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Correspondence to Peter Krieg: p.krieg{at}dkfz.de
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This study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.
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50% amino acid identity. Their differentiation-dependent expression pattern in epithelial tissues suggests a common physiological role in the regulation of proliferation and differentiation of epithelial cells, especially keratinocytes. The epidermal 12R-LOX and eLOX-3 differ from all other mammalian LOX in their unique structural and enzymatic features (Boeglin et al., 1998; Krieg et al., 1999; Kinzig et al., 1999). Both proteins contain an extra domain located at the surface of the catalytic subunit. 12R-LOX represents the only mammalian LOX that forms products with R-chirality, and, unlike all other LOX, eLOX-3 does not exhibit dioxygenase activity, but functions as a hydroperoxide isomerase (Yu et al., 2003). Both enzymes act in sequence to convert arachidonic acid via 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) to the corresponding hepoxilin-like epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosatrienoic acid. This sequence has been hypothesized to be part of a novel LOX pathway in skin that plays an important role in terminal differentiation (Jobard et al., 2002; Yu et al., 2003).
Recent genetic studies have identified mutations in the coding regions of 12R-LOX and eLOX-3 genes in patients with autosomal recessive congenital ichthyosis (ARCI), linking for the first time mutations of a LOX gene to the development of a disease (Jobard et al., 2002; Eckl et al., 2005). ARCI is a clinically and genetically heterogeneous group of skin disorders that is associated with hyperkeratosis and impaired skin barrier functions (Traupe, 1989). We and others recently showed that the point mutations found in the LOX genes of the ARCI patients completely eliminated the catalytic activity of the LOX enzymes, indicating that mutational inactivation of either 12R-LOX or eLOX-3 is causally linked to the ARCI phenotype (Eckl et al., 2005; Yu et al., 2005).
To investigate the physiological role of 12R-LOX and to analyze the molecular mechanisms that underlie the ichthyosiform skin phenotype, we developed mice with targeted inactivation of the 12R-LOX gene. Examination of the resulting phenotype has revealed a crucial role of 12R-LOX in the development of epidermal barrier function, demonstrating for the first time an indispensable function of a LOX isoform for postnatal survival of mice.
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30% of their body weight within 3 h, whereas their heterozygous and wild-type littermates maintained their weight (Fig. 3 B). Transepidermal water loss (TEWL) of homozygous mutant mice was increased by a factor of 8 compared with wild-type and heterozygous littermates (Fig. 3 C). Thus, the lethal phenotype of the 12R-LOX–deficient mice most likely resulted from water loss as a result of impaired epidermal barrier function. Barrier formation that starts around E16 in a patterned fashion (Hardman et al., 1998) was measured with a whole-mount skin toluidine blue penetration assay. In wild-type animals, the staining pattern reflects the decrease of skin permeability from embryonic day (E) 16.5 to E17.5, when the barrier development proceeds in a dorsal to ventral pattern, up to E18.5, which is when skin has become completely impermeable. Skin of homozygous mutant mice, in contrast, remained permeable, as indicated by intense staining (Fig. 4 A). We then assessed the permeability of the newborn epidermal barrier from outside using the fluorescent dye Lucifer yellow. In skin of 12R-LOX–deficient mice, the dye was found to penetrate throughout the stratum corneum, whereas it was retained in the very top layers in the skin of wild-type mice (Fig. 4 B). These findings clearly indicate that both the inside-out and the outside-in water barrier function were severely affected in the epidermis of 12R-LOX–deficient mice. We also assessed the barrier function of tight junctions by injecting newborn mice subcutaneously with biotin and measuring its diffusion into the epidermis. Prevention of diffusion was observed in the upper granular cells of the skin of homozygous mutant mice, as well as in wild-type mice, indicating that 12R-LOX deficiency did not affect the barrier function of tight junctions (unpublished data).
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-hydroxyceramides and -hydroxy-fatty acids, called the lipid envelope. The exact function of the lipid envelope still remains unclear, but there is evidence that interactions of protein-bound lipids with free intercellular lipids contribute to the patterned organization of the lamellae seen in EM (Madison, 2003). It has been shown that alterations in lipid composition of free or protein-bound lipids impair barrier function of the skin and lead to an increased TEWL (Meguro et al., 2000; Macheleidt et al., 2002). We thus determined the levels of these lipids in the skin of wild-type and mutant mice. The levels of total fatty acids, cholesterol, and total free ceramides were not substantially different between control and 12R-LOX–deficient mice (not depicted). In the free ceramide fraction, we found mainly ceramide EOS, NS, and NP, as well as two ceramide AS species (Fig. 9).
