A 12 R -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 [10 R -3 H] and [10 S -3 H]-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 <50% identity to other human lipoxygenases. 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 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.

15-Lipoxygenase-2 (15-LOX-2) Is Expressed in Benign Prostatic Epithelium and Reduced in Prostate Adenocarcinoma

“…Whereas a strong immunoreactive band was obtained with purified 15-LOX-2 protein (5 ng), no immunoreactivity was seen with purified 15-LOX-1 or 5-LOX (5 or 50 ng) or a sample of partially purified 12S-LOX (not shown). Weak immunostaining was seen with a purified preparation of a recently identified 12R-LOX, 13 which may be expected, given the greater percentage of identity between 15-LOX-2 and 12R-LOX (approximately 50%) than between 15-LOX-2 and 5-LOX, 12S-LOX, and 15-LOX-1 (approximately 40%). 8,13 However, unlike 12S-LOX, which has been reported in prostate (see Discussion), 12R-LOX has not been identified in prostate.…”

“…15-LOX-1, 15-LOX-2, and 12R-LOX 13 were prepared by bacterial expression, each including an amino-terminal histidine tag. The proteins were recovered using a nickel affinity column (Qiagen, Chatsworth, CA) essentially according to the manufacturer's instructions.…”

Identification of hydroxyeicosatetraenoic acid components of schistosomal hemozoin

“…Chiral purity of biologically derived hydroxylated fatty acids is typically attributed to the lipoxygenase (Lox) family [24][25][26][27][28][29][30][31] with preference usually shown for S-stereoisomer products. In the absence of Lox, however, free-radical mediated peroxidation of AA can yield a racemic mixture of each hydroxylated isomer.…”

Eicosanoids in skin inflammation

“…12-LOX activity is mediated by the highly abundant cytosolic leukocyte-type 12-LOX, the microsomal platelet-type 12-LOX, and the unique to mammalian skin cells, 12R-LOX [75]. The epidermis-type lipoxygenase-3 (eLOX-3) is acting in sequence with 12R-LOX contributing to the terminal differentiation of keratinocytes and integrity of the epidermal barrier [76,77].…”

“…12-HETE is the predominant eicosanoid found greatly up-regulated in psoriasis, whilst increases in the levels of PGE 2 and PGF 2α are less prominent [3,96] and 15-HETE is decreased in uninvolved psoriatic skin compared to healthy epidermis [97]. Chiral analysis has revealed the prevalence of 12R-HETE in psoriatic scales pointing at the involvement of 12R-LOX [75]. 13-(R,S)-HODE have also been found in psoriatic skin [98] suggesting not only an upregulation of LOX reactions but also the potential formation of racemic hydroxyfatty acid mixtures due to oxidative stress, another feature of this disease [99].…”