schnetz E, Fartasch M, Sandhoff K, Magin TM: Normal ultrastructure, but altered stratum corneum lipid and protein composition in a mouse model for epidermolytic hyperkeratosis. J Invest Dermatol 113:329±334, 1999 Roop D: Defects in barrier. Science 267:474±475, 1995 Rothnagel JA, Dominey AM, Dempsey LD, et al: Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis. Science (Wash DC) 257:1128±1130, 1992 Schreiner V, Gooris GS, Pfeiffer S, et al: Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imagining. J Invest Dermatol 114:654±660, 2000 Suga Y, Duncan KO, Heald PW, Roop DR: A novel helix termination mutation in keratin 10 in annular epidermolytic ichthyosis, a variant of bullous congenital ichthyosiform erythroderma. J Invest Dermatol 111:1220±1223, 1998 Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT: Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest Dermatol 92:251±257, 1989 Traube H: The Ichthyosis Springer Verlag, Berlin, 1989, pp 139±144 Wang F, Man MQ, Elias PM: A lipid mixture improves skin hydration in ichthyosis vulgaris. Int J Dermatol 36:876±877, 1997 Wiegmann K, SchuÈtze S, Machleit T, Witte D, KroÈnke M: Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005±1015, 1994 Williams ML: The ichthyoses ± pathogenesis and prenatal diagnosis: a review of recent advances. Pediatr Dermatol 1:1±24, 1983 Wood LC, Jackson SM, Elias PM, Grunfeld C, Feingold KK: Cutaneous barrier pertubation stimulates cytokine production in the epidermis of mice. J Clin Invest 90:482±487, 1992 Yang JM, Yoneda K, Morita E, Imamura S, Nam K, Lee ES, Steinert PM: An alanine to proline mutation in the 1A rod domain of the keratin 10 chain in epidermolytic hyperkeratosis. J Invest Dermatol 109:692±694, 1997 Zettersten E, Man M-Q, Sato J, et al: Recessive X-linked ichthyosis: role of cholesterol-sulfate accumulation in the barrier abnormality. J Invest Dermatol 111:784±790, 1998
Point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) in humans have been shown to be the cause of the congenital ichthyosis epidermolytic hyperkeratosis. Recently, a K10 de®cient mouse model was established serving as a model for epidermolytic hyperkeratosis. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes developed hyperkeratosis with age. To see whether phenotypic abnormalities in the mouse model were associated with changes in skin barrier function and skin water content we studied basal transepidermal water loss and capacity for barrier repair after experimental barrier disruption as well as stratum corneum hydration. Also, we determined the activities of acid and neutral sphingomyelinase key enzymes of the tumor necrosis factor and interleukin-1 signal transduction pathways generating the ceramides most important for epidermal permeability barrier homeostasis. Neonatal homozygotes showed an 8-fold increase in basal transepidermal
water loss compared with wild type controls. Adult heterozygotes exhibited delayed barrier repair after experimental barrier disruption. Stratum corneum hydration was reduced in homozygous and heterozygous mice. Acid sphingomyelinase activity, which is localized in the epidermal lamellar bodies and generates ceramides for extracellular lipid lamellae in the stratum corneum permeability barrier, was reduced in homozygous as well as heterozygous animals. Neutral sphingomyelinase activity, which has a different location and generates ceramides involved in cell signaling, was increased. The reduction in acid sphingomyelinase activity may explain the recently described decreased ratio of ceramides to total lipids in K10 de®cient mice. In summary, our results demonstrate the crucial role of the keratin ®lament for permeability barrier function and stratum corneum hydration. Key words: ceramide/epidermolytic hyperkeratosis/skin barrier function/skin hydration/sphingomyelinase. J Invest Dermatol 115:708±713, 2000
E
