Center for Matrix Biology - Vanderbilt University and Medical Center

Diane S. Keeney, Ph.D.
Adjunct Assistant Professor in the Department of Medicine

	Office Address:	607 Light Hall
	Mailing Address:	607 Light Hall Nashville, TN 37232-0146
	Lab address:	F320 Acre Bldg; mail only: 607 Light Hall Vanderbilt University Nashville, TN 37232-0146
	Phone:	615-322-3318
		email

Secondary appointment in the Biochemistry department.

Research Summary

Stratified squamous epithelia are self-renewing and directly interface the environment. In mammals, they comprise competent barriers that provide protection against hostile environmental elements; consequently, they are primary sites of disease, including cancer. Proliferating keratinocytes from different stratified squamous epithelia have many similarities, but their terminal differentiation programs are quite distinct. For example, the outer cell layer of the epidermis is anucleated, dry, and relatively impermeable; that in oral epithelium is nucleated, wet, and relatively permeable. Phenotype reversal at these sites would be fatal. We are studying mechanisms that regulate these disparate phenotypes during terminal differentiation and role for cytochrome P450-derived metabolites in these processes.
Skin expresses many cytochromes P450 (CYP genes) having critical roles in endogenous and exogenous substrate metabolism (e.g., therapeutic drugs, xenobiotics, fatty acids, eicosanoids, sterols, steroids, vitamins A & D). We characterized three homologous P450s in rodent skin (CYP2B19, 2B15, 2B12). Mouse CYP2B19 and rat CYP2B12 were found to be arachidonic acid epoxygenases, which generate epoxyeicosatrienoic (EET) acids. These P450 enzymes were detected only in differentiated keratinocytes comprising the outer epithelial cell layers, and this cell-specific transcriptional regulation suggested important roles for these enzymes in the production and/or disposition of molecules affecting keratinocyte differentiation and epidermal functions.
EETs are natural epoxy fatty acids formed in cutaneous tissues and in cultured keratinocytes. In keratinocyte cultures, the CYP2B19 metabolite 14,15-EET activated transglutaminase enzyme activities and promoted cell cornification. In present studies, we aim to understand the mechanisms how P450-derived epoxy fatty acids regulate these cellular processes, which are critical to establishing a competent epidermal barrier. In other ongoing projects, we aim to identify functional homologues in human epidermis responsible for generating epoxy fatty acids and to develop a Cyp2b19 null mouse. These in vitro and in vivo studies will generate new insight into how endogenously formed lipid mediators (EETs) regulate functions of epidermal keratinocytes that are responsible for forming the stratum corneum and the water permeability barrier in mammals.
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Research Keywords

Biochemistry, Toxicology,

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Associated initiative(s):

Biomaterials

Wound Repair

Career opportunities:
None currently available

Selected Publications

» Du, Liping, Hoffman, Susan M G, Keeney, Diane S Epidermal CYP2 family cytochromes P450 Toxicol Appl Pharmacol 2004. 195:278-87

» Ladd, Patricia A., Du, Liping, Capdevila, Jorge H., Mernaugh, Raymond, Keeney, Diane S. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J Biol Chem 2003. :

» Wang, H., Donley, K.M., Keeney, D.S., and Hoffman, S.M.G. Organizaton and evolution of the CYP2 gene cluster on mouse chromosome 7, and a comparison to the syntenic human cluster. Environ. Health Perspec. Toxicogenomics 2003. in press:

» Du, L., Hoffman, S.M.G., and Keeney, D.S. Epidermal CYP2 family cytochromes P450. Toxicol. Appl. Pharmacol. 2003. in press:

» Hoffman, Susan M G, Keeney, Diane S Fine-scale mapping of CYP gene clusters: an example from human CYP4 family. Methods Enzymol 2002. 357:36-44

» Holla, V R, Adas, F, Imig, J D, Zhao, X, Price, E, Olsen, N, Kovacs, W J, Magnuson, M A, Keeney, D S, Breyer, M D, Falck, J R, Waterman, M R, Capdevila, J H Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension. Proc Natl Acad Sci U S A 2001. 98:5211-6

» Shappell, S B, Keeney, D S, Zhang, J, Page, R, Olson, S J, Brash, A R 15-Lipoxygenase-2 expression in benign and neoplastic sebaceous glands and other cutaneous adnexa. J Invest Dermatol 2001. 117:36-43

» Schneider, C, Keeney, D S, Boeglin, W E, Brash, A R Detection and cellular localization of 12R-lipoxygenase in human tonsils. Arch Biochem Biophys 2001. 386:268-74

» Hoffman, S M, Nelson, D R, Keeney, D S Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 2001. 11:687-98

» Keeney, D.S Radiolabeled cRNA and In Situ Hybridization Systematic Approach (Sundberg, J.P. and Bogess, D., eds.) 2000. :145-167

» Keeney, D.S. and Waterman, M.R Two novel sites of expression of NADPH cytochrome P450 reductase during murine embryogenesis: limb mesenchyme and developing olfactory neuroepithelia Develop. Dynamics 1999. 216:511-517

» Keeney, D S, Waterman, M R Two novel sites of expression of NADPH cytochrome P450 reductase during murine embryogenesis: limb mesenchyme and developing olfactory neuroepithelia. Dev Dyn 1999. 216:511-7

