Báo cáo khoa học: The hepoxilin connection in the epidermis docx

MINIREVIEW The hepoxilin connection in the epidermis Alan R. Brash, Zheyong Yu*, William E. Boeglin and Claus Schneider Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA Introduction A relatively quiet area of eicosanoid research, the oxi- dative metabolism of polyunsaturated fatty acids in skin, recently jumped to prominence with the discovery of a genetic connection between two lipoxygenase (LOX) genes and a rare form of inherited ichthyosis [1]. The findings gave life to a LOX enzyme thought to be inactive and linked its function to the second LOX, both of which appear to be essential in creating the normal permeability barrier of the skin. Our recent analysis of the catalytic activities of these proteins, 12R-LOX and eLOX3, suggested that the formation of epoxy-hydroxy fatty acid derivatives (hepoxilins) could be an integral part of the biological pathway disrupted in the LOX-related form of ichthyosis and, by infer- ence, responsible for the wellbeing of the epidermis in all normal subjects [2]. From the discovery of essential fatty acids (EFAs) around 1930, it has been clear that there is some con- nection between these particular lipids and the proper functioning of the water-impermeable barrier of the epidermis [3,4]. One of the hallmark symptoms of EFA deficiency is the development of a scaly skin phe- notype, and this is cured by topical application of lino- leic, arachidonic and other EFAs [4–8]. Although LOX enzymes were not identified in animal tissues until the mid-1970s, thereafter several lines of evidence sugges- ted that the ameliorative effects of EFAs in the EFA- deficient animal involve the LOX-catalyzed conversion Keywords arachidonic acid; epidermis; epoxyalcohol; essential fatty acid; hepoxilin; ichthyosis; linoleic acid; lipoxygenase; psoriasis; trioxilin Correspondence A. R. Brash, Department of Pharmacology RRB Room 510, Vanderbilt University Medical Center, 23rd Ave at Pierce, Nashville, TN 37232-6602, USA Fax: +1 615 3224707 Tel: +1 615 343 4495 E-mail: alan.brash@vanderbilt.edu *Present address Howard Hughes Medical Institute, Washing- ton University, St Louis, MO, USA (Received 13 October 2006, accepted 12 March 2007) doi:10.1111/j.1742-4658.2007.05909.x The recent convergence of genetic and biochemical evidence on the activit- ies of lipoxygenase (LOX) enzymes has implicated the production of hep- oxilin derivatives (fatty acid epoxyalcohols) in the pathways leading to formation of the water-impermeable barrier of the outer epidermis. The enzymes 12R-LOX and eLOX3 are mutated in a rare form of congenital ichthyosis, and, in vitro, the two enzymes function together to convert arachidonic acid to a specific hepoxilin. Taken together, these lines of evi- dence suggest an involvement of these enzymes and their products in skin barrier function in all normal subjects. The natural occurrence of the speci- fic hepoxilin products, and their biological role, whether structural or sign- aling, remain to be defined. Abbreviations AA, arachidonic acid; EFA, essential fatty acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; KETE, ketoeicosatetraenoic acid; LOX, lipoxygenase; NCIE, nonbullous congenital ichthyosiform erythroderma. 3494 FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS of the fatty acid substrate to oxygenated products [9,10]. This is now considered to include further trans- formation of the primary LOX products to epoxy- alcohols (hepoxilins), triols and possibly also x-hydroxylated derivatives. There may or may not be a direct mechanistic connection between the effects of EFAs and the recent findings on ichthyosis, but together they reinforce the fact that there is an obliga- tory involvement of EFAs and their products in the normal process of forming the water-impermeable barrier in skin. Herein is reviewed the connection of EFAs, LOXs, hepoxilins and the structure–function of the human epidermis. The six human LOX genes Of the six different LOXs in the human genome, the best known by far is the 5-LOX of leukocytes, the enzyme that gives rise to the leukotrienes, the inflam- matory mediators implicated in asthma [11–13]. The role of the other LOXs is less certain, although they are known to be specific to certain cell types and to produce distinct fatty acid hydroperoxide products [12,14–16]. The prototypical cells of expression of one group of LOX enzymes are distinct blood cell types, namely the 5-LOX in leukocytes, the 12S-LOX in platelets and the 