title: Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes.
abstract: The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis. In cultured keratinocytes, CYP2B19 mRNAs are up-regulated coordinately with those of profilaggrin, another granular cell-specific marker. We investigated effects of the CYP2B19 metabolites 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on keratinocyte transglutaminase activities and cornified cell envelope formation. Keratinocytes were differentiated in vitro in the presence of biotinylated cadaverine. Transglutaminases cross-linked this substrate into endogenous proteins in situ; an enzyme-linked immunosorbent assay was used to quantify the biotinylated proteins. Exogenously added or endogenously formed 14,15-EET increased transglutaminase cross-linking activities in cultured human and mouse epidermal keratinocytes in a modified in situ assay. Transglutaminase activities increased approximately 8-fold (p < or = 0.02 versus mock control) in human keratinocytes transduced with adenovirus particles expressing a 14S,15R-EET epoxygenase (P450 BM3v). The physiological transglutaminase substrate involucrin was preferentially biotinylated in situ, determined by immunoblotting and mass spectrometry. P450 BM3v-induced transglutaminase activation was associated with increased 14,15-EET formation (p = 0.002) and spontaneous cell cornification (p < or = 0.001). Preferential involucrin biotinylation and the increased cornified cell envelope formation provided evidence that transglutaminases mediated the P450 BM3v-induced cross-linking activities. These results support a physiological role for 14,15-EET epoxygenases in regulating epidermal cornification, and they have important implications for epidermal barrier functions in vivo.
authors.affiliation: Department of Medicine, Division of Dermatology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Ladd
authors.forename: Liping
authors.initials: L
authors.lastname: Du
authors.forename: Jorge H
authors.initials: JH
authors.lastname: Capdevila
authors.forename: Raymond
authors.initials: R
authors.lastname: Mernaugh
authors.forename: Diane S
authors.initials: DS
authors.lastname: Keeney
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D019921:Biotinylation; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000072467:Cytochrome P450 Family 2; D004789:Enzyme Activation / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D012867:Skin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011498:Protein Precursors; C032253:involucrin; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C033914:Cyp2b19 protein, mouse; D000072467:Cytochrome P450 Family 2; D011503:Transglutaminases; D015126:8,11,14-Eicosatrienoic Acid; D002103:Cadaverine