Cotovio, J., F. Fiat, C. Ferraris, M. Baur, A. Hertzeisen, and P. Catroux.� Effect of Calcium on In Vitro Differentiation of Immortalised Adult Human Kerationcytes DK7-NR in Immersed Conditions:� Comparison with Cultured Normal Human Keratinocytes.� ATLA 1999.� 27:� 298.

 

Critical events associated with commitment of normal keratinocytes to terminal differentiation are not fully understood. Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes.� Similarly, constitutive activities such as PAH-responsive monooxygenases in keratinocytes may be modulated as a function of the calcium gradient established during cell differentiation. Differentiation-promoting culture conditions, such as serum and calcium, have also been reported to be required for P4501A1 mRNA induction in human keratinocytes (Berghard et al. Journal of Biological Chemistry 265:� 21, 086-21,090, 1990) and to enhance oxidative stress defenses.� We studied the effect of calcium concentration in the culture medium on the immortalized human keratinocytes, DK7-NR (Baur et al. in preparation) compared to normal human keratinocytes (NHK).� 3-Methylcholanthrene induction of ethoxyresorufin-O-deethylase (EROD), mainly supported by CYP1A1, appeared to be dependent on high Ca2+ and culture time.� Maximum inductions were obtained after 3 days in 1.5 mM Ca2+ for DK7-NR, while NHK only needed one day.� In addition, DK7-NR was more sensitive to high calcium (Ca+) than were NHK (respectively 50 times and 2 times level increase as compared to 0.11 mM Ca2+).� Early differentiation markers (keratin K1/10) and late differentiation markers (involucrin, filaggrin, loricrin), as well as transglutaminase were detected by immunochemistry.� Switching confluent cells from low to high Ca2+ for 72 hours showed a strong up-regulation of these markers.� The proliferation status was also evaluated by labeling the cells with Ki67 (nuclear antigen expressed in proliferating cells).� Under low calcium condition, almost all the cells were positive.� Switching to high calcium did not significantly affect this marker.� Electron microscopy images of DK7-NR revealed, at low Ca2+, the presence of flat and spread cells with wide intercellular spaces.� Cell-cell connections involved microvilli.� Only immature desmosomes were present, as well as unstratified monolayer with cells partially attached to the coated surface.� 72 hours after switching to high Ca2+, a multilayered epithelium with at least three layers was observed, which had spread keratin filaments, abundant well-differentiated mature desmosomes and numerous associated bundles of tonofilaments.� Few differences between DK7-NR and NHK ultrastructure were observed under identical culture conditions.� One difference is the ability of NHK to stratify (e to 4 layers) even at low Ca2+ concentration, however, desmosal structures remain immature.� In conclusion, these results demonstrate that immortalized DK7-NR keratinocytes can reach differentation and stratify under controlled in vitro calcium concentration, even in immersed conditions.� Similarity to NHK makes these cells a useful in vitro model for pharmacotoxicological studies.