Epoxyeicosatrienoic Acids Activate Transglutaminases in Situ and Induce Cornification of Epidermal Keratinocytes

• Published in: The Journal of biological chemistry 2003-09, Vol.278 (37), p.35184-35192
• Main Authors: Ladd, Patricia A., Du, Liping, Capdevila, Jorge H., Mernaugh, Raymond, Keeney, Diane S.
• Format: Article
• Language: English
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - pharmacology; Adenoviridae - genetics; Adenovirus; Amino Acid Sequence; Animals; Aryl Hydrocarbon Hydroxylases - metabolism; Biotinylation; Cadaverine - analogs & derivatives; Cadaverine - pharmacology; Cell Differentiation - drug effects; Cells, Cultured; Cytochrome P450 Family 2; Enzyme Activation - drug effects; Genetic Vectors; Humans; Keratinocytes - cytology; Keratinocytes - drug effects; Keratinocytes - physiology; Mice; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Peptide Fragments - chemistry; Protein Precursors - chemistry; Protein Precursors - metabolism; Skin - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Transglutaminases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M301666200

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 ∼8-fold (p ≤ 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 ≤ 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.