UVB radiation induces neutral and acidic sphingomyelinases in normal human keratinocytes: trigger for ceramide pathway.

The sphingomyelin pathway is an ubiquitous, evolutionary-conserved signaling system which transduces extracellular signal into the cell. Cleavage of sphingomyelinase (SMases), evoked by different stimuli, causes the release of ceramide, which can act as a second messenger for mediating apoptosis. Sphingomyelin can be hydrolysed by two types of SMasewith neutral or acidic pH optimum, respectively. We have shown previously that treatment with exogenous ceramides analogues was sufficient for induction of apoptosis in normal human keratinocytes. The aim of the present study was to investigate the role of neutral and acidic SMases and ceramide in the intracellular signalling generated after UVB exposure of normal human keratinocytes(NHK). NHK were cultivated with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium and, at preconfluency were irradiated with a UVB dose of 100mJ/cm2.At different times after UVB irradiation, cells were harvested for lipid extraction and for in vitro measurement of neutral and acidic SMaese enzymatic activity.Exposure to UVB radiation results in a rapid in vivo sphingomyelin hydrolysis and generation of ceramide as measured by TLC analysis. The ceramide accumulation peaked at 15 min after UVB irradiation.In vitro measurement of neutral SMases activity from UVB-treated NHK extracts, using labelled sphingomyelin as substrate, showed an induction of both neutral and acidic SMase with slightly different kinetics. These data indicate that UVB can act on cellular membranes, inducing sphingomyelin hydrolysis and ceramide production through both neutral and acidic SMases.