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.
Highlights
The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis
The 14,15-epoxyeicosatrienoic acids (EETs) Epoxygenase P450 BM3v Activates Keratinocyte Transglutaminases—We examined whether keratinocyte transglutaminases were activated by 14,15-EET biosynthesized by an epoxygenase overexpressed in epidermal keratinocytes
The cytochrome P450 CYP2B19 is a P450 epoxygenase originally discovered in fetal mouse skin [11]
Summary
The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis. 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. Cornification involves extensive, transglutaminase-mediated crosslinking of scaffold proteins and keratin intermediate filaments, to form a ϳ10 –15-nm-thick cornified cell envelope (CE) just beneath the plasma membrane [8] This insoluble structure acquires functional significance as granular cells undergo programmed cell death. Their pleiotrophic effects include regulation of mitogenesis and cell survival [15,16,17,18,19], cell adhesion [20], vascular tone, ion channel function, and concentrations of intracellular calcium ions [21,22,23]
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