Gene targeting at the mouse K10 locus resulted in mice with different genotypes in the homozygotes and heterozygotes, both of which exhibit similarities to speci®c clinical signs of EHK. Homozygotes revealed very fragile skin with small blisters and large areas of erosions and died within several hours after birth. Heterozygotes showed no clinical phenotype at birth, but developed hyperkeratosis and ¯aking in adults. Morphologically in both genotypes, alterations in the programme of epidermal differentiation, abnormal aggregation of cytokeratin intermediate ®laments, and changes in cytokeratin expression were observed. In addition, increased expression of K16 and K17 in the de®cient mice has been described (Porter et al, 1996, 1998). A reduced skin barrier function has been described in EHK and several other types of ichthyosis (Frost et al, 1968; Lavrijsen et al, 1993; 1995). The permeability barrier of the skin is localized in the stratum corneum, consists of corneocytes and lipid-enriched intercellular domains, and develops during epidermal differentiation. The intercellular domains mainly contain cholesterol, free fatty acids, and, most important, ceramides. Several studies by Elias, Feingold and coworkers have shown that an increase in the synthesis of these lipids in the nucleated epidermis occurs after experimental barrier disruption (reviewed by Elias, 1983; Feingold, 1991). These lipids or the respective precursors, glucosylceramides and phospholipids including sphingomyelin, are stored in the epidermal lamellar bodies, extruded, and converted to ceramides
pidermolytic hyperkeratosis (EHK, also known as bullous congenital ichthyosiform erythroderma Brocq) is caused by point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992; Yang et al, 1997; Suga et al, 1998). Clinically EHK includes localized or extensive blistering and erosions in severe cases. Adults show hystrix-like hyperkeratosis. The disease is complicated by erythroderma and secondary infections. Widespread disease can be very debilitating and dis®guring. Histologic features are cytokeratin ®lament aggregation, vacuolization of spinous and granular layer keratinocytes, cytolysis, and alteration of nuclear shape (Traube, 1989; DiGiovanna and Bale, 1994). Manuscript received November 25, 1999; revised June 7, 2000; accepted for publication July 7, 2000. Reprint requests to: Dr. Ehrhardt Proksch, Department of Dermatology, University of Kiel, Schittenhelmstr. 7, 24105 Kiel, Germany. Email: [email protected] 1Part of this study was presented at the 66th Annual Meeting of the Society for Investigative Dermatology (SID), April 23±27, 1997, Washington, DC. 2Present address: The Harvard Skin Disease Research Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Abbreviation: TEWL, transcutaneous/transepidermal water loss. 0022-202X/00/$15.00
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and fatty acids during the transition from the stratum granulosum to the stratum corneum. We recently showed that ceramides for permeability barrier function are generated not only using a synthetic pathway but also by the hydrolytic enzyme acid sphingomyelinase (A-SMase), localized in the endosomal lamellar body compartment (Jensen et al, 1999). In response to acute and chronic permeability barrier disruption A-SMase hydrolyzes sphingomyelin to ceramides. Ceramides are not only structural components for the physical permeability barrier, but they are also important second messengers in cell signaling. We showed recently that the neutral sphingomyelinase (N-SMase), which is localized in the cell membranes, is involved in cell signaling for permeability barrier repair (Jensen et al, 1999; Kreder et al, 1999). A- and NSMase are activated by tumor necrosis factor (TNF) and interleukin-1 (Il-1) during distinctive signal transduction pathways (Kolesnik and Golde, 1994; Wiegmann et al, 1994). We described previously that experimental barrier disruption in hairless mouse skin induces an increase in epidermal proliferation and changes in epidermal differentiation. We found an increase in the expression of the hyperproliferation- and in¯ammationassociated keratins K6, K16, and K17, but also in the suprabasal keratin K10, and a premature expression of involucrin after experimental barrier disruption in hairless mice (Proksch et al, 1991; Ekanayake-Mudiyanselage et al, 1998). Therefore, we and colleagues suggested that keratins and corni®ed envelope proteins are important for permeability barrier function and repair (Bickenbach et al, 1995; Roop, 1995). Reduced hydration of the stratum corneum and aggregated desquamating corneocytes appearing as white scales are the hallmarks of dry skin. Dry skin is common in otherwise healthy persons but is often observed in atopic dermatitis and in different types of ichthyosis (Grif®ths, 1982; Fartasch et al, 1989; Mukherjee and Gupta, 1994; Rabinowitz and Esterly, 1994; Johansen et al, 1995). The cause of dry skin