Aloxe3 Knockout Mice Reveal a Function of Epidermal Lipoxygenase-3 as Hepoxilin Synthase and Its Pivotal Role in Barrier Formation

ORIGINAL ARTICLE

Aloxe3 Knockout Mice Reveal a Function of Epidermal Lipoxygenase-3 as Hepoxilin Synthase and Its Pivotal Role in Barrier Formation Peter Krieg1, Sabine Rosenberger1, Silvia de Juanes1, Susanne Latzko1, Jin Hou2, Angela Dick2, Ulrich Kloz3, Frank van der Hoeven3, Ingrid Hausser4, Irene Esposito5, Manfred Rauh2,6 and Holm Schneider2,6 Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOXknockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway. Journal of Investigative Dermatology (2013) 133, 172–180; doi:10.1038/jid.2012.250; published online 26 July 2012

INTRODUCTION Autosomal recessive congenital ichthyosis (ARCI) are a clinically and genetically heterogeneous group of severe keratinization disorders with an estimated prevalence of 1 in 100,000 newborns (Oji and Traupe, 2006). Most of the patients are born as collodion babies. Later, the main symptoms are widespread scaling of the skin and varying degrees of erythema. To date, mutations in eight different genes have been identified

1

Genome Modifications and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany; 2Department of Pediatrics, University of Erlangen-Nu¨rnberg, Erlangen, Germany; 3Transgenic Service, German Cancer Research Center, Heidelberg, Germany; 4Department of Dermatology, University Clinic, Heidelberg, Germany and 5Institute of Pathology, Technische Universita¨t Mu¨nchen, Munich, Germany

6

These authors contributed equally to this work.

Correspondence: Peter Krieg, Genome Modifications and Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 280,D-69120 Heidelberg, Germany. E-mail: [email protected] Abbreviations: ARCI, autosomal recessive congenital ichthyosis; CE, cornified envelope; CLE, corneocyte lipid envelope; eLOX-3, epidermal LOX-3; EOS, esterified omega-hydroxyacyl-sphingosine; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HPLC, high-performance liquid chromatography; LOX, lipoxygenase; MS/MS, tandem mass spectrometry; OS, omega-hydroxyacyl-sphingosine; TEWL, transepidermal water loss; TLC, thin-layer chromatography Received 28 April 2012; revised 6 June 2012; accepted 24 June 2012; published online 26 July 2012

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to cause the described phenotype, among them are the lipoxygenase (LOX) genes ALOX12B and ALOXE3. The corresponding proteins have been proposed to be involved in the formation of the permeability barrier of the skin, an important protection against water loss and physical injury. ALOX12B and ALOXE3 encode the lipoxygenases 12RLOX and epidermal LOX-3 (eLOX-3), respectively. Both are members of the epidermal subfamily of mammalian LOX with preferential expression in the skin and several other epithelial tissues (Krieg et al., 2002). LOXs constitute a widespread family of non-heme iron dioxygenases that insert molecular oxygen into polyunsaturated fatty acids, producing highly active lipid mediators (Brash, 1999). 12R-LOX and eLOX-3 differ from all other LOXs by unusual structural and enzymatic features. 12R-LOX represents the only mammalian LOX that forms products with R-chirality, and eLOX-3 functions as a hydroperoxide isomerase by exhibiting only latent dioxygenase activity (Kinzig et al., 1999; Krieg et al., 1999; Yu et al., 2003; Zheng and Brash, 2010). Both enzymes act in sequence to convert fatty acid substrates via Rhydroperoxides to specific epoxyalcohol derivatives. With arachidonic acid as substrate, the 12R-LOX/eLOX-3 pathway results in the production of a specific R-epoxyalcohol of the hepoxilin family, members of which are known to function as signaling molecules (Brash et al., 2007). Recent studies suggested a structural role of the 12R-LOX/eLOX-3 pathway & 2013 The Society for Investigative Dermatology