» Brash, A R, Jisaka, M, Boeglin, W E, Chang, M S, Keeney, D S, Nanney, L B, Kasper, S, Matusik, R J, Olson, S J, Shappell, S B Investigation of a second 15S-lipoxygenase in humans and its expression in epithelial tissues. Adv Exp Med Biol 1999. 469:83-9

» Keeney, D S, Skinner, C, Wei, S, Friedberg, T, Waterman, M R A keratinocyte-specific epoxygenase, CYP2B12, metabolizes arachidonic acid with unusual selectivity, producing a single major epoxyeicosatrienoic acid. J Biol Chem 1998. 273:9279-84

» Keeney, D S The novel skin-specific cytochrome P450 Cyp2b19 maps to proximal chromosome 7 in the mouse, near a cluster of Cyp2 family genes. Genomics 1998. 53:417-9

» Keeney, D S, Skinner, C, Travers, J B, Capdevila, J H, Nanney, L B, King, L E, Waterman, M R Differentiating keratinocytes express a novel cytochrome P450 enzyme, CYP2B19, having arachidonate monooxygenase activity. J Biol Chem 1998. 273:32071-9

» Keeney, D.S., Skinner, C., Travers, J.B., Capdevila, J.H., Nanney, L.B., King Jr., L.E., and Waterman, M.R Differentiating keratinocytes express a novel cytochrome P450 enzyme, CYP2B19, having arachidonate oxygenase activity J. Biol. Chem. 1998. 273:32071-32079

» Keeney, D.S., Skinner, C., Wei, S., Friedberg, T., and Waterman, M.R A keratinocyte-specific epoxygenase, CYP2B12, metabolizes arachidonic acid with unusual selectivity, producing a single major epoxyeicosatrienoic acid J. Biol. Chem. 1998. 273:9279-9284

» Keeney, D.S The novel skin-specific cytochrome P450, Cyp2b19, maps to proximal Chromosome 7 in the mouse, near a cluster of Cyp2 family genes Genomics 1998. 53:417-419

» Mason, J I, Keeney, D S, Bird, I M, Rainey, W E, Morohashi, K, Leers-Sucheta, S, Melner, M H The regulation of 3 beta-hydroxysteroid dehydrogenase expression. Steroids 1997. 62:164-8

» Str??mstedt, M, Keeney, D S, Waterman, M R, Paria, B C, Conley, A J, Dey, S K Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis Mol Reprod Dev 1996. 43:428-36

» Waterman, M R, Keeney, D S Signal transduction pathways combining peptide hormones and steroidogenesis. Vitam Horm 1996. 52:129-48

» Funk, C D, Keeney, D S, Oliw, E H, Boeglin, W E, Brash, A R Functional expression and cellular localization of a mouse epidermal lipoxygenase. J Biol Chem 1996. 271:23338-44

» Keeney, D S, Ikeda, Y, Waterman, M R, Parker, K L Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1. Mol Endocrinol 1995. 9:1091-8

» Keeney, D S Perspectives in steroid hydroxylase gene expression: novel sites of expression during embryonic development Endocr Res 1995. 21:103-7

» Keeney, D S, Jenkins, C M, Waterman, M R Developmentally regulated expression of adrenal 17 alpha-hydroxylase cytochrome P450 in the mouse embryo. Endocrinology 1995. 136:4872-9

» Jarow, J P, Keeney, D S, Robaire, B, Ewing, L L, Zirkin, B R In vivo model for chronic direct intratesticular drug administration. J Androl 1994. 15:456-61

» Keeney, D S, Murry, B A, Bartke, A, Wagner, T E, Mason, J I Growth hormone transgenes regulate the expression of sex-specific isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse liver and gonads. Endocrinology 1993. 133:1131-8

» Keeney, D S, Waterman, M R Regulation of steroid hydroxylase gene expression: importance to physiology and disease. Pharmacol Ther 1993. 58:301-17

» Keeney, D S, Naville, D, Milewich, L, Bartke, A, Mason, J I Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver. Endocrinology 1993. 133:39-45

» Keeney, D S, Mason, J I Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats. Endocrinology 1992. 130:2007-15

» Waterman, M R, Keeney, D S Genes involved in androgen biosynthesis and the male phenotype. Horm Res 1992. 38:217-21

» Naville, D, Keeney, D S, Jenkin, G, Murry, B A, Head, J R, Mason, J I Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase. Mol Endocrinol 1991. 5:1090-100

» Keeney, D S, Ewing, L L Effects of hypophysectomy and alterations in spermatogenic function on Leydig cell volume, number, and proliferation in adult rats. J Androl 1990. 11:367-78

» Keeney, D S, Sprando, R L, Robaire, B, Zirkin, B R, Ewing, L L Reversal of long-term LH deprivation on testosterone secretion and Leydig cell volume, number and proliferation in adult rats. J Endocrinol 1990. 127:47-58

» Mendis-Handagama, S M, Keeney, D S, Hardy, M P, Ewing, L L Application of the disector method to enumerate cells in the testis. Ann N Y Acad Sci 1989. 564:86-98

» Juniewicz, P E, Keeney, D S, Ewing, L L Effect of adrenocorticotropin and other proopiomelanocortin-derived peptides on testosterone secretion by the in vitro perfused testis. Endocrinology 1988. 122:891-8