is only known in part: using a commercially available corneometer or hygrometer, a reduced stratum corneum water content was detected in dry skin. Several studies on dry skin and ichthyosis showed changes in lipid content and lipid distribution, especially in ceramides (Paige et al, 1994; Lavrijsen et al, 1995). Also ®laggrin, ®laggrin degradation to natural moisturizing factors, and amino acid content are important for water binding in the stratum corneum (reviewed by Rawlings et al, 1994). In addition, in dry skin of healthy persons we previously found changes in epidermal differentiation including a decreased expression in K1 and K10 (Engelke et al, 1997). In this report we asked if the genetically determined cytokeratin intermediate ®lament abnormalities in the K10 de®cient mouse model result in changes in epidermal permeability barrier homeostasis and repair as well as in skin hydration and A- and N-SMase activity. MATERIALS AND METHODS Biochemicals and radiochemicals Biochemicals were purchased from Sigma-Aldrich Chemie (Munich, Germany). 14C-sphingomyelin (CFA566, 47.0 mCi per mmol) was purchased from Amersham Pharmacia Biotech Europe (Braunschweig, Germany). Experimental protocols Transepidermal water loss (TEWL) and stratum corneum hydration in homozygous and heterozygous mice TEWL and stratum corneum hydration in homozygous, heterozygous, and wild type K10 de®cient mice bred on a hairless background (Porter et al, 1996) (kindly provided by T.M. Magin and J. Reichelt, Bonn, Germany) were determined on ¯ank skin with the Tewameter water analyzer and the Corneometer (both by Courage & Khazaka, Cologne, Germany). Stratum corneum hydration was recorded as units (U) (Hashimoto-Kumasaka et al, 1993). Wild type animals served as control. Measurements in newborn mice were performed 2-6 h after birth because homozygous K10 de®cient mice died within 24 h (Porter et al, 1996). TEWL was studied in skin with normal appearance but also in sites of erosions in homozygous animals. In addition, TEWL and stratum corneum hydration were determined in adult (6±12 wk of age) hetero-
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zygous and wild type mice. Ambient room temperature was 22°C 6 2°C, and humidity was 45% 6 2%. Integrity of the stratum corneum and capacity in skin barrier repair in heterozygous mice Experimental disruption of the permeability barrier was induced in heterozygous hairless K10 de®cient mice (age 6±12 wk) by tape stripping (cellophane tape, Scotch type) until a 5±6-fold increase in TEWL was achieved (Tewameter) (Zettersten et al, 1998). The number of tape strips to disrupt the permeability barrier was counted and barrier recovery was monitored by TEWL at 1, 3, 5, 7, 24, and 48 h after treatment. In vitro assay of A- and N-SMase activity Flank skin samples (about 4 cm2) were excised. The epidermis isolated by incubation of the skin in 10 mM ethylenediamine tetraacetic acid (EDTA) at 37°C for 30 min was homogenized in a glass homogenizator on ice with a Potter S (Braun, Melsungen, Germany) at 500 rpm in 400 ml. For measuring A-SMase, buffer A containing 0.2% Triton-X 100 was used, and for N-SMase, buffer B containing 20 mM HEPES, 10 mM NaF, 2 mM EDTA, 10 mM MgCl2, 30 mM p-nitrophenyl phosphate, 100 mM sodium vanadate, 10 mM bglycerophosphate, 1 mM phenylmethylsulfonyl ¯uoride, 1 mM pepstatin A, leupeptin, antipain (PLA), 750 mM adenosine-5¢-triphosphate, and 0.2% Nonidet P-40 was used. The cell debris and nuclei were removed by lowspeed centrifugation at 1500 3 g for 10 min. The supernatants were used for the in vitro assay as described by Wiegmann et al (1994): 50 mg protein from the supernatants were incubated for 2 h at 37°C in reaction buffer A or B (50 mg ®nal volume), for A- or N-SMase, containing 2.25 ml of [Nmethyl-14C]-sphingomyelin (0.2 mCi per ml, speci®c activity 56.6 mCi per Mol, Amersham) as substrate. The reactions were stopped by addition of 800 ml chloroform:methanol (2:1, vol/vol) and 250 ml of H2O. [14C]phosphorylcholine, produced from [14C]-SM, was extracted from the aqueous phase, identi®ed by thin-layer chromatography, and routinely determined by liquid scintillation counting. Protein determination The protein content in epidermal homogenates was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as the standard (BCA-protein assay). Statistics Statistical signi®cance was determined using the two-tailed Student t test.
