DECREASED PHOSPHOLIPASE-D (PLD) ACTIVITY IN CERAMIDE-INDUCED APOPTOSIS OF HUMAN KERATINOCYTE CELL-LINE HACAT

Ceramide is recognized as an intracellular lipid second messenger, whi ch induces various kinds of cell function including apoptosis. To eval uate the competence of ceramide on the keratinocyte apoptosis, we exam ined effects of a cell-permeable ceramide, N-acetylsphingosine (C-2-ce ramide), on a human keratinocyte cell Line, HaCaT. C-2-ceramide induce d a distinct apoptosis in HaCaT cells in a time-dependent manner, as i nferred by morphologic hallmarks of apoptosis such as bleb formation, cell body shrinkage, nuclear chromatin condensation, and internucleoso mal DNA fragmentation. In sharp contrast, an inactive C-2-ceramide, di hydroC(2)-ceramide, which lacks the 4-5trans double bond, failed to in duce the apoptosis. The apoptotic HaCaT cells induced by C-2-ceramide showed a significant suppression of phospholipase D (PLD) activity, re gardless of the presence or absence of guanosine 5'-O-(3-thiotriphosph ate) (GTP gamma S). This indicates that C-2-ceramide inhibits both GTP gamma S dependent and GTP gamma S independent PLD. The membrane assoc iated GTP gamma S dependent PLD activity was stimulated by recombinant adenosine diphosphate-ribosylation factor. The adenosine diphosphate- ribosylation factor dependent and independent PLD activities were inhi bited by C-2-ceramide in a concentration dependent manner, but not by the inactive C-2-ceramide. The concentration of C-2-ceramide to inhibi t the membrane associated PLD activity was comparable with that requir ed for apoptosis induction in HaCaT cells. It was thus suggested that downregulation of PLD activity may be involved in the mechanism underl ying C-2-ceramide induced keratinocyte apoptosis.