Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope

Abstract

A barrier to water loss is vital to maintaining life on dry land. Formation of the mammalian skin barrier requires both the essential fatty acid linoleate and the two lipoxygenases 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3), although their roles are poorly understood. Linoleate occurs in O-linoleoyl-ω-hydroxyceramide, which, after hydrolysis of the linoleate moiety, is covalently attached to protein via the free ω-hydroxyl of the ceramide, forming the corneocyte lipid envelope, a scaffold between lipid and protein that helps seal the barrier. Here we show using HPLC-UV, LC-MS, GC-MS, and (1)H NMR that O-linoleoyl-ω-hydroxyceramide is oxygenated in a regio- and stereospecific fashion by the consecutive actions of 12R-LOX and eLOX3 and that these products occur naturally in pig and mouse epidermis. 12R-LOX forms 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide, further converted by eLOX3 to specific epoxyalcohol (9R,10R-trans-epoxy-11E-13R-hydroxy) and 9-keto-10E,12Z esters of the ceramide; an epoxy-ketone derivative (9R,10R-trans-epoxy-11E-13-keto) is the most prominent oxidized ceramide in mouse skin. These products are absent in 12R-LOX-deficient mice, which crucially display a near total absence of protein-bound ω-hydroxyceramides and of the corneocyte lipid envelope and die shortly after birth from transepidermal water loss. We conclude that oxygenation of O-linoleoyl-ω-hydroxyceramide is required to facilitate the ester hydrolysis and allow bonding of the ω-hydroxyceramide to protein, providing a coherent explanation for the roles of multiple components in epidermal barrier function. Our study uncovers a hitherto unknown biochemical pathway in which the enzymic oxygenation of ceramides is involved in building a crucial structure of the epidermal barrier.

FIGURE 1.

Esterified ceramides of the mammalian epidermal barrier. A, EOS in the outer epidermis is esterified mainly with linoleic acid (C18:2) and is glucosylated at C-1 of the sphingosine (Glc-EOS) early in differentiation. After hydrolysis of the glycosidic and ester bonds, the resulting OS is esterified by transglutaminase (TGase) to glutamines in the cross-linked proteins of the CE (9). The ester linkage in the resulting glutamates bonds the monomolecular lipid coating, the CLE, to the outer face of the CE. ω-Hydroxy-VLFA (OAcid, shown on the right side) are also ester-linked components of the CLE. B, the CE is a highly cross-linked protein coat at the cell periphery, bonded with the extracellular lipid milieu via the CLE, with a detailed view illustrated in the segment below.

FIGURE 2.

Transformation of Glc-EOS by 12R-LOX and eLOX3 in vitro. A, RP-HPLC analysis of the oxygenation of Glc-EOS by 12R-LOX. The Glc-EOS substrates containing esterified linoleate (and differing in chain length and number of double bonds of the amide linked fatty acid) elute at ∼6.5–11 min and are detected by UV recording at 205 nm (lower trace, blue) and by APCI-MS of the [M+H]⁺ ions at the m/z values indicated. The oxygenated products elute at ∼3.5–5 min and are detected at 235 nm (upper trace, red). Inset, UV spectrum of the products. B, combined mass spectra of the substrates (lower panel) and products (upper panel). C, normal phase HPLC analysis (left panel) and chiral HPLC (right panel) of the reduced and transesterified 12R-LOX products as methyl esters. D, analysis of the reaction of eLOX3 with the 9R-hydroperoxides of Glc-EOS. Normal phase HPLC of the transesterified products reveals a keto derivative at 4 min of retention time (detected at 270 nm) and the major epoxyalcohol product eluting at 13.6 min (detected at 205 nm); the epoxyalcohol was identified by co-chromatography with authentic standard on HPLC (cf. Fig. 3C) and by GC-MS of the methyl ester trimethylsilyl ether derivative (E). E, GC-MS analysis of the main epoxyalcohol product from the reaction of eLOX3 with 9R-HPODE-Glc-EOS. Prior to the analysis, the product was transesterified to the methyl ester and derivatized to the TMS ether.

SCHEME 1

FIGURE 3.

Identification of oxygenated EOS ceramides in pig epidermis. A, normal phase HPLC with UV detection reveals KODE-EOS (270 nm, black trace), containing a keto derivative of linoleate, HODE-EOS (235 nm, red trace) containing hydroxy-linoleate, and EpOH-EOS (205 nm, blue trace) containing an epoxyalcohol derivative of linoleate. Unmodified EOS and other known ceramides are also detected at 205 nm (5–7 min). B, following transesterification, the main hydroxy product is identified as 9-HODE, and chiral HPLC reveals that it is predominantly 9R-HODE. C, transesterified EpOH-EOS shows a single peak (top panels), corresponding to a standard of 9,10-trans-epoxy-13-hydroxy-octadeca-11E-enoate methyl ester (details in SI Methods). Chiral HPLC analysis of the natural diastereomer (top panels) shows that it corresponds exclusively to the 9R,10R,13R enantiomer of a racemic standard (bottom panels).