RESULTS Impaired basal permeability barrier homeostasis in newborn homozygous and heterozygous K10 de®cient mice As homozygous K10 de®cient mice exhibit severe skin fragility with small blisters and erosions we determined TEWL as a marker of barrier function in skin with normal appearance and in focally denuded skin at 2-6 h after birth. In skin with normal appearance TEWL was increased 8-fold compared with wild type animals (p < 0.001, n = 6) (Fig 1). In focally denuded skin areas there was a totally disrupted permeability barrier: TEWL was extremely high and out of range for the Tewameter instrument (not shown). In newborn heterozygous mice TEWL was slightly increased compared with the wild type (+21.1%, p < 0.025, n = 5, Fig 1). In parallel with the development of severe scales in adult heterozygous mice basal TEWL was slightly lower than in wild type mice (heterozygotes, 6.88 6 0.74, n = 14; wild type, 8.57 6 0.36, n = 34; ±20%, p < 0.05). By comparing newborn and adult mice we noted that TEWL was slightly increased in adult heterozygous and wild type mice. These results show an impaired basal skin permeability barrier function in newborn homozygous and heterozygous K10 de®cient mice. Delayed permeability barrier repair after experimental barrier disruption, but no changes in stratum corneum integrity (tape stripping) in adult heterozygous K10 de®cient mice Because in heterozygous mice we found a small increase in basal TEWL in the newborns only, we performed functional studies and determined permeability barrier repair after experimental disruption by tape stripping (Zettersten et al, 1998). These studies could only be performed in adult mice because of the small size of newborn mice. The physical forces necessary to disrupt the permeability barrier in adult heterozygous K10 de®cient mice did not differ from those in wild type controls. In both types of animals six to eight tape strips were necessary to obtain a 5±6-fold
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Figure 1. TEWL is signi®cantly increased in homozygous K10 de®cient mice. TEWL was determined in newborn homozygous (skin with normal appearance) and heterozygous K10 de®cient mice as well as in adult heterozygotes compared with wild type controls of the some age [*p < 0.05; newborn, n = 6 (homozygotes), n = 5 (heterozygotes and wild type); adult, n = 14 (heterozygotes), n = 34 (wild type)].
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Figure 3. Signi®cant reduced stratum corneum hydration in newborn homozygous and adult heterozygous mice. Hydration of the stratum corneum on ¯ank skin was determined using the Corneometerq in newborn homozygous mice (in skin with normal appearance) and heterozygous mice and also in adult heterozygous animals [*p < 0.025; newborn, n = 5 (homozygotes), n = 34 (heterozygotes), n = 18 (wild type); adult, n = 42 (heterozygotes) and n = 51 (wild type)].
newborn heterozygous mice hydration was only slightly reduced (54.3 6 1.7 U, n = 34, ±3%, NS). In adult mice, however, in parallel with the progress in scaliness a signi®cant reduction in stratum corneum hydration occurred [adult heterozygotes, 78.3 6 0.8 U; adult wild type, 84.0 6 0.6 U; ±7%, p < 0.005, n = 42 (heterozygotes), n = 51 (wild type)] (Fig 3). Remarkably, hydration in adult animals was higher than in young animals. The results exhibit a reduced skin hydration in newborn homozygous and adult heterozygous K10 de®cient mice compared with wild type mice of the same age.
