Bcl-2 transfected HaCaT keratinocytes resist apoptotic signals of ceramides, tumor necrosis factor alpha and 1 alpha,25-dihydroxyvitamin D-3

During the last few years increasing evidence has shown that sphingolipid m etabolites are highly bioactive compounds that play important roles in cell ular regulation. The induction of ceramide signalling in primary human kera tinocytes and HaCaT keratinocytes has recently been demonstrated using 1 al pha,25-dihydroxyvitamin D-3. The data obtained indicate that approximately one-third of the proapoptotic effect of 1 alpha,25-dihydroxyvitamin D-3, is mediated by an intracellular ceramide increase induced via tumor necrosis factor a. expression and autocrine stimulation of sphingomyelin hydrolysis. In the present study the role of bcl-2 in this process was investigated. H aCaT keratinocytes were transfected with bcl-2 and the effects of C-2-ceram ide, tumor necrosis factor ex and 1 alpha,25-dihydroxyvitamin D-3 on HaCaT keratinocytes stably overexpressing bcl-2 were determined. Apoptosis was me asured by detection of soluble DNA-histone complexes using the ELISA techni que. In situ analysis of apoptotic cells was also carried out by detecting phosphatidylserine flip using the annexin V method and by detecting DNA fra gmentation using the TUNEL assay. The results obtained showed that apoptosi s induced by C-2-ceramide, tumor necrosis factor alpha and1 alpha,25-dihydr oxyvitamin D-3 occurred in a rector-transfected clone but not in a bcl-2-tr ansfected HaCaT clone. This indicates the important role of bcl-2 in the re gulation of ceramide-mediated signalling pathways in human keratinocytes an d supports the involvement of ceramide as a signalling molecule in 1 alpha, 25-dihydroxyvitamin D-3-induced biological responses.