15-LOX-1 in reticulocytes. Three other LOX enzymes are epithelial cell-specific – and two of these three (15-LOX-2 and 12R-LOX) were dis- covered in human skin by our group [17,18]. The third, cloned initially from mouse skin, is called eLOX3 [19]. In contrast to other family members, it is incapable of forming fatty acid hydroperoxides [2,19] and therefore has no ‘5-’, ‘12-’, or ‘15-’ designation to its name. It is simply named from the fact that it was the third epi- thelial LOX to be discovered – eLOX3. It is one of the two LOX enzymes that are central to this review, the other being 12R-LOX. Ichthyosis has multiple genetic origins The name ichthyosis comes from the Greek word for fish. Taken together, the ichthyoses are a group of der- matological conditions caused by genetic abnormalities and characterized by a scaly skin phenotype. The dif- ferent mutations typically cause problems associated with construction of the water-impermeable barrier in the outermost cornified layer of the epidermis. Ichthy- osis vulgaris is the most common type (with an inci- dence worldwide of 1 in 250), as implied by its name, and is associated with mutations in the filaggrin gene [20]; this leads to defects in formation of the cornified cell envelope, an important component of the barrier layer. Other types of ichthyosis typically have inci- dences of 1 in 100 000 or less, and mutations in a number of genes involved in barrier function have been implicated [21]. Among the more recent additions to the expanding list are the genes encoding 12R-LOX and eLOX3. 12R-LOX and eLOX3 mutations: the connection to ichthyosis A major breakthrough in understanding the role of LOX enzymes in skin was provided by the genetic study of Fischer and colleagues in 2002 [1]. The authors pinpointed a previously reported locus of inherited ichthyosis on chromosome 17p [22] with mutations in the coding regions of either the 12R-LOX (ALOX12B) or eLOX3 (ALOXE3) genes [1]. The phe- notype is classified as nonbullous congenital ich- thyosiform erythroderma (NCIE, in layman’s terms translating as a nonblistering, inherited, scaly red skin). An independent study later extended these find- ings following the identification of 17 additional famil- ies with mutations in the same two LOX genes [23]; for classification of the disease, these authors used the more general designation of autosomal-recessive con- genital ichthyosis, of which NCIE could be considered a subdivision [21]. Because one or the other LOX genes was mutated in the affected families, producing a similar phenotype, Fischer and colleagues speculated that the two enzymes operate in the same metabolic pathway [1]. This served to revamp our thinking on the potential catalytic activities of the apparently non- functional oxygenase, eLOX3. LOX enzyme expression in epidermis All the LOX genes are expressed in skin, as detected by activity, immunohistochemistry and⁄ or PCR of the mRNA. The activity of 12R -LOX, detected as 12R- hydroxyeicosatetraenoic acid (12R-HETE) formation, is quite low in normal human epidermis [24], in which the dominant LOX activities are 12S-LOX and 15-LOX [24–27]. The synthesis of 12R-HETE is strongly elevated in the inflammatory and proliferative skin disease of psoriasis [28,29]. (The pro-inflammatory bioactivity of 12R-HETE is quite weak [30,31] and we speculate that its elevated synthesis is a result of the keratinocyte hyperproliferation of psoriasis.) For many years, the enzyme making 12R-HETE was unknown. Then, in 1998, we reported the discovery of a 12R- LOX in human skin and showed that it can account for the selective formation of 12R-HETE [18]. Others found that the mouse 12R-LOX is first expressed on A. R. Brash et al. Hepoxilins in the epidermis FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS 3495 embryonic day 15.5 at the location and time that the epidermis is being formed [32]. The second LOX gene implicated in NCIE, eLOX3, is strongly expressed in the epidermis, as indicated by RT-PCR [33,34]. In addition, Krieg and coworkers detected expression of the human mRNA in additional tissues such as placenta, pancreas, ovary, testis, brain and some secretory epithelia. In general, the expression pattern of human and mouse eLOX3 was paralleled by the expression of 12 R -LOX and was highest in the skin [34]. eLOX3 as a hydroperoxide isomerase (hepoxilin synthase) The genetic findings concerning 12R-LOX and eLOX3 mutations in ichthyosis are intriguing from the bio- chemical point of view, partly because the eLOX3 