FIGURE 4.

Oxygenated EOS ceramides of mouse epidermis; absence in 12R-LOX^−/− mice. A, normal phase HPLC of the ceramides extracted from wild-type (top panel) and 12R-LOX-deficient mouse epidermis (bottom panel). Ceramides identified by MS include EOS, non-hydroxy-fatty acid sphingosine (NS), α-hydroxy-fatty acid sphingosine (AS), and non-hydroxy-fatty acid hydroxysphingosine (NH). B, normal phase HPLC of mouse epidermal lipids recovered from a final soak for 24 h at room temperature after exhaustive chloroform-methanol extractions at 0 °C, from wild-type epidermis (top panel) and 12R-LOX^−/− (bottom panel). Top panel, inset, typical UV spectrum of products I and II in normal phase HPLC solvent. C, because of its instability during transesterification, the epoxy-ketone derivative in the ceramides was first reduced using NaBH₄ and then analyzed as the resulting epoxyalcohol diastereomers. D, normal phase HPLC separation of the NaBH₄ reduction products. Chiral HPLC analysis shows that the first eluting diastereomer is 9R,10R-epoxy-11E-13R-hydroxy-octadecenoate, identical to the epoxyalcohol product of 12R-LOX and eLOX3 observed in pig epidermis and in vitro (cf. Fig. 2D and supplemental Fig. S2).

FIGURE 5.

Analysis of free oxygenated fatty acid metabolites from wild-type mouse epidermis (A) and from 12R-LOX^−/− mouse epidermis (B). Left panels, RP-HPLC analysis of free HODE and HETE. Middle panels, HODE was collected and further analyzed by normal phase HPLC. Right panels, 9-HODE was collected and further analyzed by chiral HPLC Free HODE and HETE eluted between 2 and 4 min in the normal phase HPLC analysis of total lipid extract as in Fig. 4A. Therefore, the fraction from 2 to 4 min was collected and then subjected to the RP-HPLC analysis shown here. Chiral HPLC analysis indicated that 12-HETE from both wild-type and 12R-LOX^−/− epidermis was in S configuration (data not shown). Identical aliquots were injected in this wild type versus 12R-LOX^−/− comparison. Shown are representative chromatograms from three independent experiments on three sets of wild-type and 12R-LOX^−/− littermates. Note the 4- or 10-fold more sensitive y axis absorbance scale on the 12R-LOX^−/− chromatograms.

FIGURE 6.

Covalently bound ω-hydroxyceramides in mouse epidermis. A, top panel, the structure of the most prominent OS species (M + 1 ion at m/z 805) released by mild alkaline hydrolysis from the proteins of wild-type epidermis. Bottom panel, APCI-MS of the mixture of all OS species. B, top panel, normal phase HPLC-APCI-MS analysis of bound ceramides from wild-type epidermis reveals a main peak of OS (ω-hydroxysphingosine) and minor OH (ω-hydroxy-hydroxysphingosine). Bottom panel, the profile of the OS species at m/z 805. C, the corresponding analysis of bound ceramides from 12R-LOX^−/− epidermis. The height of the OS peak compared with wild type was 1.3% in this experiment and in four independent experiments averaged 0.8 ± 0.3% (S.E.).

FIGURE 7.

12R-LOX^−/− corneocytes lack visible CLE. Although corneocytes from 12R-LOX^−/− mice display normal CE, they lack externally apposed corneocyte lipid envelopes. The inset shows typical CLEs surrounding corneocytes in a wild-type (wt) littermate. Scale bar, 100 nm.

FIGURE 8.

Proposed model linking ceramides, EFA, and LOX in skin barrier formation. Early events (not illustrated) involve fusion of lipid-containing lamellar granules with the corneocyte plasma membrane, extruding the lipid lamellar discs extracellularly and combining the granule limiting membrane (comprised of Glc-EOS) with the cell plasma membrane, initiating formation of the CLE (shown greatly expanded). Progression toward the mature barrier entails LOX-catalyzed oxygenation of the linoleate. The resultant “oxygen signal” permits esterase-catalyzed hydrolysis of the oxidized linoleate, freeing the ceramide ω-hydroxyl for transglutaminase-catalyzed covalent coupling of the lipids to the cross-linked proteins of the CE, thus forming the CLE and helping to seal the barrier. Lipoxygenase-catalyzed oxygenation of Glc-EOS may be initiated earlier in the process than illustrated here.