Modulation of Constitutive Cytochrome P-450 Expression In Vivo and In Vitro in Murine Keratinocytes as a Function of Differentiation and Extracellular Ca2+Concentration

A procedure was developed for the per cell estimation of cytochrome P-450-dependent monooxygenase activities in cultures and whole cell suspensions of murine epidermal keratinocytes (MEKs). Murine keratinocytes cultured in medium containing ≤0.04 mM Ca2+can be induced to differentiate by raising medium Ca2+concentrations to 1.2 mM. The per cell activities of the monooxygenases 7-ethoxyresorufin O-deethylase (7-ER) and 7-ethoxycoumarin O-deethylase (7-EC) were elevated ≥2090% and ≈460%, respectively, within 13-24 hr of Ca2+shift. These increases could be completely suppressed by supplementation of culture medium with actinomycin D or cycloheximide immediately prior to Ca2+shift. After prolonged culture in low Ca2+medium, some MEKs detached from the monolayer. These detached cells had the characteristics of differentiating MEKs but did not have elevated 7-EC or 7-ER activities. Percoll gradient centrifugation of freshly isolated dorsal skin MEKs was used to prepare four subpopulations that differed in their stages of terminal differentiation. 7-EC and 7-ER activities varied among these subpopulations and correlated with the degree of MEK differentiation. Specifically, the lowest and highest per cell activities (>7-fold difference) were in the basal and most differentiated spinous cell populations, respectively. Collectively, the current studies demonstrate that in vivo P-450 activities are markedly different in proliferating and differentiating MEKs and suggest that constitutive P-450 expression may be modulated as a function of changes in Ca2+concentration that occur during keratinocyte terminal differentiation.