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Figure 1. Loss of epidermal barrier function in epidermal lipoxygenase-3 (eLOX-3)-deficient mice. (a) Dehydration over time. Data are presented as percentage of initial body weight of wild-type ( þ / þ ), heterozygous ( þ /), and homozygous (/) mutant mice (n ¼ 5 for each genotype). Only homozygous mutants showed a weight loss of B3% per hour. (b) Transepidermal water loss (TEWL) measured on the lateral surface of newborn wild-type ( þ / þ , n ¼ 9), heterozygous ( þ /, n ¼ 18), and homozygous (/, n ¼ 16) mutant mice. (c) Barrier-dependent dye exclusion assay on wild-type and eLOX-3 mutant mice at the ages indicated. In control embryos, a decrease of skin permeability was observed from E16.5 to E18.5 as the barrier is acquired in the dorsal to ventral pattern, whereas eLOX-3-knockout embryos remained completely stained. ****Pp0.0001.

via processing of o-hydroxy acylceramides as a prerequisite for the formation of the corneocyte lipid envelope (CLE) (Zheng et al., 2011). Mutations in ALOX12B or ALOXE3 have been detected in B15% of ARCI patients and represent the second most common cause of ARCI (Eckl et al., 2009; Fischer, 2009). Studies of our group and others demonstrated that such mutations may completely abolish the catalytic activity of the respective LOX enzymes (Eckl et al., 2005; Yu et al., 2005). In previous studies, we and others showed that inactivation of 12R-LOX in mice leads to neonatal death due to a severely impaired permeability barrier function (Epp et al., 2007; Moran et al., 2007). Our studies showed that the defective barrier function of neonatal 12R-LOX-knockout skin is associated with ultrastructural anomalies in the upper granular layer, impaired processing of profilaggrin to filaggrin monomers, and disordered composition of ester-bound ceramide species. The mature 12R-LOX-deficient mouse skin, as analyzed in skin grafts, developed an ichthyosiform phenotype that closely resembled the phenotype observed in ichthyosis patients (de Juanes et al., 2009). In this study, we generated and analyzed Aloxe3-null mice that display a lethal phenotype associated with severe skin barrier dysfunction, which is similar to that observed in 12RLOX-knockout mice. The pivotal role of the 12R-LOX/eLOX3 pathway for the establishment of the epidermal barrier function can be traced back to the oxygenation of linoleatecontaining ceramides, which constitutes an important step in the construction of CLE and barrier formation. Moreover, the data indicate an additional role of eLOX-3 in epidermal eicosanoid signaling.

RESULTS Postnatal death and loss of skin barrier function in mice lacking eLOX-3

For targeting the Aloxe3 gene, we chose a conditional genetargeting approach utilizing the Cre-loxP and flp-FRT systems. A targeting construct was prepared and eLOX-3null mice were generated as described in Supplementary Figure S1a online and under Supplementary Materials and Methods online. The genotypes of the eLOX-3 null mice were determined by PCR and Southern blot analysis (Supplementary Figure S1a–c online). RT-PCR, western blot analysis, and immunofluorescence revealed no expression of eLOX-3 wildtype mRNA and the absence of eLOX-3 protein in eLOX-3/ mice (Supplementary Figure S1d and e online, Figure 3). On the basis of proposed close functional relationship of both enzymes in barrier formation, we expected the eLOX-3deficient mice to show a similar lethal phenotype as observed earlier in12R-LOX-knockout mice (Epp et al., 2007). Whereas heterozygous animals (eLOX-3 þ /) were phenotypically indistinguishable from wild-type mice and reproduced normally, eLOX-3/ mice were born at the expected Mendelian ratio, but died within 5–12 hours after birth owing to defective skin barrier function. Weighing revealed that eLOX-3/ mice lost around 2.5% of body weight per hour, whereas their heterozygous and wild-type littermates maintained weight (Figure 1a). Dehydration through the skin was assessed by measuring transepidermal water loss (TEWL). Homozygous mutant mice showed approximately 4fold increased TEWL compared with control littermates (Figure 1b). www.jidonline.org

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We assessed outside-in barrier acquisition during embryonic development by performing a whole-mount skin toluidine blue penetration assay (Hardman et al., 1998). In wild-type animals, the staining reflects a dorsal to ventral pattern of decreasing skin permeability with proceeding barrier development between embryonic day 16.5 and 17.5, coming to a halt on embryonic day 18.5 when the skin has become completely impermeable. Skin of homozygous mutant mice, in contrast, remained permeable as indicated by intense staining (Figure 1c). We also investigated the barrier function of tight junctions by injecting biotin subcutaneously into newborn mice and measuring its diffusion into the epidermis. Prevention of diffusion was observed in the upper granular cell layer of the skin of both homozygous mutant mice and wild-type mice, indicating that eLOX-3 deficiency did not affect the barrier function of tight junctions (not shown).