pro- tein has been expressed and studied in at least two laboratories, including our own, and no oxygenase activity was detectable with any of a selection of potential fatty acid substrates [2,19]. This conundrum, the association of eLOX3 mutations with an ichthyosis phenotype in the apparent absence of any LOX activ- ity in the expressed eLOX3 protein, led us to examine the possibility that the primary products of other LOX enzymes were substrates for eLOX3. We found that, indeed, eLOX3 will metabolize fatty acid hydroperox- ides, although not through oxygenation as is typical of LOX enzymes. eLOX3 reacts with the hydroperoxide moiety and induces an isomerization of the hydro- peroxide to specific epoxyalcohol (hepoxilin-type) products and a ketoeicosatetraenoic acid (KETE) [2]. Among the three arachidonate-derived hydroperoxides that are most likely to be found in human epidermis [12S-hydroperoxyeicosatetraenoic acid (HPETE), 15S- HPETE and 12R-HPETE – Fig. 1 shows the structures of the main products] the best substrate was 12R- HPETE. As this is formed by 12R-LOX, the other gene implicated in the LOX-related form of NCIE, this sets up a potential biochemical rationalization of the genetic findings in NCIE [2]. An unusual aspect of eLOX3 catalysis is that its activity is stimulated by typical LOX-reducing inhibi- tors such as nordihydroguaiaretic acid. The explan- ation is that, in contrast to LOX enzymes acting as dioxygenases, the active form of the enzyme utilizes the reduced, ferrous, form of the iron (Fig. 2). The hepoxilin product contains both the original oxygen atoms of the hydroperoxide substrate (Fig. 2) and thus eLOX3 functions as a hydroperoxide isomerase [2]. It was implicit in the report on the link between mutations in 12R-LOX and eLOX3 in NCIE that the enzyme activities would be compromised [1]. It has now been demonstrated experimentally that these and more recently identified mutations in both 12R-LOX and eLOX3 inactivate the enzymes [23,35]. Several O HO O HO 2 C HO O HO HO 2 C HO 2 C O HO HO 2 C From 12 R -HPETE :- + 12-KETE + 12-KETE + 15-KETE From 12 S -HPETE :- From 15 S -HPETE :- Fig. 1. Structures of the epoxyalcohol products of eLOX3. The structural analysis is given in a previous publication [2]. NDGA 12R-HPETE OOH 128 12-KETE O 3 1 4 2 O O OH product Epoxyalcohol Fe 2+ Fe 3+ Fe 3+ -OH Fe 3+ -OH + H 2 O O Fig. 2. Proposed mechanism for eLOX3 catalysis. The Fe 2+ enzyme initiates homolytic cleavage of the O–O bond of the fatty acid hydroperoxide, forming an Fe 3+ –OH complex and a substrate alk- oxyl radical (RO • ) (step 1). The alkoxyl radical instantly reacts with the adjacent double bond, forming an epoxyallylic carbon radical (step 2); this is hydroxylated by oxygen rebound from the Fe 3+ –OH complex, thus completing the catalytic cycle (step 3). KETE is formed as a minor by-product (step 4). Hepoxilins in the epidermis A. R. Brash et al. 3496 FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS mutations are remote from the LOX active site, and we surmise that they may destabilize the protein, as the mutants failed to accumulate during heterologous expression in Escherichia coli [35]. A primer on hepoxilins and their formation from HPETEs As for the prostaglandins, leukotrienes and lipoxins, it is useful to have a group name for the fatty acid epoxyalcohols. The name ‘hepoxilin’, coined by Pace- Asciak, serves a useful function in this regard, the first three letters of the name (hep) standing for hydroxy- epoxy; trioxilins are the corresponding trihydroxy hydrolysis products [36–38]. Although applied strictly only to derivatives of 12-HPETE, the hepoxilin termin- ology is useful for describing two general classes of epoxyalcohol, hepoxilin A-type and B-type (Fig. 3). The terms hepoxilin A 3 or hepoxilin B 3 (‘3¢ for the three double bonds in hepoxilins derived from arachidonic acid) each refer to any of a mixture of diastereomers and⁄ or enantiomers, and therefore the hepoxilin nomenclature runs into difficulties when pre- cise definition is required. In this review, individual molecules are named with the hydroxyl and epoxide configurations specified, as in the 12R-LOX–eLOX3 product, 8R-OH,11R,12R-epoxy-hepoxilin A 3 . The transformation of fatty acid hydroperoxides to epoxyalcohols is a facile nonenzymatic reaction, the chemistry of which has been studied extensively and found to be complex (reviewed in ref. 39). Free heme or transition metals will initiate the reaction. For any one fatty acid hydroperoxide, for example 12-HPETE, there