ABSTRACT
During the last few years increasing evidence has shown that sphingolipid metabolites are highly bioactive compounds that play important roles in cellular regulation. The induction of ceramide signalling in primary human keratinocytes and HaCaT keratinocytes has recently been demonstrated using 1 alpha,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. expression and autocrine stimulation of sphingomyelin hydrolysis. In the present study the role of bcl-2 in this process was investigated. HaCaT keratinocytes were transfected with bcl-2 and the effects of C(2)-ceramide, tumor necrosis factor alpha and 1 alpha,25-dihydroxyvitamin D(3) on HaCaT keratinocytes stably overexpressing bcl-2 were determined. Apoptosis was measured by detection of soluble DNA-histone complexes using the ELISA technique. In situ analysis of apoptotic cells was also carried out by detecting phosphatidylserine flip using the annexin V method and by detecting DNA fragmentation using the TUNEL assay. The results obtained showed that apoptosis induced by C(2)-ceramide, tumor necrosis factor alpha or 1 alpha,25-dihydroxyvitamin D(3) occurred in a vector-transfected clone but not in a bcl-2-transfected HaCaT clone. This indicates the important role of bcl-2 in the regulation of ceramide-mediated signalling pathways in human keratinocytes and supports the involvement of ceramide as a signalling molecule in 1 alpha,25-dihydroxyvitamin D(3)-induced biological responses.
Subject(s)
Apoptosis , Calcitriol/pharmacology , Genes, bcl-2 , Keratinocytes/drug effects , Sphingosine/analogs & derivatives , Tumor Necrosis Factor-alpha/pharmacology , Cell Line , DNA Fragmentation , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Genetic Vectors , Humans , Keratinocytes/physiology , Phosphatidylserines/analysis , Sphingosine/pharmacology , TransfectionABSTRACT
The generation of lipid second messengers via phosphatidylcholine (PC)-specific phospholipase D (PLD) has emerged as an important step leading to transduction of extracellular signals. In the present investigation the expression of human cytosolic PLD isoenzymes in the immortalized human keratinocyte cell line HaCat was determined. At the mRNA level we found the expression of hPLD1b and for the first time in human cells also the expression of hPLD2. For further analysis of enzyme expression at the protein level, hPLD1 peptide fragments were synthesized and specific antibodies were generated (rabbit) to be used for detection of hPLD1 in Western blot experiments. Furthermore, small G-proteins were found to be involved in the regulation of PLD activity in HaCaT cells using the guanine nucleotide analogue GTPgammaS.