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The skin of eLOX-3/ newborns closely resembled the phenotype observed earlier in 12R-LOX-knockout mice. The stratum corneum appeared to be more tightly packed than in control skin (Figure 2a and b), whereas no significant differences were observed in the basal, spinous, or granular layers. At the ultrastructural level, electron microscopy revealed hyperkeratosis of the skin of eLOX-3-deficient mice as indicated by the presence of very compact horny layers (Figure 2d). Furthermore, we noticed an increased occurrence of transition cells underlying the stratum corneum and occasionally irregular tubulus-like structures in the horny layers, indicating defects or delay of the desquamation process in the skin of eLOX-3-knockout animals (Figure 2e and f). The characteristic vacuole-like structures observed earlier in the granular layers of neonatal 12RLOX-deficient skin, however, could not be detected in the skin of eLOX-3/ mice. Epidermal differentiation

To unveil defects in epidermal differentiation, we studied the distribution as well as the expression levels of differentiation markers. Immunofluorescence staining was performed on the back skin of newborn mice using the following markers: keratin 14, keratin 10, keratin 6, filaggrin, involucrin, loricrin, and repetin. No obvious alterations of the expression pattern could be detected for keratins 10 and 14, involucrin, or repetin. Keratin 6 staining, however, which was found to be restricted to hair follicles in wild-type mice, was strongly increased in basal keratinocytes of eLOX-3-deficient animals (Figure 3). The nearnormal expression of the early and late differentiation markers was confirmed by western blot analysis, which also indicated that processing of profilaggrin to filaggrin monomers was not disturbed in eLOX-3-knockout skin—in contrast to neonatal 12R-LOX-deficient skin that completely lacks mature filaggrin monomers (not shown). In summary, except for keratin 6 none of the early, intermediate, and late differentiation markers analyzed were altered in eLOX-3deficient mice. 174

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Figure 2. Structural skin abnormalities of epidermal lipoxygenase-3 (eLOX-3)-deficient mice. Representative skin sections from (a) newborn wild-type and (b) eLOX-3/ littermates stained with hematoxylin–eosin. The staining shows a more tightly packed stratum cormeum but otherwise normally stratified organization of keratinocytes in knockout mouse skin. Electron microscopy of skin from (c) control and (d–f) eLOX-3/ neonates. (d) Stratum corneum of mutant skin. Note the markedly piled-up cornified cells. (e) Accumulation of transition cells (asterisk) that underlay the stratum corneum. (f) Irregular tubular-like structures in the horny lamellae (arrow) may indicate defects or delay of the desquamation process. Bars ¼ 100 mm for a and b; 2,500 nm for c–e; and 250 nm for f.

Altered lipid composition

As lipid analysis of the skin from 12R-LOX-deficient mice had revealed a significant decrease of protein-bound o-hydroxyceramides, whereas other lipid components had remained unchanged, we assumed that loss of these covalently bound ceramide species, which are the major constituents of the CLE and thus essential for permeability barrier function, would be mainly responsible for epidermal barrier failure. Bringing forward a new model of the assumed role of the 12R-LOX/eLOX-3 pathway in skin barrier function, Brash and colleagues recently suggested that oxygenation of the linoleate-containing esterified omega-hydroxyacyl-sphingosine (EOS) ceramides by 12R-LOX and eLOX-3 is required to facilitate hydrolysis and subsequent covalent linkage of the o-hydroxyceramides to proteins of the cornified envelope