are three separate routes for conversion to epoxyalcohols: the new hydroxy group can be formed by rearrangement of the two hydroperoxide oxygens, by the reaction of intermediates with O 2 , or by reac- tion with water [39–42]. Nonenzymic reactions give a mixture of hepoxilin A-type and B-type products with a cis or trans epoxide and with R or S in stereochemis- try of the hydroxyl group. From this knowledge of the extensive nonenzymic possibilities for reaction, it is easy to see why the appearance of a single distinct epoxyalcohol isomer is taken as one hallmark denoting the potential involvement of an enzyme. The hepoxilin A-type epoxides are much more sensi- tive to acid-catalyzed hydrolysis than the B-type (Fig. 3), and they may also be more readily hydrolyzed enzymatically. Accordingly, in biological extracts the hepoxilin B-type epoxides are often detectable, whereas the A-type are recovered as their trihydroxy hydrolysis products. Detection of hepoxilins and triols in the epidermis Nugteren and coworkers were the first to provide evi- dence that LOX-derived epoxyalcohol and triol fatty acids are important to the structure–function of the epidermal water barrier [9]. They applied different 14 C-labeled unsaturated fatty acids onto the skin of live fatty acid-deficient rats and followed the metabolic fate over the course of 1–4 days. The radiolabeled sub- strates [linoleic acid, or its trans ⁄ cis, cis ⁄ trans and trans ⁄ trans isomers, or arachidonic acid (AA)] were transformed through multiple pathways [9], including incorporation into complex acylceramide lipids that are a characteristic of the epidermal barrier layer [43,44]. Formed specifically from arachidonic and lino- leic acids (and not the trans isomers) were polyhydrox- ylated fatty acid derivatives (epoxyalcohols and triols). Their synthesis in vivo was blocked by co-application of the LOX inhibitor eicosatetraynoic acid, and this paralleled its inhibition of the ameliorative effects of the applied EFA. The authors speculated that these OOH hepoxilin B-type (more stable) hepoxilin A-type (easily hydrolyzed) O HO 8 12 8 R ,11 R ,12 R - hepoxilin A 3 O HO O HO O HO O HO O HO O HO O HO O HO O HO Fatty acid hydroperoxide Fig. 3. General structures of hepoxilin A-type and B-type. Nonenzy- mic transformation from racemic hydroperoxide could lead to for- mation of all the individual isomers shown (plus the corresponding cis-epoxides, not shown). 12R-lipoxygenase (12R-LOX) and eLOX3 form exclusively the 8R-OH,11R,12R-epoxy-hepoxilin A 3 (boxed). A. R. Brash et al. Hepoxilins in the epidermis FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS 3497 LOX-derived products contributed to the formation of the lamellar lipid phase that helps constitute the water- impermeable barrier [44], or that they serve as a signal to promote differentiation [9]. Notably, these LOX- derived products could not be detected in normal epi- dermis using a similar approach [45]. It remains an open debate of whether LOX-derived products have any structural role and ⁄ or act as specific signaling mol- ecules in contributing to the epidermal water barrier. Also awaiting clarification is linoleic acid metabolism by 12R-LOX, the primary LOX enzyme strongly impli- cated through genetic evidence as being involved in the skin barrier function. The biosynthesis of hepoxilin-type products and their triol derivatives from 14 C-AA has been reported in isolated human epidermal fragments [46]. Vila and colleagues showed a predominant 12-LOX pathway of metabolism leading to both hepoxilin A 3 -derived triols and hepoxilin B 3 products. The results are of special interest, not only for the characterization of specific products, but also for the finding of predominant 12-LOX metabolism. By contrast, cultured human ker- atinocytes are found typically to convert AA mainly via a 15-LOX pathway [47,48]. In fact ‘12-LOX’ would encompass 12S-LOX and 12R-LOX and ‘15-LOX’ could reflect 15-LOX-1 and ⁄ or 15-LOX-2. Each is rep- resented in human epidermis [18,27,49] and the relative proportions probably reflect differences in the site of epidermis collection and the stage(s) of differentiation of the keratinocytes. Another significant finding was the well-documented synthesis of a single predominant hepoxilin B 3 product in epidermal fragments and in the microsomal fraction [46,50]. This hepoxilin B 3 product had the same GC-MS characteristics as the synthetic standard of 10R-hydroxy-11S,12S-hepoxilin B 3 [50], which is a product we identified as