Figure 2. Reduced capacity in permeability barrier repair in heterozygous K10 de®cient mice. In adult heterozygous K10 de®cient mice and in wild type controls the permeability barrier was experimentally disrupted by tape stripping and barrier repair was monitored at different time points after treatment [*p < 0.0001, n = 5 (heterozygotes and wild type)].
increase in TEWL. Permeability barrier repair in de®cient mice compared with wild type mice, however, was signi®cantly delayed at all time points after treatment (1 h, ±58%; 3 h, ±44%; 5 h, ±38%; 7 h, ±27%; and until 24 h, ±12%; p < 0.05, n = 5 at each time point). The time delay to achieve the same degree in barrier repair (same TEWL) in the de®cient mice compared with wild type was about 3±4 h (Fig 2). At 48 h after treatment there was still a small delay in barrier repair in the K10 de®cient mice (NS, data not shown). These studies reveal a signi®cantly reduced capacity for permeability barrier repair in adult heterozygous K10 de®cient mice. Reduced hydration of the stratum corneum in homozygous and heterozygous K10 de®cient mice Dry skin is a wellknown clinical feature of ichthyosis in humans (Williams, 1983; Traube, 1989). Therefore, we examined stratum corneum hydration in our K10 de®cient mouse model. In newborn mice at the age of 2 h we found a signi®cant reduced hydration of the ¯ank skin in homozygous compared with wild type mice [newborn homozygotes, 42.6 6 5.8 U; newborn wild type, 55.8 6 1.5 U; ±24%, p < 0.025, n = 5 (homozygotes), n = 18 (wild type)]. In
Reduced A-SMase in homozygous and heterozygous K10 de®cient mice We recently showed that the hydrolytic enzyme A-SMase generates ceramides from sphingomyelin with structural functions in the lipid intercellular layers of the stratum corneum permeability barrier (Jensen et al, 1999). In this study we examined epidermal A-SMase activity in K10 de®cient mice. A-SMase activity in homozygous de®cient mice compared with wild type was decreased by 35% [p < 0.005, n = 6 (homozygous), n = 18 (wild type)]. Also, in age-matched heterozygous animals a decrease in the activity of epidermal A-SMase was noted (±32%, p < 0.005, n = 18) (Fig 4a). These ®ndings show a reduced activity of the enzyme ASMase in K10 de®cient mice. Increased N-SMase activity in homozygous and heterozygous K10 de®cient mice Several cell culture studies have revealed the importance of N-SMase in cell signaling (Kolesnik and Golde, 1994; Wiegmann et al, 1994). In addition, we have shown that N-SMase activity is involved in cell signaling for permeability barrier repair in vivo (Kreder et al, 1999). Here we found that the absolute activity for N-SMase in each model was much lower compared with A-SMase (about a thirtieth). N-SMase activity was signi®cantly increased in age-matched homozygous and heterozygous K10 de®cient mice compared with wild type animals (homozygotes, +273%, p < 0.05, n = 6; adult heterozygotes, +232%, p < 0.01, n = 6) (Fig 4b). The results reveal increased N-SMase activity in K10 de®cient mice. DISCUSSION We found that K10 de®ciency in hairless mice leads to profound changes in permeability barrier function. Baseline TEWL in skin with normal appearance of newborn homozygous K10 de®cient mice was increased 8-fold compared with wild type controls. In newborn heterozygous animals baseline TEWL was slightly increased and in adult heterozygotes TEWL was slightly reduced in parallel with the increase in hyperkeratosis (Porter et al, 1996). In functional studies after experi-
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Figure 4. Reduced A-SMase and increased N-SMase in K10 de®cient mice. Epidermal samples from newborn homozygous and heterozygous K10 de®cient mice as well as wild type mice were isolated and A- and N-SMase activity was determined by speci®c enzyme assays: (a) A-SMase (*p < 0.05), n = 6 (homozygotes), n = 18 (heterozygotes and wild type); (b) N-SMase, n = 6, n = 12 (heterozygotes and wild type).
mental barrier disruption by tape stripping, the capacity for barrier repair was reduced in adult heterozygous mice. Barrier repair was signi®cantly delayed at 1, 3, 5, 7, and 24 h after treatment. These results show a relationship between keratin ®lament abnormalities (K10 de®ciency) and permeability barrier function. The recently described mild abnormalities in keratin ®laments in the heterozygous mice lead to a diminished repair capacity only, whereas profound changes in keratin ®laments in homozygous mice (Porter et al, 1996) lead to a signi®cant reduction in baseline barrier function.3 Our results are in accordance with studies of the human disease EHK, where a reduced barrier function is known. These patients show a 2±5fold increase in TEWL (Frost et al, 1968). Also in another type of congenital ichthyosis, lamellar ichthyosis (a nonbullous 3Jensen J-M, Proksch E, Reichelt J, Magin T, Swensson O: Disturbed barrier function and delayed corni®cation in keratin 10 knockout mice. J Invest Dermatol 108: 593, 1997 (abstr.)
