Yasushi Kikuta

Cloning and expression of a novel form of leukotriene B4 ω-hydroxylase from human liver

We have isolated and sequenced a cDNA for human liver LTB4 ω‐hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA‐deduced amino acid sequence showed 87.3% homology to that of human polymorphonuelear leukocytes (PMN) LTB4 ω‐hydroxylase (CYP4F3). Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN. The microsomes from yeast cells transfected with the cDNA catalysed the ω‐hydroxylation of LTB4 with a K m of 44.8 μM. These results clearly show that a new form of the CYP4F LTB4 ω‐hydroxylase exists in human liver.

Prostaglandin and leukotriene ω-hydroxylases

Abstract Omega and subterminal hydroxylations of prostaglandins (PGs), leukotriene B 4 (LTB 4 ) and some related eicosanoids are catalyzed by the cytochrome P450 (CYP) enzymes belonging to the CYP4A and CYP4F subfamilies. CYP4A4, which is induced in pregnant rabbits, is the only elucidated PGE ω-hydroxylase within the CYP4A subfamily. CYP4F3 is the most tissue specific and most efficient LTB 4 ω-hydroxylase, judging from its restricted localization in human polymorphonuclear leukocytes (PMN) and its very low K m value for LTB 4 . CYP4F2 is widely distributed in human liver and other tissues, and catalyzes ω-hydroxylation of various lipoxygenase-derived eicosanoids as well as LTB 4 , with relatively comparable and high K m values. CYP4F3B is very similar to CYP4F2 in its tissue localization and its K m value for LTB 4 . Human seminal vesicle CYP4F8 is the first elucidated hydroxylase with substrate specificity for PG endoperoxides, whereas ram seminal vesicle CYP4F12 is the only elucidated PGE ω-hydroxylase within the CYP4F subfamily. Rat CYP4F1, CYP4F4 and CYP4F5, and mouse Cyp4f14 have LTB 4 ω-hydroxylase activity. Three additional human, four mouse, and one fish members of the CYP4F subfamily have been identified.

Inflammation resolved by retinoid X receptor-mediated inactivation of leukotriene signaling pathways

Leukotrienes are implicated in the pathogenesis of diverse, inflammation‐driven diseases. Metabolic inactivation of leukotriene signaling is an innate response to resolve inflammation, yet little is known of mechanisms regulating disposition of leukotrienes in peripheral tissues afflicted in common inflammatory diseases. We studied leukotriene hydroxylases (CYP4F gene products) in human skin, a common target of inflammation and adverse drug reactions. Epidermal keratinocytes express at least six CYP4F enzymes;the most highly expressed and highly regulated is CYP4F3A—the main neutrophil leukotriene hydroxylase. Differentiation‐specific factors and retinoids are positive CYP4F regulators in vitro, effecting increased leukotriene B4 hydroxylation (inactivation). CYP4F expression is up‐regulated in situ in hyperproliferative dermatoses—an innate mechanism to repair and restore epidermal barrier competency—and after retinoid therapy. Enhanced CYP4F‐mediated inactivation of leukotriene signaling is a previously unrecognized antiinflammatory property of therapeutic retinoids mediated by preferential interactions between retinoid X receptors and CYP4F promoter elements in epidermal cells.—Kalsotra, A., Du, L., Wang, Y., Ladd, P. A., Kikuta, Y., Duvic, M., Boyd, A. S., Keeney, D. S., Strobel, H. W. Inflammation resolved by retinoid X receptor‐mediated inactivation of leukotriene signaling pathways. FASEB J. 22, 538–547 (2008)

PURIFICATION AND CHARACTERIZATION OF TWO NEW CYTOCHROME P-450 RELATED TO CYP2C SUBFAMILY FROM RABBIT SMALL INTESTINE MICROSOMES

Two forms of cytochrome P-450, designated P-450id and P-450ie, were purified to specific contents of 14.3 and 15.0 nmol of P-450/mg of protein, respectively, from small intestine mucosa microsomes of rabbits. P-450id and P-450ie showed apparent molecular weights of 50 and 49 kDa, respectively, on SDS-PAGE. Both P-450s catalyzed N-demethylation of nitrosodimethylamine. The NH2-terminal amino acid sequence (first 19 residues) of P-450id exhibited 74-90% identity with those of six members of the rabbit P-450 2C subfamily, except for P-450 2C3. Similarly, the NH2-terminal sequence (first 22 residues) of P-450ie showed 73-86% identity with those of the same members of the rabbit P-450 2C subfamily. The peptide mapping patterns of the two P-450s were quite different from each other. In addition, P-450id did not cross-react with the guinea-pig antibodies against P-450ie. The results indicate that rabbit small intestine mucosa contain two new distinct forms of P-450s, both of which may be classified into the 2C subfamily.