specifically formed from 12S- HPETE by eLOX3 [2]. (Note, however, that Anto ´ n& Vila’s method could not distinguish between this hep- oxilin B 3 and its enantiomer, 10S-hydroxy-11R,12R- hepoxilin B 3 .) The product was formed from AA in epidermal microsomes and at a much lower yield using recombinant platelet-type 12S-LOX. Formation from 12S-HPETE could not be demonstrated. In the absence of other candidate enzymes (the activity of eLOX3 being unknown at the time), the authors con- cluded that 12S-LOX is probably the hepoxilin B 3 syn- thase [50]. This work ranks as one of the very few in which a single hepoxilin diastereomer has been shown to be produced in mammalian cells or tissue. Produc- tion of a single diastereomer (as opposed to an equi- molar mixture of a diastereomeric pair) probably denotes its enzymatic synthesis. Vila and colleagues went on to use GC-MS to demonstrate the presence of CO 2 H CO 2 H CO 2 H CO 2 H OOH CO 2 H OH CO 2 H O HO O HO HO OH + Arachidonic acid 12R-HPETE 12R-HETE 8R,11R,12R-epoxyalcohol 12-KETE 8,11,12-triol 12R-LOX eLOX3 epoxide hydrolase peroxidase mutations mutations CH 2 OH omega-oxidation products CYP 4F22 ? ? CYP 4F22 Fig. 4. Proposed metabolism of arachidonic acid (AA) in human epidermis through the 12R-lipoxygenase (12R-LOX)–eLOX3 path- way. The putative x-hydroxylase, CYP4F22, was able to react at other points on the pathway [e.g. with AA or 12R- hydro(pero)xyeicosatetraenoic acid (12R- HPETE)], or with unrelated fatty acids (see the text under Recent developments). Hepoxilins in the epidermis A. R. Brash et al. 3498 FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS elevated levels of endogenous hepoxilins and trioxilins in human psoriatic scales [50,51]. One aspect of these observations that should be revisited is the occurrence of a novel hepoxilin isomer in psoriatic scales. This putative hepoxilin B 3 isomer was separated from the authentic standards on GC-MS [51]. The finding of the product specifically in psoriasis is intriguing and worthy of further study. eLOX3 converts fatty acid hydroperoxides to a KETE byproduct in addition to the hepoxilins (Figs 1, 2 and 4) [2]. This type of unsaturated ketone is highly reactive with cellular nucleophiles, such as glutathione [52], and consequently it may not appear as a peak of KETE upon chromatography of cell extracts. Although reaction with glutathione is usually considered a pathway of inactivation, some derivatives are bioactive (e.g. leukotrienes); the fate and potential bioactivity of the eLOX3-derived ketones remains to be evaluated. Recent developments: further aspects of the LOX pathway Two independent reports describe a dramatic pheno- type associated with deletion of the 12R-LOX gene in mice [53,54]. The homozygous– ⁄ – neonates die within hours of birth because of excessive transepidermal water loss. As is typical for gene defects that disrupt barrier function, the phenotype is more severe in the mouse compared with the human as a result of the much larger surface to volume ratio, but generally is consistent with the findings in the ichthyosis patients. Although the gross morphology of the epidermis was not affected, the upper granular layer of the skin in the knockout animals showed evidence of disruption of the normal processing of the lipid-rich lamellar bod- ies that play a crucial role in formation of the water- impermeable barrier. These studies add a convincing new line of evidence for a key role of 12R-LOX in the normal functioning of the epidermis and, furthermore, provide models in which the mechanism of action can be investigated. The findings do, however, present a conundrum for the proposed role of hepoxilins in maintaining the epidermal water barrier. Whereas mouse eLOX3 has the required activity with fatty acid hydroperoxides, mouse 12R-LOX is very unusual in apparently lacking oxygenase activity with AA or any other polyunsaturated lipid tested to date [55–57]. Arachidonate methyl ester is metabolized to the corres- ponding 12R-hydroperoxide, but the methyl ester is not naturally occurring. Thus, there remains an open question of whether mouse 12R -LOX could generate a fatty acid hydroperoxide with a suitable natural substrate, or whether the protein functions in some other way to promote the correct differentiation of the epidermis. Meanwhile, the geneticists continue to provide pro- vocative new insights. The association of mutations in a putative membrane protein giving a similar pheno- type as in the LOX-related form of NCIE led Fischer and