These results are in accordance with previously published results (Doering et al., 1999, 2002). However, in the protein-bound fraction, five different species could be detected on HPTLC (B1–5). The exact identities of these species still have to be elucidated, but B4 is possibly ceramide OS. Significant differences were found in the subfractions of ester-bound lipids between wild-type and knockout mice. Whereas species B1 was found significantly increased, three other species (B2, B4, and B5) were almost completely absent in knockout epidermis (Fig. 9).
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The epidermis is a self-renewing stratified epithelium that serves as a protective barrier against mechanical, chemical, and biological insults; it is also a water-impermeable barrier that prevents excessive loss of body fluids. This function is critical for the survival of all terrestrial vertebrates and is established during late embryonic development. Identification of the molecular nature of the barrier is still under investigation. There is consent, however, that specialized structures in the stratum corneum, the CE, and extracellular lipid lamellae, as well as tight junctions in the granular layers, play essential roles in the development of the skin barrier function (Tsuruta et al., 2002; Segre, 2003).
The stratum corneum is formed from granular cells during terminal differentiation as keratinocytes ascend from the proliferative cell type in the basal layer through the spinous and granular layers to end up as flat, dead corneocytes within the cornified layer. The CE is assembled underneath the plasma membrane by sequential incorporation and transglutaminase-mediated cross-linking of precursor proteins, followed by the covalent attachment of extracellular lipids. At the granular layer–stratum corneum interface, the lamellar bodies that are thought to be elements of the tubulovesicular TGN fuse with the cell membrane and extrude their contents to form a multilamellar lipid complex that fills most of the intercellular space.
Pathological abnormalities in the stratum corneum, with the subsequent breakdown of epidermal barrier function, are observed in various skin diseases, which are referred to as ichthyoses. The loss of barrier function can be caused by several defects in the molecular mechanisms involved in the proper assembly of the CE or the intercorneocyte lipids. Several defective genes have been identified in ichthyosiform skin disorders so far, including genes coding for CE components (keratins, loricrin, and filaggrin) and proteins involved in the assembly and protein turnover (transglutaminase 1 and LEKTI), and, most frequently, genes coding for enzymes involved in lipid metabolism (e.g., fatty aldehyde dehydrogenase, steroid sulfatase, glucocerebrosidase, ATP-binding cassette transporter, (for review see Richard, 2004). Recent studies from our group and others have linked inactivating mutations in the genes of 12R-LOX and eLOX-3 to the development of ARCI (Jobard et al., 2002; Eckl et al., 2005).
12R-LOX is a member of the LOX multigene family, exhibiting, among mammals, a unique R-stereospecificity of oxygen insertion. The enzyme is found almost exclusively in skin. In mouse epidermis, a predominant mRNA expression was observed in the differentiated keratinocytes (Sun et al., 1998; Heidt et al., 2000). By immunofluorescence analyses, we could now localize the 12R-LOX protein at the surface of the keratinocytes in the stratum granulosum, indicating a function in late epidermal differentiation. Interestingly, an almost identical expression pattern was observed for eLOX-3 (not depicted), suggesting a colocalization of both LOX in the plasma membranes of the stratum granulosum. The implication of 12R-LOX and eLOX-3 in ARCI has brought forth the concept that both enzymes function in the same metabolic pathway to convert arachidonic acid via 12R-HPETE to hepoxilin- and trioxilin-like metabolites that are critically involved in keratinocyte differentiation (Jobard et al., 2002; Eckl et al., 2005; Yu et al., 2005; Lefevre et al., 2006). As shown for enzymes of the leukotriene synthesis, which form multimeric complexes in the nuclear membrane (Mandal et al., 2004), a coordinated membrane organization of these two enzymes, probably together with other enzymes and/or accessory proteins, may be a prerequisite for full enzyme activity and the proper generation of the bioactive lipid products of the 12R-LOX–eLOX-3 pathway in skin.