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Figure 3. Detection of epidermal lipoxygenase-3 (eLOX-3), 12R-LOX, and epidermal differentiation markers in the skin from control and eLOX-3-deficient mice. Skin sections from control and eLOX-3/ mice were immunostained for the indicated proteins. Nuclei are counterstained with Hoechst 33258. Specific staining (arrows) for eLOX-3 is observed in keratinocytes of the granular layers of wild-type mice but absent in mutants, whereas a similar staining pattern for 12R-LOX is unaffected. Note that except for keratin 6 (K6) where staining is seen in basal layers of mutant skin but restricted to hair follicles in wild-type mice, keratin 14 and 10 (K14, K10), filaggrin (Flg), involucrin (Ivl), and repetin (Rptn) staining is found at the typical localizations and at similar intensities in control and mutant skin. s.b., stratum basale; s.c., stratum corneum; s.g., stratum granulosum; s.s., stratum spinosum. Bars ¼ 20 mm.

(CE) (Zheng et al., 2011). To test this hypothesis in our eLOX3-knockout model, we analyzed the ceramide composition of the epidermis of control and mutant mice by thinlayer chromatography (TLC) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). TLC analysis revealed no differences in the levels of free lipids (Figure 4a), whereas the subfractions of ester-bound ceramides differed significantly between control and knockout mice: they were strongly reduced in eLOX-3-deficient epidermis but completely lost in 12R-LOX-knockout mice (Figure 4b). We next evaluated the total amount of ohydroxyacyl-sphingosine ceramides Cer[OS (18)] by HPLCMS/MS (Figure 4c). Both in eLOX-3/ and 12R-LOX/ mice omega-hydroxyacyl-sphingosine (OS) formation was clearly lower than in the wild type. However, 12R-LOX-deficient animals showed a more distinct reduction. Cer[OS (18)]

levels of heterozygous animals did not differ significantly from those of wild-type mice. The observed difference between eLOX-3/ and 12R-LOX/ mice became more evident when looking at different ceramide species that vary in the length of their long-chain fatty acid (Figure 4d). In eLOX-3/ mice, levels of ceramides with fatty acids containing 30 or 32 carbon atoms were not significantly lower than in heterozygous or control animals, whereas 12RLOX-deficient mice showed a marked reduction of all OS species investigated. To clarify the impact of eLOX-3 on eicosanoid signaling, relevant free lipids of the epidermis of newborn mutant mice were further investigated by HPLC-MS/MS (Figure 4e and f). Knockout of eLOX-3 had no detectable influence on the concentrations of the linoleic acid–derived LOX products, 9hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic www.jidonline.org

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Figure 4. Lipid analysis of the epidermis of epidermal lipoxygenase-3/ (eLOX-3/) and 12R-LOX/ mice. (a, b) Thin-layer chromatography (TLC) analysis of free and ester-linked lipids from epidermis samples of newborn eLOX-3- or 12R-LOX-deficient and control mice. The ceramide nomenclatures used in this figure are as suggested previously (Motta et al., 1993). The positions of comigrating standards are indicated. (c, d) Protein-bound ceramides were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Results were normalized to epidermal tissue weight and displayed in relation to the wild-type mice. (c) Peak areas of ceramide species were combined and normalized to epidermal weight. (d) Amounts of individual OS species that differ in the length of the fatty acid attached. (e, f) Levels of free LOX products in the epidermis of eLOX-3/ mice. (e) Relative amounts of different linoleic acid– and arachidonic acid–derived LOX products were determined by HPLC-MS/MS. The results were normalized to epidermal tissue weight. (f) Epidermis samples were further analyzed by chiral HPLC, which indicated that 12-hydroxyeicosatetraenoic acid (12-HETE) was exclusively in S-configuration. AS, ceramide AS; Chol, cholesterol; c.p.s, counts per second; HODE, hydroxyoctadecadienoic acid; NS, ceramide NS; OS, ceramide OS. **Pp0.01; ***Pp0.001.

acid, and the arachidonic acid–derived LOX product, 15hydroxyeicosatetraenoic acid (15-HETE), whereas higher amounts of 12-HETE were detected in eLOX-3/ epidermis than in the samples from heterozygous or wild-type mice (Figure 4e). As mouse 12R-LOX does not metabolize free arachidonic acid, but only esterified substrates (Siebert et al., 176