colleagues to speculate that it constitutes a hepoxi- lin or trioxilin receptor [58]. They named this new gene ichthyin. Another new gene implicated in ichthyosis by the same group encodes a cytochrome P450 [59]; it is classified as CYP4F22 [60], an uncharacterized member of the CYP4 family, which are generally fatty acid x-hydroxylases. Again, based on the similarity to the ichthyosis phenotype, the genetics group questioned if this potential x-hydroxylase is involved in producing the biologically active end-product of the LOX path- way. There is biochemical precedent for the P450 x-hydroxylases having substrate specificity for an oxygenated fatty acid: thus, CYP4F8 efficiently x-hydroxylates the prostaglandin endoperoxide PGH 2 , whereas AA and PGE 2 are comparatively feeble sub- strates [61]. Similarly, several CYP4A isoforms more efficiently x-hydroxylate epoxyeicosatrienoic acids than AA [62]. Perhaps the active principal of the epidermal LOX pathway is x-hydroxylated hepoxilin or trioxilin (Fig. 4). Incidentally, the linoleate-containing acylcera- mide of the epidermal barrier layer is composed of sphingosine in amide linkage to the carboxyl group of a very long x-hydroxy acid (mainly C 30 ,C 32 ,orC 34 chain length), which in turn is esterified to the carb- oxyl group of linoleate; the enzyme responsible for the x-hydroxylation of the long-chain acid is uncharacter- ized and could be the P450 enzyme that Lefevre et al. have implicated in ichthyosis [59]. Interestingly, the hepoxilin derived via 12R-LOX and eLOX3 is hydrolyzed specifically in keratinocytes to a single triol, tentatively identified as 8R,11S,12R- trihydroxyeicosa-5Z,9E,14Z-trienoic acid formed by S N 2 hydrolysis of the epoxide at C-11 [35]. In human keratinocytes, this hydrolase activity may be a down- stream enzyme in the pathway consisting of 12R-LOX and eLOX3 to form an active mediator in the regula- tion of keratinocyte differentiation (Fig. 4). A bioac- tive trioxilin is precedented: in vascular endothelial cells a specific triol is implicated as one of the endo- thelium-derived hyperpolarizing factors [63]. Concluding remarks ) unresolved issues Linoleate is usually considered to be a structural component of the ceramides in the stratum corneum A. R. Brash et al. Hepoxilins in the epidermis FEBS Journal 274 (2007) 3494–3502 ª 2007 The Authors Journal compilation ª 2007 FEBS 3499 [6,8], whereas arachidonate is viewed currently as the initial substrate of the hepoxilin signaling pathway [1,2,23]. So, it is questionable as to whether the EFA- deficiency phenotype is attributable to lack of the LOX products that are missing in the LOX-depend- ent ichthyosis. This highlights the two poles in cur- rent views on the activities of EFA in the epidermis: structural and ⁄ or signaling. Part of this debate con- cerns whether hepoxilin-type derivatives of linoleate itself might function in a critical role in the epidermal barrier function. The recent 12R-LOX knockout studies in mice leave little doubt about the crucial involvement of this LOX gene, but raise issues of its proposed metabolic coup- ling with eLOX3 (which itself is known to be critical in human genetic studies [1,23]). Identification of a natural substrate for the mouse 12R-LOX or condi- tions under which it exhibits oxygenase activity will be necessary to substantiate the hepoxilin connection to epidermal differentiation. With regard to the LOX-dependent phenotype, the nature, occurrence and bioactivity of the active prod- ucts remain to be defined. Currently, the lack of authentic hepoxilins is a significant holdup. The prime candidates for assay and for pharmacological testing include the hepoxilin derivatives of eLOX3, the corres- ponding trihydroxy hydrolysis products and their x-hydroxylated derivatives. Regarding mechanism of action, this is also currently an open issue. Over 20 years ago the main proponents of the LOX connec- tion in barrier function noted either a structural [44] or a signaling function in differentiation [9], and we are not much further advanced in defining the mechanism today. Genetic analyses of mutant skin phenotypes have paved the way for unraveling the LOX ⁄ hepoxilin pathway in the epidermis, and they continue to pro- vide fresh impetus with identification of the putative receptor protein, ichthyin. Defined candidates are on the table and the search is on to determine their involvement. Acknowledgements This work was supported by NIH grant AR51968 to ARB. 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