Indeed, under cell-free conditions, the recombinant human 12R-LOX exhibits only low catalytic activity converting arachidonic acid to 12R-HPETE, whereas the mouse enzyme does not metabolize free arachidonic acid, but only esterified substrates, including arachidonic acid and linoleic acid methyl esters (Siebert et al., 2001). This has raised the question as to the nature of the endogenous substrate and the functional homology of the mouse and human enzyme (Krieg et al., 1999; Yu et al., 2005, 2006). The results of this paper demonstrate an essential role of 12R-LOX in the development of epidermal barrier function in mice, documenting a functional homology of the mouse and human enzyme in skin.
12R-LOX–deficient mice exhibited the most severe phenotype regarding water barrier dysfunction reported so far. All knockout mice died within 3–5 h after birth as a result of severe dehydration. The mice lost 10% of their weight per hour. Other knockout mouse models with epidermal barrier defects, including mice deficient in KLF4 (Segre et al., 1999), claudin (Furuse et al., 2002), E-cadherin (Tunggal et al., 2005), LEKTI (Descargues et al., 2005), CAP1 (Leyvraz et al., 2005), and FATP4 (Herrmann et al., 2003), exhibited substantially less weight loss, resulting in a longer life span of the transgenic mice. Analyses of dye permeability and of TEWL clearly demonstrated a severely defective inward and outward epidermal barrier function in 12R-LOX–deficient mice, while the barrier function of tight junctions was unaffected. The knockout mice failed to develop a functional epidermal barrier, which was acquired in wild-type mice around E17. At this time point, expression of 12R-LOX, which starts in embryonic skin at E15.5, was shown to reach high levels that persists at later embryonic stages and in newborn skin (Sun et al., 1998).
Defective skin barrier function typically results in compensatory mechanisms involving epidermal hyperproliferation, hyperkeratosis, and/or parakeratosis, which are observed frequently in ichthyosiform human skin and various mouse models (Elias, 2004). 12R-LOX–deficient mice did not display such an obvious cutaneous phenotype, which may not develop because of the early neonatal lethality. In fact, markers of keratinocyte proliferation and terminal differentiation appeared to be unaffected, with the exception of filaggrin.
This late terminal differentiation marker is the result of a complex proteolytic processing of profilaggrin by several enzymes, including protein phosphatases, proteases, and protease inhibitors (Resing et al., 1984). Only mature filaggrin aggregates keratin filaments to form macrofibrils that crisscross the cornified cells of the stratum corneum, and it is an integral part of CE that contributes to its structural integrity. Thus, the decreased mechanical strength of CE from Alox12b–/– mice may be caused by the reduced profilaggrin processing. Furthermore, filaggrin monomers are degraded and provide free amino acids that, together with derivatives of amino acids and specific salts, constitute the natural moisturizing factor that is involved in the hydration of the stratum corneum (Rawlings et al., 1994). Thus, reduction of filaggrin monomers might explain the more densely packed stratum corneum, which could contribute to the impairement of the barrier function in the mutant mice. In fact, disturbance of the proteolytic profilaggrin processing by gene inactivation has been shown to be associated with impairment of barrier function in several mouse models (Presland et al., 2000; List et al., 2002, 2003; Leyvraz et al., 2005; Descargues et al., 2005). In humans, lack of proteolytically processed filaggrin monomers caused by loss-of-function mutations have been shown to underlie ichthyosis vulgaris and discussed to be a major predisposing factor for atopic dermatitis (Sandilands et al., 2006; Smith et al., 2006).
An important component of the epidermal barrier is the arrangement of intra- and extracellular lipid accumulation in the stratum granulosum and stratum corneum, in particular the processing of intracellular lipids and the process of their extrusion into the intercellular space. Ultrastructural analysis revealed structural anomalies in the upper granular layers of the skin of 12R-LOX knockout mice that may reflect defects in the lipid metabolism associated with the observed phenotype. The features of the abnormalities are reminiscent of characteristic alterations found in a subgroup of ichthyosis congenital patients (Anton-Lamprecht, 1992). They include electron-lucent vesicles of variable size with lamellar structures reminiscent of the content of lamellar bodies adhering to the surrounding membrane. The appearance of these structures suggests that they may originate from defects in the assembly and/or extrusion of lamellar bodies, probably caused by aberrant lipid processing. Electron microscopic examination with ruthenium tetroxide postfixation to preserve lipid structure did not reveal major disturbances of the intercellular lipid lamellae. However, we presently cannot exclude more subtle local disturbances of these structures. Nevertheless, this analysis refers to a specific defect in lipid content and organization that may contribute to the barrier impairment observed in Alox12b–/– mice. Although the levels of total fatty acids, cholesterol, and free ceramides did not show substantial differences between control and mutant mice, we observed significant alterations in ester-bound lipids from Alox12b–/– mice. Ceramides covalently attached to involucrin and other CE peptides are major constituents of the cornified lipid envelope that surrounds the corneocyte and have been discussed to be critical components of the barrier function (Elias et al., 2000; Doering et al., 2002). The identity of the lipid species altered in the 12R-LOX–deficient epidermis remains to be elucidated.