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2001), we speculated that 12-HETE detected in mouse skin is not a 12R-LOX product. Indeed, chiral analysis revealed this to be exclusively 12(S)-HETE, indicating that loss of eLOX-3 activity results in an accumulation of this eicosanoid derived from the 12(S)-LOX pathway (Figure 4f). The 12-LOX pathway is bifurcated at the level of its primary product

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Figure 5. Dual signaling and structural functions of epidermal lipoxygenase-3 (eLOX-3) in the mouse epidermis. Left panel: involvement of eLOX-3 in eicosanoid signaling. 12S-hydroperoxyeicosatetraenoic acid (12S-HPETE) generated via the 12S-LOX pathway is converted by eLOX-3 to hepoxilin A3 and hepoxilin B3, which are further metabolized to the corresponding trioxilins. Right panel: involvement of eLOX-3 in ceramide processing. The linoleate moiety of esterified omega-hydroxyacyl-sphingosine (EOS) ceramide is oxidized by 12R-LOX to the 9R-hydroperoxide derivative that is subsequently converted by eLOX3 to a 9R,10R epoxy-13R-hydroxy-epoxyalcohol derivative. LOX-mediated oxygenation of EOS is required to facilitate hydrolysis and subsequent covalent linkage of the o-hydroxyceramides to proteins of the cornified envelope (CE). The covalent coupling of OS ceramides as major constituents of the corneocyte lipid envelope (CLE) is an important step in establishing the water barrier. 9R(10)R, 13R-EpHOE, 9R(10R)-epoxy-13R-hydroxy-octadecaenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid.

12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can either be reduced to 12-HETE or converted to hepoxilins. Metabolites of this pathway such as hepoxilin B3, trioxilin A3, and trioxilin B3 were found to be strongly reduced in eLOX-3/ mice (Figure 6a). In 12R-LOX-deficient epidermis, in contrast, no alterations in the levels of hepoxilin metabolites were detected (data not shown). This indicates an important role of eLOX-3 in the respective hepoxilin signaling cascade and reveals an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase (Figure 5). DISCUSSION Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 are among the most frequent causes of ARCI. Knockout of these LOX genes in the mouse models described here and in previous reports confirm a close functional relationship of the corresponding LOX enzymes, 12R-LOX and eLOX-3, and their essential role in skin barrier formation. In both models, the mice die soon after birth owing to severe TEWL. Our data show that eLOX-3-deficient mice exhibit similar but somewhat less severe barrier defects than reported

earlier for 12R-LOX-knockout mice (Epp et al., 2007), indicated by weight loss (2.5% per hour vs 10%), increase in TEWL (4-fold vs 8-fold), and a maximum life span of about 12 vs 3 hours. The typical histological phenotype of skin from ichthyosis patients is characterized by variable epidermal hyperplasia and marked hyperkeratosis, and is thought to result from a physical compensation for the defective cutaneous permeability barrier required for survival in a terrestrial environment. The macroscopic appearance of both 12RLOX/ and eLOX-3/ mouse skin resembles that of mutant human skin albeit at different degrees. Indeed, previous skin grafting studies confirmed that matured 12R-LOXdeficient skin developed an ichthyosiform phenotype similar to that seen in severe ichthyosis (de Juanes et al., 2009). Although our electron microscopy studies revealed less pronounced structural abnormalities in the skin of eLOX-3deficient mice than in 12R-LOX/ skin, they clearly indicated the onset of compensatory hyperkeratosis and defects or delay of the desquamation process. Similarly, ectopic interfollicular expression of keratin 6 in neonatal www.jidonline.org