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congenital ichthyosis), which involves defects in structural proteins caused by mutations in transglutaminase (Huber et al, 1995), a reduced skin barrier function and abnormal stratum corneum lipid organization have been described (Lavrijsen et al, 1993; 1995; Paige et al, 1994). The reduced permeability barrier function in the K10 de®cient mouse model may be explained by changes in epidermal differentiation and in lipids. It has been described that K10 is tightly bound to the corni®ed envelope (Ming et al, 1994). Reduced expression of K10 leads to changes in the expression of the corni®ed envelope protein involucrin in the de®cient mouse model, as has been shown by Reichelt et al (1999). The corni®ed envelope proteins involucrin, envoplakin, and periplakin are covalently linked to w- hydroxyceramides (Marekov and Steinert, 1998; Nemes et al, 1999), which serve as a scaffold for the attachment of further lipids forming multilamellar intercellular layers crucial for permeability barrier function (Swartzendruber et al, 1989; Downing, 1992). A decreased ratio of ceramides, especially ceramides 1, 3, 4, and 5, to total stratum corneum lipids in the K10 de®cient mouse model has been described recently (Reichelt et al, 1999). Therefore we suggest that the disturbed epidermal differentiation and corni®cation, including changes in keratins, corni®ed envelope proteins, and lipids, is functionally related to impaired permeability barrier homeostasis in K10 de®cient mice. Recently, Elias et al described a normal barrier repair in K10 de®cient heterozygous mice. In contrast to our studies, they used hairy mice that were clipped and shaved prior to barrier disruption (Elias et al, 2000). In our experience these pretreatments lead to irritation of the skin and therefore in¯uence permeability barrier repair. Therefore, we used hairless de®cient mice only. Hydration of the stratum corneum was signi®cantly reduced in newborn homozygous mice at the age of 2-6 h compared with wild type mice. In adult heterozygous mice a signi®cant reduction in hydration compared with wild type animals occurred in parallel with the appearance of an ichthyosis-like scaliness. The K10 de®cient mouse model therefore exhibits dry skin, which is well known by clinical signs and by measurements of stratum corneum hydration in different types of ichthyosis (Johansen et al, 1995; Wang et al, 1997; Ganemo et al, 1999). Our results suggest a relationship between K10 ®lament abnormalities and dry skin conditions. This relationship has also been described in our previous studies in healthy humans with dry and aged dry skin conditions where we found a reduced expression of the differentiation-related keratins K1 and K10 (Engelke et al, 1997). Also, a reduced skin hydration (and a reduced barrier function) accompanied by decreased expression of K1 and K10 has been reported in psoriasis lesional skin (Hagemann and Proksch, 1996). K10 de®ciency is therefore associated with dry skin conditions from different causes. Reduced water content in the stratum corneum of K10 de®cient mice may be explained by pertubations in keratins and lipids. Leveque et al (1987) described a direct role of keratins in water binding: water molecules are bound to the keratin chain up to a water content of 12%. Stratum corneum hydration could also be in¯uenced indirectly by changes in the lipid content shown in the K10 de®cient model. Stratum corneum lipids serve a water-holding function through the formation of lamellar structures within the stratum corneum (Imokawa et al, 1991; Rawlings et al, 1994). Lipids protect water-soluble substances and therefore in¯uence stratum corneum hydration (Middleton, 1968). Also, the water content in¯uences the ordering of stratum corneum lipids (Bouwstra et al, 1991; Pechthold et al, 1998; Schreiner et al, 2000). The reduced stratum corneum hydration in our K10 de®cient mice could therefore be a direct effect related to changes in keratins or an indirect effect related to changes in lipids.
