Abstract
Ceramide, representing the central molecule in the sphingomyelin cycle, serves as a second messenger for cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis [Geilen et al., 1997a].
In the present study the effect of ceramide on proliferation, apoptosis and expression of apoptosis related genes was investigated in human keratinocytes in serum-free medium .
Because natural long-chain ceramides poorly penetrate the cell membrane, short-chain ceramide analogues were used which mimic the effects of natural intracellular ceramides. The human keratinocyte cell line HaCaT served as cell culture system.
First the effect of the ceramide analogue C2-ceramide on proliferation and apoptosis of HaCaT cells was tested in serum-free medium. The results showed that C2-ceramide inhibits cell proliferation and induces apoptosis in a concentration dependent manner. Moreover it could be demonstrated that the biologically inactive ceramide analogue C2-dihydroceramide was also able to induce apoptosis and to inhibit cell proliferation in HaCaT cells, but to a much lesser extent.
Furthermore the effect of C2-ceramide (30 µmol/l) on the expression of several apoptosis-related genes was investigated using RT-PCR.
At the mitochondrial level proteins like Bax which is pro-apoptotic and Bcl-2 which is anti-apoptotic play a major role in apoptosis. Although we measured a slightly elevated bcl-2-expression and a concurrent unaltered bax-expression, ceramide-induced apoptosis was not prevented.
The expression of p53 which is involved in controlling the cell cycle, was unchanged.
The expression of c-myc, a transcription factor which is likewise involved in control of cell cycle, was slightly elevated after 2 h, 6 h and 24 h.
c-fos and c-jun encoding two subunits of the transcription factor AP-1 (activating protein-1) showed distinct changes in gene expression. The expression of c-jun was induced within 24 h up to a 5-fold increase. In contrast c-fos was downregulated after 24 h to 50 % of the control. These data suggest that c-Jun rather than c-Fos contributes to the formation of AP-1 in this system.
In order to prove the formation and activation of AP-1 and to confirm ceramide-mediated induction of AP-1, transactivating-assays were carried out. After 24 h incubation with C2-ceramide a 8-fold increase of AP-1-activity was observed.
In conclusion the participation of AP-1 in cermide-induced apopotosis could be established on a functional level. These data confirm that transcription factor AP-1 regulates the expression of differentiation and apoptosis related genes in mammals and the proof for a direct link between an increase of intracellular ceramide and the inhibition of cell growth as well as induction of apoptosis could be presented. These findings complete the signalling pathway of vitamin D3 which we previously showed to inhibit cell growth and to induce apoptosis in keratinocytes [Geilen et al., 1997b].
In summary the following possible signal transduction pathway can be concluded:
1?,25-dihyroxyvitamin D3 -> TNF? -> Ceramide -> AP-1 -> inhibition of cell growth and induction of apoptosis. |
