A Biosynthetic Pathway Generating 12-Hydroxy-5,8,14-eicosatrienoic Acid from Arachidonic Acid Is Active in Mouse Skin Microsomes

  1. Liping Du,
  2. Valery Yermalitsky,
  3. David L. Hachey,
  4. Setti G. Jagadeesh,
  5. John R. Falck and
  6. Diane S. Keeney
  1. Departments of Biochemistry (L.D., V.Y., D.S.K.), Pharmacology (D.L.H.), and Medicine/Dermatology (D.S.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (S.G.J., J.R.F.); and Veterans Administration Tennessee Valley Healthcare System, Nashville, Tennessee (D.S.K.)
  1. Address correspondence to:
    Dr. Diane S. Keeney, Department of Medicine/Dermatology and Biochemistry, Vanderbilt University, 607 Light Hall (0146), Nashville, TN 37232-0146. E-mail: diane.keeney{at}vanderbilt.edu

Abstract

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-14C]arachidonic acid. The majority of the 14C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal 14C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Footnotes

  • This work was supported in part by the Department of Veterans Affairs and Public Health Service National Institute of Arthritis & Musculoskeletal & Skin Diseases, National Institutes of Health Grants AR45603 and AR47357 (to D.S.K.), National Institute of General Medical Sciences Grant GM31278 and the Robert A. Welch Foundation (to J.R.F.), and by the Center in Molecular Toxicology at Vanderbilt (National Institute of Environmental Health Sciences, National Institutes of Health Grant P30 ES00267).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.105.093922.

  • ABBREVIATIONS: P450, cytochrome P450; HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; 12-HETrE, 12-hydroxy-5,8,14-eicosatrienoic acid; HPLC, high-performance liquid chromatography; 12-oxo-ETrE, 12-oxo-5,8,14-eicosatrienoic acid; DiHETrE, dihydroxyeicosatrienoic acid; LC, liquid chromatography; MS, mass spectrometry; ESI, negative electrospray ionization; 15-HETrE, 15-hydroxy-8,11,13-eicosatrienoic acid; 12-oxo-ETE, 12-oxo-5,8,10,14-eicosatetraenoic acid; 12-HpETE, 12-hydroperoxy eicosatetraenoic acid; NP, normal phase; RP, reversed phase.

    • Received August 5, 2005.
    • Accepted September 14, 2005.
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  1. Published online before print September 16, 2005, doi: 10.1124/jpet.105.093922 JPET January 2006 vol. 316 no. 1 371-379
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