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The role of ®laggrin and ®laggrin degradation to natural moisturizing factors for stratum corneum hydration has been described in several studies (reviewed by Rawlings et al, 1994). Increased staining of ®laggrin has been found for K10 de®cient mice (Fuchs et al, 1992) and for EHK (Ishida-Yamamoto et al, 1994), though hydration is reduced in both cases. Electron microscopy revealed a pertubation of keratin±®laggrin interactions. Instead of normal K1-K10 association, predominantly K6/K16± ®laggrin interactions were observed (Magin, 1998). Besides keratins and ceramides, these altered keratin±®laggrin interactions might be the cause of the reduced skin hydration, to which ®laggrin contributes. Sphingomyelinase, generating ceramide from sphingomyelin, is involved in Il-1a and TNF cell signaling (Kolesnik and Golde, 1994; Wiegmann et al, 1994). The activated keratinocytes in response to epidermal injury, e.g., wound healing and barrier disruption, release Il-1 and TNF (Wood et al, 1992; Nickoloff and Naidu, 1994; Komine et al, 1995; Geilen et al, 1997). We very recently showed the importance of the TNF signaling pathway and SMases generating ceramides for skin barrier repair in normal hairless mice (Jensen et al, 1999). ASMase is colocalized with lipids including sphingomyelin and other hydrolytic enzymes in the lamellar bodies of the upper stratum spinosum and the stratum granulosum. Activation of ASMase is involved in early barrier repair, generating ceramide with structural function (Jensen et al, 1999). In the homozygous and heterozygous K10 de®cient mice in this report we found a decreased A-SMase activity. These results are in accordance with the decreased ratio of ceramides to total lipids in K10 de®cient mice recently described by Reichelt et al (1999). Very recently Uchida et al showed that stratum corneum ceramide 2 and most important ceramide 5 in mouse and human skin are derived by sphingomyelin hydrolysis.4 Our results suggest that the reduced ratio of ceramides to the total amount of lipids is related to a decreased activity in the enzyme A-SMase. The cell-membrane-associated N-SMase activity was signi®cantly increased in homozygous and heterozygous K10 de®cient mice, in contrast to A-SMase activity. But the activity of NSMase was a thirtieth of that of A-SMase. This activity is too low to generate an essential part of the ceramides for structural function in the stratum corneum lipid bilayers. Therefore we recently proposed during our studies on barrier function in normal hairless mice that N-SMase is most probably involved in cell signaling for skin barrier repair (Jensen et al, 1999). The relationship between N-SMase and epidermal proliferation was very recently shown in the FAN-de®cient mouse model, too. De®ciency in the FAN-adapter protein of the TNF receptor p55/N-SMase pathway leads to a decreased epidermal proliferation after stimulation (Kreder et al, 1999). Increase in N-SMase activity as described in this report is most probably related to the known increase in epidermal proliferation in the disease EHK and in K10 de®cient animals (Fuchs et al, 1992; Kanitakis et al, 1993; Bickenbach et al, 1996; Porter et al, 1998). Because of ongoing keratin ®lament abnormalities in the K10 de®cient mice the increase in N-SMase activity and in epidermal proliferation does not lead to normal barrier function or barrier repair. In summary, we found a disrupted permeability barrier in homozygous K10 de®cient mice and a delayed permeability barrier repair in heterozygous K10 de®cient mice. Also, a reduced stratum corneum hydration was noted in both types of de®ciency. The defects are related in part to changes in A- and N-SMase activities generating the ceramides most important for structural and signaling functions in the skin. Our results show 4Uchida Y, Hara M, Nishio H, Inoue S, Ohtuka F, Elias PM, Holleran WM, Hamanaka S: Epidermal sphingomyelins are precursors for speci®c ceramides in the stratum corneum. J Invest Dermatol 114: 878, 2000 (abstr.)
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the crucial role of keratin ®lament expression and organization for permeability barrier function and skin hydration. We would like to thank T. Magin and J. Reichelt, Institute of Molecular Genetics, University of Bonn, Germany, for providing the K10 de®cient mice and for helpful discussions. J.-M. Jensen is a recipient of a grant from the European Academy of Dermatology (EADV and Yamanouchi Europe), and the Deutsche Forschungsgemeinschaft (DFG). This work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 415) given to E. Proksch and S. SchuÈtze.
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