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 ∼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.