Dioxin-induced increase in leukotriene B4 biosynthesis through the aryl hydrocarbon receptor and its relevance to hepatotoxicity owing to neutrophil infiltration

Dioxin and related chemicals alter the expression of a number of genes by activating the aryl hydrocarbon receptors (AHR) to produce a variety of disorders including hepatotoxicity. However, it remains largely unknown how these changes in gene expression are linked to toxicity. To address this issue, we initially examined the effect of 2,3,7,8-tetrachrolodibenzo-p-dioxin (TCDD), a most toxic dioxin, on the hepatic and serum metabolome in male pubertal rats and found that TCDD causes many changes in the level of fatty acids, bile acids, amino acids, and their metabolites. Among these findings was the discovery that TCDD increases the content of leukotriene B4 (LTB4), an inducer of inflammation due to the activation of leukocytes, in the liver of rats and mice. Further analyses suggested that an increase in LTB4 comes from a dual mechanism consisting of an induction of arachidonate lipoxygenase-5, a rate-limiting enzyme in LTB4 synthesis, and the down-regulation of LTC4 synthase, an enzyme that converts LTA4 to LTC4. The above changes required AHR activation, because the same was not observed in AHR knock-out rats. In agreement with LTB4 accumulation, TCDD caused the marked infiltration of neutrophils into the liver. However, deleting LTB4 receptors (BLT1) blocked this effect. A TCDD-produced increase in the mRNA expression of inflammatory markers, including tumor-necrosis factor and hepatic damage, was also suppressed in BLT1-null mice. The above observations focusing on metabolomic changes provide novel evidence that TCDD accumulates LTB4 in the liver by an AHR-dependent induction of LTB4 biosynthesis to cause hepatotoxicity through neutrophil activation.

Expression and induction of cytochrome P450s in rabbit parotid glands.

Earlier, we isolated and purified five different P450 isoforms from rabbit kidney cortex microsomes, three of which are members of the CYP4A subfamily (CYP4A5, CYP4A6, and CYP4A7), with the others being CYP2B4 and CYP1A1. In contrast, P450s in parotid glands were unknown. The fact that the parotid glands bear a marked morphological and functional resemblance to kidney tissue prompted us to investigate P450s in these glands. The present study was undertaken to determine which P450 isoforms are expressed in this tissue. Microsomes from parotid glands of untreated rabbits were found to contain 42.3 pmol of P450/mg protein and to catalyze the omega-hydroxylation of laurate. Administration of di(2-ethylhexyl) phthalate (DEHP) resulted in a 7-fold increase of laurate omega-hydroxylation. This enzyme activity was greatly inhibited by pretreatment with antibodies against CYP4A5. Furthermore, parotid gland CYP4A5, CYP4A6, and CYP4A7 mRNAs were identified by RT-PCR. Moreover, the CYP4A enzymes were demonstrated immunohistochemically to be localized exclusively in the ducts of these glands. In addition to the CYP4A enzymes, immunoblot analysis revealed that CYP2B4 is constitutively present, and that CYP1A1 is induced in these glands by treatment with 3-methylcholanthrene. Taken together, we can conclude that the P450 isoforms expressed in rabbit kidney cortex and parotid glands are identical in composition.

Catalytic properties of two human leukotriene B4 ω-hydroxylase P450s

Abstract We have investigated the catalytic properties of recombinant proteins of two human leukotriene B4 (LTB4) ω-hydroxylases (CYP4F2 and CYP4F3) as well as the gene structures of these closely related enzymes. When expressed in yeast cells and purified to homogeneity, both enzymes metabolized not only LTB4, but also other lipoxygenase-dependent arachidonic acid metabolites such as 12-HETE. However, there were some striking differences between CYP4F2 and CYP4F3: (1) the Km of CYP4F2 for LTB4 (60 μM) was 94-fold higher than that of CYP4F3 (0.64 μM); (2) CYP4F3 preferentially ω-hydroxylated lipoxin B4 over lipoxin A4, whereas CYP4F2 was only capable of metabolizing lipoxin A4; (3) in contrast to CYP4F3, CYP4F2 was very unstable for storage. The CYP4F2 and CYP4F3 genes displayed a marked structural similarity. However, the structure of exon III was quite different. Variation in exon III resulted in a low homologous region (27%) within the primary structure of the enzymes.