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A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.

ABSTRACT: A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can account for the biosynthesis. Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12R-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12S-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10R-3H] and [10S-3H]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydrogen from the 10-carbon of arachidonate. This result is compatible with 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a P450-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share 98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.

SUBMITTER: Boeglin WE

PROVIDER: S-EPMC22619 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

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A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.

Boeglin W E WE Kim R B RB Brash A R AR

Proceedings of the National Academy of Sciences of the United States of America 19980601 12

A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can ...[more]

PMID: 9618483

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Project description:BACKGROUND:Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R-lipoxygenase (12R-LOX) and other enzymes before transglutaminase (TG) attaches ?-hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross-linked by TG that has been activated by cathepsin D (CathD). OBJECTIVES:The primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R-LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH. METHODS:The first and ninth tape strip of photo-exposed (PE) cheek and photo-protected (PP) post-auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors. RESULTS:Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R-LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R-LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN-27219. However, a protease inhibitor mix reversed the negative effect of overhydration. CONCLUSION:The study adds to the understanding of the roles of 12R-LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

Dataset Information

A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.

Publications

A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.

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