It also remains to be established weather 12R-LOX is directly involved in the enzymatic lipid processing or in the generation of lipid metabolites that are involved in the regulation of lipid metabolism. It is of interest to note that the epoxyalcohol metabolites produced by the 12R-LOX–eLOX-3 pathway are able to transactivate peroxisome proliferator-activated receptors (PPARs; Yu, 2005). Recent studies provide evidence for a role of PPARs in the regulation of terminal keratinocyte differentiation, including lipid synthesis and processing (Di Poi et al., 2004; Elias, 2005). Moreover, it was recently reported that PPAR activators are able to accelerate permeability barrier recovery after acute barrier disruption (Man et al., 2006).
In summary, this study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.
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To convert the targeted allele into a mutant allele structurally lacking the essential exon 8 of the Alox12b gene, F3 Alox12b+/flox heterozygotes were crossed with CMV-Cre transgenic mice exhibiting ubiquitous Cre expression (Schwenk et al., 1995). Complete excision of the resistance cassette and exon 8 in offspring mice was confirmed by Southern blot and PCR analyses (Fig. 1 C). Heterozygous mutant mice (Alox12b+/–) were bred with 129S6, and their heterozygous offspring were intercrossed to obtain homozygous mutant mice (Alox12b–/–). Genotyping was performed by PCR using 100 ng DNA isolated from tail and organs as a template and the primers ol1231 (5'-ACCCTCCCCTGCTGCTGTTGC-3') and ol709 (5'-AGAGACCTCCCTTGTTGAGAAG-3') to distinguish the 459-bp mutant allele band from the 1,075-bp wild-type band (Fig. 1 D).
RNA isolation and RT-PCR
Total RNA was isolated from epidermis as previously described (Kinzig et al., 1999) and mRNA was reverse transcribed with MuLV reverse transcriptase using the SuperScript II first strand synthesis system (Invitrogen). The resulting cDNA was subjected to PCR with the primer pairs ol230 (5'-CTGTGCCCCGATGTGCTTGCTG-3') and ol709 (3'-AGAGACCTCCCTTGTTGAGAAG-5') for 12R-LOX. As a control, ß-actin cDNA was amplified as a housekeeping gene.
Antibodies
Rabbit polyclonal antipeptide antibodies against 12R-LOX and eLOX-3 have been previously described (Eckl et al., 2005). Mouse anti–12R-LOX mAb were raised using GST-12R-LOX fusion protein as immunogen. Other primary antibodies used were goat anti-actin (Santa Cruz Biotechnology), rabbit anti–Claudin-1, rabbit anti-occludin (both from Invitrogen), mouse anti-filaggrin (Monosan), rabbit anti-keratin 5, rabbit anti-keratin 10, rabbit anti-involucrin, and rabbit anti-Ki67 (all from CRP). Secondary antibodies used were Alexa Fluor 488 goat anti–mouse IgG (Invitrogen), CY3 anti–rabbit IgG (BD Biosciences), and anti–goat antibodies (Santa Cruz Biotechnology).
Epidermal protein extraction and Western blot analysis
Trunk epidermis of newborn mice was separated mechanically from the dermis after incubation for 30 s at 56°C in PBS. Epidermal proteins were extracted as described elsewhere (Tunggal et al., 2005; Leyvraz et al., 2005), and Western blot analysis was performed as previously described (Eckl et al., 2005).
Histological and immunofluorescence analysis
For light microscopic observation, samples were fixed for 24 h with 4% formalin in PBS, dehydrated in 70% ethanol, and embedded in paraffin. 5-µm sections were mounted on slides, dewaxed, rehydrated, and stained with hematoxylin and eosin. For methylene blue staining, skin was fixed with 1% glutaraldehyde for 24 h and embedded in epon. 1-µm sections were stained with methylene blue. For immunofluorescence microscopy, cryosections (3-µm thick) were fixed in acetone for 10 min at –20°C and permeabilized with 0.05% Triton X-100 in PBS and flushed with PBS. The slides were blocked in 1% BSA in PBS for 1 h and incubated with the primary antibody for 1 h. After washing three times (10 min each) in PBS, samples were incubated with a fluorescent dye coupled with antibody and Hoechst 33258 diluted in blocking buffer for 30 min and washed three times. Sections were embedded in mounting medium (DakoCytomation) and examined by light microscopy using a photomicroscope (Axioplan 2) with a 25x Plan-Neofluar objective (both Carl Zeiss MicroImaging, Inc.). Images were acquired with a high sensibility digital black/white AxioCam (Carl Zeiss MicroImaging, Inc.).
