Characterization of the Cytochrome P450 CYP2J4: Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal

doi:10.1006/abbi.1998.0654

Archives of Biochemistry and Biophysics

Volume 353, Issue 2, 15 May 1998, Pages 257-264

https://doi.org/10.1006/abbi.1998.0654 Get rights and content

Abstract

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. ApparentK_mvalues for the formation of retinoic acids were 54 and 49 μM, respectively, and apparentV_maxvalues were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestinein vivo.

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Interestingly, low levels of Aldh1a1 expression were detected in SPL-DCs from BALB/c mice but not in those from B10.D2 mice. The ALDH1A-independent RA-producing enzymes, CYP1B1, and CYP2J6 (Chambers et al., 2007; Zhang et al., 1998), were also undetectable in DCs and CD11c− cells in the secondary lymphoid organs (Yokota et al., 2009). Therefore, RALDH2 is likely to be the most critical enzyme for RA production by DCs.
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Recent discoveries have changed our view of the evolutionary history of retinoic acid (RA) machinery. It is no longer considered a vertebrate or chordate invention but rather a common genetic toolkit of diverse lineages of metazoans. In particular, the basic machinery of RA-metabolizing enzymes, retinoid-binding proteins and RA-binding nuclear receptors has been identified in protostome and deuterostome lineages. Moreover, the retinoid content and the effects of RA treatment have been described in a number of invertebrates, although the physiological role of RA signaling outside vertebrates is still not fully understood. This review summarizes the evidence gathered over many years on the invertebrate RA system, highlighting the ancient origin of the RA genetic machinery and a basic role in neuronal differentiation. Comparison of invertebrate and vertebrate RA toolkits suggests some innovations in the RA machinery of vertebrates that might have contributed to improving the physiological control of retinoid homeostasis, compensating for vitamin A fluctuations in this lineage. Analysis of the RA machinery in invertebrates also reveals independent losses of RA components during evolution, which might be related to changes in embryonic developmental modes and the absence of the temporal collinearity of hox clusters. Additional studies analyzing the biochemical and functional characteristics of the invertebrate RA genetic machinery are warranted to lend experimental support to the hypotheses sketched in this review. These hypotheses open, however, new perspectives toward understanding how the RA genetic machinery evolved to suit the physiological and developmental requirements of metazoans.
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It will be interesting to determine whether EPA or DHA are specifically oxidized by CYP2Ps, which could suggest similar involvement by the CYP2Js and may support physiological roles for EPA and DHA metabolites. The CYP2Ps also may oxidize retinoids, suggested by the finding that CYP2Js catalyze oxidation of retinoids [56]. In summary, we have identified three new P450s defining a new vertebrate CYP2 subfamily, the fish CYP2Ps.
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^☆: This investigation was supported in part by Grants ES06258 (to L.S.K.), DK10339 (to M.J.C.), and ES07462 (to X.D.) from the National Institutes of Health.

^☆☆: Sporn, M. B.Roberts, A. B.Goodman, D. S.

²: Current address: Department of Chemistry, University of North Carolina at Greensboro, Greensboro, NC 27412.

³: To whom correspondence should be addressed at Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. Fax: 518-486-1505. E-mail:[email protected].

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Regular ArticleCharacterization of the Cytochrome P450 CYP2J4: Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal☆,☆☆

Abstract

Arch. Biochem. Biophys.

Cell

Biochim. Biophys. Acta

Biochem. Pharmacol.

J. Immunol. Methods

Anal. Biochem.

J. Biol. Chem.

Crit. Rev. Toxicol.

Drug Metab. Dispos.

The Retinoids: Biology, Chemistry, and Medicine

Regular Article
Characterization of the Cytochrome P450 CYP2J4: Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal☆,☆☆