A 12R-LIPOXYGENASE IN HUMAN SKIN - MECHANISTIC EVIDENCE, MOLECULAR-CLONING, AND EXPRESSION

A recognized feature of psoriasis and other proliferative dermatoses i s 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-lipoxyge nase and heretofore in mammals is known only as a product of cytochrom e 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 rete ntion 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 th e 12-keto derivative. Secondly, analysis of product formed from [10(R) -H-3] and [10(S)-H-3]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydr ogen from the 10-carbon of arachidonate. This result is compatible wit h 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a p45O -catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxyge nase from human keratinocytes; the cDNA and deduced amino acid sequenc es share less than or equal to 50% identity to other human lipoxygenas es. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA b y Northern analysis of keratinocytes. Identification of this enzyme ex tends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psori asis.