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.