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eLOX-3/ skin as shown by immunofluorescence may be indicative of hyperproliferation. This study provides insights into the mechanisms by which loss of eLOX-3 function might affect epidermal barrier and skin homeostasis. Unlike all other members of the LOX family, eLOX-3 displays only latent dioxygenase activity but exhibits a dominant hydroperoxide isomerase activity that converts fatty acid hydroperoxides, the usual LOX products— preferentially products of 12R-LOX—to epoxyalcohols and ketones. Although initially a function of the 12R-LOX/eLOX-3 enzyme sequence in eicosanoid signaling was hypothesized, a recent study linked the 12R-LOX/eLOX-3 pathway to skin ceramide processing and consequently to the formation of the CLE (Zheng et al., 2011). The study showed that linoleatecontaining EOS ceramide is a natural substrate for the 12R-LOX/eLOX-3 pathway being oxygenated by consecutive actions of 12R-LOX and eLOX-3 to a 9R-hydroperoxideand a corresponding 9R,10R-epoxy-13R-hydroxy-derivative. The model suggests that the LOX-catalyzed oxygenation facilitates hydrolysis of the oxidized linoleate moiety resulting in a free o-hydroxyl on the ceramides for coupling to the cross-linked proteins of the CE. Cross-linked OS ceramides and o-hydroxy very long-chain fatty acids are the major constituents of the CLE that surrounds the corneocyte, and the covalent coupling is an important step in sealing the water barrier. The results of our study are in line with the model of Zheng et al. and provide further evidence for a consecutive oxygenation of the linoleate esterified in EOS by 12R-LOX and eLOX-3. Lipid analysis showed a significant decrease of covalently bound OS ceramide species in eLOX-3-deficient skin, with a reduction of the total amount of bound ceramides to half of the wild-type level, whereas in 12R-LOX-deficient mice a reduction to less than 10% of the wild-type level was observed. The less pronounced loss of ester-bound ceramides in eLOX-3/ mice may be explained by a 12R-LOXmediated partial oxidation of the linoleate moiety to a 9Rhydroperoxide derivative, which may permit a reduced hydrolysis of the linoleate and subsequent covalent linkage of resultant o-hydroxy ceramides, whereas interruption of the first oxidation step by 12R-LOX deficiency completely prevents hydrolysis of the EOS ceramides. The loss of bound ceramides is accompanied by barrier defects of increasing severity in the different mouse models, indicating that loss of ceramide binding may be the main cause of epidermal barrier failure in the mutant mice. It remains to be elucidated whether the free oxygenated cleavage products may have signaling functions in barrier formation or other aspects of skin physiology beyond those hypothesized recently (Brash et al., 2007). Analysis of the free oxygenated fatty acid metabolites furthermore revealed strongly reduced levels of different hepoxilin metabolites (the trihydroxy hydrolysis product of the unstable hepoxilin A3 as well as hepoxilin B3 and its corresponding triols) in eLOX-3-deficient skin, indicating an additional function of eLOX-3 as a hepoxilin synthase in mammalian skin. We report on the presence of hepoxilin and trioxilin in mouse skin, which to our knowledge is previously 178

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unreported. Anton and Vila (2000) previously reported elevated levels of endogenous hepoxilins and trioxilins in human psoriatic scales and the biosynthesis of hepoxilin-type products and their triol derivatives in isolated human epidermal fragments (Anton et al., 1998). Although the exact steric identification of the hepoxilin metabolites in mouse skin remains to be established, the hepoxilin B product had the same gas chromatography–mass spectrometry characteristics as the synthetic standard of 10R-hydroxy-11S,12S-hepoxilin B3, which is the product of eLOX-3-catalyzed transformation of 12S-HPETE (Yu et al., 2003). Furthermore, reduction of hepoxilin metabolite levels in eLOX-3-deficient epidermis was accompanied by increased levels of 12-HETE, which was shown by chiral analysis to be exclusively in the S-configuration. Given the aforementioned inactivity of mouse 12R-LOX toward free arachidonic acid, we conclude that the hepoxilin metabolites are derived from 12S-HPETE formed by platelet-type 12S-LOX and/or epidermal 12S-LOX, both of which are expressed in the mouse epidermis (Heidt et al., 2000). Hepoxilins and their triol derivatives have been shown to have a role in a variety of physiological processes, including inflammation and neurotransmission. Most functions appear to be mediated by influencing intracellular calcium concentrations (Pace-Asciak, 2009). In the skin, various potential mechanisms of action have been discussed for these metabolites including effects on a putative membrane receptor, ichthyin (NIPAL4), implicated through genetic studies in ichthyosis patients or through activation of PPAR receptors (Lefevre et al., 2004; Yu et al., 2007). Further research is required to determine the exact mode of action and the physiological role of hepoxilin-type metabolites in the epidermis. Taken together, our data indicate signaling and structural functions of eLOX-3 in the epidermis, as summarized in Figure 5. In addition to its function in the skin, eLOX-3 has also been proposed to have a role in adipose tissue (Fu¨rstenberger et al., 2007). This view is supported by our previous studies demonstrating that eLOX-3 is critically involved in adipocyte differentiation in vitro. The data suggest that eLOX-3, in synergistic action with xanthine oxidoreductase, produces hepoxilins that function as endogenous PPARg activators capable of promoting early steps in the conversion of preadipocytes into adipocytes (Hallenborg et al., 2010). In this study, histological examination of homozygous and heterozygous eLOX-3-knockout mice showed normal organ development compared with control mice (not shown). Furthermore, based on light microscopic inspection, no alterations in brown and white adipose tissues could be detected in neonatal mice, suggesting that eLOX-3 deficiency had no effect on fetal adipose tissue development. A possible role of eLOX-3 in adipose tissue in vivo at later developmental stages and in metabolism under distinct physiological conditions, however, remains to be elucidated. The availability of the floxed eLOX-3 mice generated in this study will help establish conditional knockout models with inducible tissue-specific eLOX-3 inactivation for further

