UVB-radiation induces neutral and acidic sphingomyelinases in cultured normal human keratinocytes.

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces extracellular signal into the cell. During the last year increasing evidence has shown that sphingolipids may play a role in intracellular signal transduction. The action of a ligand binding to a surface receptor results in early activation of the enzyme sphingomyelinase (SMases). SMase causes hydrolysis of membrane sphingomyelin (SM) and generation of ceramide. Activation of sphingomyelin cycle has been linked to various extracellular agents such as TNF-α and ionizing radiation. Activation of these targets is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of the present study is to investigate whether activation of SMases and generation of ceramide could be induced by UVB radiation in 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 75mJ/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 starts at 15 min after UV exposure and progressively increased up to 24h.In vitro measurement of neutral SMases activity from UVB-treated NHK extracts, using labelled C14 sphingomyelin as substrate, shows that acidic SMase peaks 15 minutes after exposure to UVB, while neutral SMase peaks at 30 minutes. TUNEL shows apoptotic cells in normal human keratinocytes after UVB radiation with a peak at 48 h. These data indicate that UVB can act on cellular membranes inducing sphingomyelin hydrolysis and ceramide production through both neutral and acidic SMases.