This application addresses the role of fatty acid metabolism in hyperproliferative skin diseases where the disease phenotype leads to abnormally high levels of eicosanoids and free fatty acids in skin lesions. While there is no unifying hypothesis explaining the roles of eicosanoids in epithelial differentiation and disease, these lipids are thought to mediate signals regulating cell proliferation and differentiation. Dr. Keeney's proposal deals with the roles of lipid mediators generated by cytochrome P450 epoxygenases in the control of epithelial cell differentiation. The applicant and colleagues have characterized keratinocyte-specific epoxygenases (CYP2B19, CYP2B12) that generate epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids from arachidonic acid. Their expression in rodent skin in epidermal keratinocyte cultures is highly differentiation-specific. Their lipid mediator (EETs) are present in normal skin, and the EETs are produced from endogenous arachidonic acid in differentiated keratinocytes. The applicant hypothesizes that P450-derived eicosanoids mediate signals regulating differentiated functions in epidermal granular cells and that ablation or overexpression of P450 epoxygenases will alter this differentiated phenotype of keratinocytes. This proposal will address 1) The endogenous CYP2B19 products that are biologically active by confirming that the EETs generated by CYP2B19 are present in murine epidermal keratinocyte cultures, coincident with CYP2B19 mRNA and protein. The applicant will prove that EETs are biologically active in murine epidermal keratinocyte cultures, coincident with mRNA and protein. 2) The applicant will demonstrate a functional relationship between endogenous EET production and the differentiated keratinocyte phenotype, and that ablation of EET production alters this phenotype. In the third specific aim, the applicant will prove that EET's are biologically active in murine epidermal keratinocyte cultures by demonstrating how the differentiation and the sensitivity of these cells to extracellular Ca2+ is altered by inappropriate overproduction of EETs. Endogenous EETs will be measured using a mass spectral assay. Chemical inhibitors and antisense cDNA will be used to ablate CYP-derived EET production. Recombinant adenoviruses expressing P450 epoxygenases (including CYP2B19) will be used to overproduce EETs intracellularly. Results of these studies will identify specific cellular pathways and granular cell functions regulated by CYP-derived eicosanoids and suggest potential mechanisms for their actions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045603-02
Application #
6375162
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Moshell, Alan N
Project Start
2000-08-08
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
2
Fiscal Year
2001
Total Cost
$213,557
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Bell, Jordan C; Strobel, Henry W (2012) Regulation of cytochrome P450 4F11 by nuclear transcription factor-?B. Drug Metab Dispos 40:205-11
Wang, Ying; Bell, Jordan C; Keeney, Diane S et al. (2010) Gene regulation of CYP4F11 in human keratinocyte HaCaT cells. Drug Metab Dispos 38:100-7
Du, Liping; Yin, Huiyong; Morrow, Jason D et al. (2009) 20-Hydroxylation is the CYP-dependent and retinoid-inducible leukotriene B4 inactivation pathway in human and mouse skin cells. Arch Biochem Biophys 484:80-6
Kalsotra, Auinash; Du, Liping; Wang, Ying et al. (2008) Inflammation resolved by retinoid X receptor-mediated inactivation of leukotriene signaling pathways. FASEB J 22:538-47
Du, Liping; Neis, Mark M; Ladd, Patricia A et al. (2006) Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells. Toxicol Appl Pharmacol 213:135-44
Du, Liping; Yermalitsky, Valery; Hachey, David L et al. (2006) A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes. J Pharmacol Exp Ther 316:371-9
Du, Liping; Neis, Mark M; Ladd, Patricia A et al. (2006) Differentiation-specific factors modulate epidermal CYP1-4 gene expression in human skin in response to retinoic acid and classic aryl hydrocarbon receptor ligands. J Pharmacol Exp Ther 319:1162-71
Du, Liping; Yermalitsky, Valery; Ladd, Patricia A et al. (2005) Evidence that cytochrome P450 CYP2B19 is the major source of epoxyeicosatrienoic acids in mouse skin. Arch Biochem Biophys 435:125-33
Wang, Haoyi; Donley, Kyle M; Keeney, Diane S et al. (2003) Organization and evolution of the Cyp2 gene cluster on mouse chromosome 7, and comparison with the syntenic human cluster. Environ Health Perspect 111:1835-42
Ladd, Patricia A; Du, Liping; Capdevila, Jorge H et al. (2003) Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J Biol Chem 278:35184-92

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