Effect of minoxidil on the mobility and differentiation of cultivated human keratinocytes.

The mode of topical action of minoxidil (U-10,858, CAS 38304-91-5) ist not entirely clear. The influence of minoxidil on the ultimate differentiation of keratinocytes was investigated. Human keratinocytes (HaCaT-cells) were incubated with minoxidil containing culture medium (10-250 micrograms/ml). Minoxidil inhibited in a concentration dependent manner cell mobility whereas the adhesion area and the cell circumference were increased. The minoxidil treated cultures had a higher desmosome density compared to parallel control cultures and they expressed the suprabasal keratins 1 and 10 (indicating progress in differentiation) earlier and to a larger extent than the controls. Keratins were revealed immunohistochemically and by two-dimensional polyacrylamide gel electrophoresis. The results suggest that minoxidil supports the differentiation of human keratinocytes.

Reactions of human keratinocytes in vitro after application of nicotine.

Nicotine is rapidly taken up by human keratinocytes (HaCaT cells) and after 3 h the uptake is approximately 50% of maximum. Cotinine, a metabolite of nicotine, was detected, thus demonstrating the metabolism of nicotine in HaCaT cells. Low nicotine concentrations (0.1-200 micrograms/ml) did not influence the incorporation rate of thymidine into DNA or amino acids into proteins. Inhibition of DNA and protein synthesis was only observed at concentrations > 200 micrograms/ml. After application of 400 micrograms/ml nicotine, the cells were vacuolated. This process was reversed after nicotine withdrawal. At low nicotine concentrations, no changes in microtubules and actin filaments could be detected. However, in the presence of nicotine (1-10 micrograms/ml), keratin filaments showed a more orderly pattern that controls, and the expression of the suprabasal keratins 1 and 10/11 was induced and increased according to the concentration of nicotine. The number of cornified envelopes also increased markedly. Nicotine concentrations > 100 micrograms/ml led to a disarrangement of keratin filaments and to a decrease in keratin expression and cornified envelope formation. Our results suggest that nicotine at concentrations up to 100 micrograms/ml is not an irritant but may induce cornification of the skin.

[The effect of hydrocortisone aceponate on proliferation, total protein synthesis and collagen synthesis in human skin fibroblasts in vitro]. / Wirkung von Hydrocortisonaceponat auf Proliferation, Gesamtprotein- und Kollagen-Synthese menschlicher Hautfibroblasten in vitro.

Effect of Hydrocortisone Aceponate on Proliferation, Total Protein Synthesis and Collagen Synthesis in Human skin Fibroblasts in vitro. Hydrocortisone aceponate, a new topical hydrocortisone derivative with esterification in positions 17 and 21, inhibits the incorporation of 3H-thymidine in DNA in human skin fibroblasts less strongly than the halogenated glucocorticosteroids betamethasone-17-valerate and clobetasol-17-propionate. Prednicarbate, a prednisolone derivative esterified in positions 17 and 21, possesses antiproliferative properties which are stronger than those of hydrocortisone aceponate, but weaker than those of the halogenated corticosteroids. The glucocorticosteroid effect on total protein and collagen synthesis of skin fibroblasts was determined by measurement of amino acid incorporation. In logarithmically growing cultures, the total protein synthesis rate was stimulated by low corticosteroid concentrations and inhibited by high concentrations. The strongest inhibition was obtained with halogenated glucocorticosteroids. In confluent, non-proliferative cell cultures, all glucocorticosteroids had a stimulating effect on total protein and collagen synthesis. Hydrocortisone aceponate and prednicarbate had a more favourable effect on collagen synthesis than the two halogenated glucocorticosteroid derivatives. According to these results, halogenation and the insertion of double bonds in the steroidal skeleton lead more readily to anti-proliferative effects than esterification in positions 17 and 21.

A new model of epidermal differentiation: induction by mechanical stimulation.

In vivo, epidermal cells are committed to terminal differentiation in which they undergo a series of morphological and biochemical changes. In vitro, keratinocytes are able to undergo some steps of this differentiation process only. In view of the fact that in vivo skin is continuously subjected to mechanical stress, we investigated the stimulation of differentiation of transformed keratinocytes by mechanical stimulation. The cells, grown in plastic culture dishes, were periodically treated with weights exerting a pressure of 0.015 Ncm-2. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, 3H-thymidine and 14C-amino acid incorporation, SDS polyacrylamide gel electrophoresis, and Western blotting. Following pressure treatment, the previously monolayered keratinocytes locally grew up to several layers, the number of horny scales increased and, after 4 days, the pattern of cytokeratin was modified. The total amount of keratin increased, forming granular accumulations, while the proliferation rate of the cells decreased. Both the 67 kDa and 49.5 kDa keratin subunits increased in stimulated cells. Moreover, a weak keratin band of 44 kDa appeared that was not present in controls. The results demonstrate that cyclic pressure promotes differentiation of cultivated epidermal cells.