Neutral sphingomyelinase is modulated by glutathione in keratinocytes UVB-induced apoptosis

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which plays 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) with consequent hydrolysis of membrane sphingomyelin (SM) and generation of ceramide. Increase in intracellular ceramide level is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of apoptosis. Recent evidences have shown that reduced glutathione (GSH), but not other antioxidative agent, is able to inhibit apoptotic cell death and necrosis induced by hypoxia in PC12 cells. This protective effect is mediated by GSH direct inhibition of neutral SMase activity and ceramide formation. In the present study we report that GSH inhibits activation of neutral SMases and generation of ceramide and partially prevents apoptotic cell death induced by UVB radiation in keratinocytes. Normal human keratinocytes were cultivated with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium. At preconfluency cells were incubated with GSH for 2h and then irradiated with a UVB dose of 75mJ per cm2. At different times after UVB irradiation, cells were harvested for in vitro measurement of neutral SMaese activity, lipid extraction and Western blot analysis. In vitro measurement of neutral SMase activity showed an early induction 15 min after exposure with a subsequent decrease to control level after 2h. Exposure to UVB radiation resulted in a rapid sphingomyelin hydrolysis and generation of ceramide as measured by TLC analysis. The ceramide accumulation started at 15 min after UV exposure and progressively increased up to 24h. Addition of GSH significantly inhibited activation of neutral SMase and generation of ceramide in UVB-treated keratinocytes. Moreover UVB-induced cleavage of PARP, a marker of the apoptotic response, was partially inhibited by GSH treatment. This data indicate that GSH plays a critical role in UVB signaling pathway regulating neutral SMase activity.