Preparation of CEs and sonication experiments
CEs were purified and sonicated at 4°C for various time points in a bath sonicator, as previously described (Koch et al., 2000).
Functional analyses of the epidermal barrier
To determine the rate of fluid loss, newborns were separated from their mother and kept at 37°C. The body weight was monitored every 30 min, until time of death of homozygous mutant mice. The rate of TEWL from the skin of newborn mice was determined by using a Tewameter (Courage + Khazaka). For penetration assays, backs of newborn mice were immersed in 1 mM Lucifer yellow in PBS at 37°C. After 1 h incubation, mice were killed and the skin was dissected out. Frozen sections were counterstained with propidium iodide and penetration of the dye was assessed by immunofluorescence microscopy.
Toluidine blue staining of mouse embryos
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 previously described (Koch et al., 2000), washed in PBS for 1 min, and stained for 30 min in 0.1% toluidine blue O/PBS. After destaining in PBS for 15 min, the embryos were photographed.
EM
All specimens were fixed for at least 2 h at room temperature in 3% glutaraldehyde solution in 0.1 M cacodylate buffer, pH 7.4, cut into pieces of 1 mm3, washed in buffer, postfixed for 1 h at 4°C in 1% osmium tetroxide or in 0.5% ruthenium tetroxide, rinsed in water, dehydrated through graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin (Glycidether 100; Merck). Ultrathin sections were treated with uranyl acetate and lead citrate and examined with an electron microscope (EM 400; Philips).
Lipid analysis
Chemicals.
Ceramide AS was purchased from Sigma-Aldrich. Ceramide NS was provided by Sederma, and ceramides EOS, EOP, NP, and AP were provided by Degussa.
Lipid extraction.
Lipid extraction followed a previously described protocol (Doering et al., 1999) with slight modifications. In brief, epidermis homogenate was extracted twice, first overnight with chloroform (methanol 1:2 at room temperature) and second with 2 ml chloroform (methanol 2:1 for 1 h at room temperature). The organic layers of both extraction steps were combined, the solvent was removed using a Christ Speed-Vac Alpha RVC/Alpha 2–4 (Christ), and the residue was redissolved in 100 µl methanol/chloroform at a 1:1 ratio.
For recovery of protein-bound lipids, the pretreated pellet was incubated with 1 ml of 1 N NaOH in methanol at a ratio of 19:1, followed by extraction with 2 ml of chloroform for 1 h at 37°C. The organic layer was removed and washed with 3 ml of PBS–buffer; after phase separation, the solvent of the organic layer was evaporated in the Speed-Vac and the residue was redissolved in 100 µl methanol/chloroform at a ratio of 1:1. Lipid extracts were stored at –20°C until use.
Lipid HPTLC.
Analysis of epidermal lipids by HPTLC followed a previously described protocol (Farwanah et al., 2002). In brief, 50 µl of a sample were applied on a prewashed TLC plate, together with reference lipids, using an Automatic TLC Sampler 4 (CAMAG). The development of the plates has been performed automatically using an AMD-2 apparatus (CAMAG). The AMD procedure used included a 17-step gradient of decreasing polarity, as previously described (Farwanah et al., 2002). After drying, the plates were sprayed with an aqueous solution of 10% CuSO4 (wt/vol), 8% H3PO4 (vol/vol), and 5% methanol (vol/vol) using a ChromaJet DS20 (Sarstedt), charred in a drying oven at 180°C for 30 min, and, finally, scanned using a TLC Scanner 3 (CAMAG). Integration and quantification based on peak areas were performed using WinCATS software (CAMAG). Quantitative results for all ceramides were related to ceramide NP.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (KR 905/5-1 and KR 905/6-1) and by a financial gift from the Selbsthilfe Ichthyose e.V.
Submitted: 20 December 2006
Accepted: 6 March 2007
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