P Krieg et al. Aloxe3 Knockout Mice

studies of the physiological role of eLOX-3 in the skin and adipose tissue. In conclusion, this study provides evidence for a structural role of the 12R-LOX/eLOX-3 pathway in skin barrier formation where LOX-catalyzed oxygenation of the linoleate moiety of ceramides constitutes an indispensable step for the covalent linkage of hydroxyl ceramides to proteins of the CE and subsequently for the formation of the CLE. Furthermore, we unveiled an additional function of eLOX-3 linked to its hepoxilin synthase activity and to the 12S-LOX pathway. The impact of this hepoxilin pathway on skin physiology remains to be elucidated. MATERIALS AND METHODS Generation and maintenance of eLOX-3-deficient mice The eLOX-3-deficient mouse line was generated by targeted gene disruptionas described in Supplementary Materials and Methods online. The animal experiments were conducted in accordance with the guidelines of the Arbeitsgemeinschaft der Tierschutzbeauftragten in Baden-Wu¨ rttemberg (Officials for Animal Welfare) and were approved by the Regierungspra¨ sidium Karlsruhe, Germany (Az. G-46/06).

Antibodies

Histological and immunofluorescence analysis For light microscopic observation, samples were fixed with 4% formalin in PBS for 24 hours, dehydrated in 70% ethanol, and then embedded in paraffin. Five-micrometer sections were mounted on slides, dewaxed, rehydrated, and stained with hematoxylin–eosin. Sections were embedded in mounting medium (DakoCytomation, Hamburg, Germany) and examined with an Axioskop 2 with a Zeiss 25  /0.8 Plan-Neofluar objective connected to an Axiocam camera using the Axiovision software (release 3.2; Zeiss, Go¨ttingen, Germany). For immunofluorescence microscopy, 3-mm cryosections were fixed in acetone for 10 minutes at 20 1C, permeabilized with 0.5% Triton X-100 in PBS, blocked with 1% BSA in PBS for 20 minutes, and incubated with the primary antibody for 1 hour. After washing three times (10 minutes each) with PBS, samples were incubated with a fluorescent dye–labeled secondary antibody and Hoechst 33258 for 30 minutes, and washed as described above. Images were acquired using a LSM 700 confocal microscope with an EC Plan-Neofluar 40  /1.30 Oil DIC objective lens (Zeiss), the software ZEN 2009 (v5.5.0.375; scan parameters 1,024  1,024 Pixel, speed 2, line average 2), and Adobe Photoshop CS3.

Functional analyses of the epidermal barrier To determine the rate of fluid loss, newborn animals were separated from their mothers and put on a heating pad set at 37 1C. Body weight was monitored every 30 minutes until homozygous mutant mice died. The rate of TEWL from the skin of newborn mice was determined with a Tewameter (Courage and Khazaka, Cologne, Germany).

Rabbit polyclonal antipeptide antibodies detecting 12R-LOX and eLOX-3, and mouse anti-12R-LOX mAb, have been described earlier (Epp et al., 2007). Rat anti-eLOX-3 mAbs were raised using His-tagged eLOX-3 protein as immunogen. Other primary antibodies used were goat anti-GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-keratin 14, rabbit anti-keratin 10, rabbit anti-keratin 6, rabbit anti-filaggrin, rabbit anti-involucrin (all Covance, Princeton, NJ), and rabbit anti-repetin (gift from D Hohl). Secondary antibodies included Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, San Diego, CA), Alexa Fluor 488 goat anti-rat IgG (Invitrogen), CY3 goat anti-rabbit IgG (Jackson ImmunoResearch, Suffolk, UK), and horseradish peroxidase–conjugated anti-rabbit IgG (BD Biosciences, Palo Alto, CA).

The developmental stage of mouse embryos was determined based on the assumption that fertilization occurred in the middle of the dark cycle the day before plugs were identified. The embryos were subjected to methanol dehydration and subsequent rehydration as described previously (Hardman et al., 1998), washed with PBS for 1 minute, and stained in 0.1% toluidine blue O/PBS for 30 minutes. After decoloration in PBS for 15 minutes, the embryos were photographed.

Epidermal protein extraction and western blot analysis

Electron microscopy

Trunk epidermis of newborn mice was separated mechanically from the dermis following submersion in hot phosphate-buffered saline (PBS) (56 1C) for 30 seconds and snap-frozen in liquid nitrogen. Epidermal tissue was disintegrated for 30 seconds in a ball mill (Micro-Dismembrator S, Sartorius, Go¨ttingen, Germany) at liquid nitrogen temperature and then homogenized in lysis buffer containing 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, 10% glycerol, 150 mM NaCl, 50 mM Tris (pH 7.4), and protein inhibitors (1 mM phenylmethylsulfonylfluorid, aprotinin 1 mg ml1 and leupeptin 1 mg ml1). Western blot analysis was performed as previously described (Epp et al., 2007).

All specimens were fixed in a solution of 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for at least 2 hours at room temperature, cut into pieces of B1 mm3, washed in buffer, postfixed for 1 hour at 4 1C in 1% osmium tetroxide, rinsed with water, dehydrated through graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin (glycidether 100). Ultrathin sections were treated with uranyl acetate and lead citrate, and then examined with an electron microscope (Philips EM 400, Eindhoven, The Netherlands).

Pathohistological evaluation Neonatal mice were killed by decapitation, fixed in 4% paraformaldehyde, and embedded in paraffin blocks for histological evaluation. One-micrometer sections were cut and stained with hematoxylin–eosin. Images of the sections were taken at  20 magnification with an automated slide scan microscope (dotSlide System, Olympus, Hamburg, Germany).

Toluidine blue staining of mouse embryos

Lipid analysis For the extraction of epidermal lipids and eicosanoids, epidermis samples were dissected and snap-frozen in liquid nitrogen. Sphingolipids and eicosanoids were extracted according to Zheng et al. (2011). High-performance thin-layer chromatography separation of lipids, HPLC-MS/MS, and LC-MS/MS analysis were performed as described earlier (Deems et al., 2007; Gelhaus et al., 2011; Jennemann et al., 2012) and in Supplementary Materials and Methods online. www.jidonline.org

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CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS

Hardman MJ, Sisi P, Banbury DN et al. (1998) Patterned acquisition of skin barrier function during development. Development 125:1541–52

We thank A Brash for generously providing ceramide standards and G Fu¨rstenberger for critical comments on the manuscript. Technical support by the DKFZ Light Microscopy Facility is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft (KR 905/ 6-2, KR 905/7-1, and SCHN 569/4-1).

Heidt M, Fu¨rstenberger G, Vogel S et al. (2000) Diversity of mouse lipoxygenases: identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern. Lipids 35:701–7

SUPPLEMENTARY MATERIAL

Kinzig A, Heidt M, Fu¨rstenberger G et al. (1999) cDNA cloning, genomic structure and chromosomal localization of a novel murine epidermistype lipoxygenase. Genomics 58:158–64

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

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