Sphingoid bases and their phosphates: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in fumonisin B sub(1) cytotoxicity

Fumonisin B sub(1), a potent inhibitor of ceramide synthase, leads to accumulation of sphinganine, and later sphingosine, in vivo and in vitro. Fumonisin B sub(1) modulates the activity of protein kinase C (PKC), however, which metabolite of disrupted sphingolipid metabolism is involved, has not been ascertained. In the present study, we evaluated the modulation of PKC by sphingolipid bases and their metabolites using exogenous sphingolipid analogues in porcine renal epithelial (LLC-PK sub(1)) cells. In preliminary studies we found that fumonisin B sub(1) (1 mu M) selectively and transiently activated PKC alpha , whereas fumonisin B sub(1) concentrations of 1-50 mu M at 48 h repressed PKC- alpha ,- delta ,- epsilon and- zeta isoforms in a concentration-dependent manner. Addition of exogenous sphinganine-1-phosphate (1 mu M for 5 min) alone stimulated cytosolic to membrane translocation of PKC alpha . Co-exposure of fumonisin B sub(1) with N, N-dimethylsphingosine, an inhibitor of sphingosine/sphinganine kinase, prevented the effects of fumonisin B sub(1) on PKC alpha . Sphinganine, sphingosine, sphingosine-1- phosphate and ceramide (all at 1 mu M) added exogenously, did not alter PKC alpha cytosolic to membrane translocation at 5 min. Fumonisin B sub(1) (10 mu M), sphinganine, sphingosine and ceramide (1 mu M each) significantly repressed PKC- alpha and- delta isoforms at 48 h, whereas all the exogenously added sphingolipids significantly repressed PKC- epsilon and zeta similar to fumonisin B sub(1). Co-exposure of myriocin with fumonisin B sub(1) prevented the delayed inhibitory effects of fumonisin B sub(1) on PKC isoforms in LLC-PK sub(1) cells. This study demonstrated that selective and transient activation of PKC alpha may be due to the fumonisin B sub(1)-induced accumulation of the bioactive sphinganine-1-phosphate, whereas the long-term repression of PKC isoforms may be predominantly due to the accumulation of sphinganine or its metabolite, and to a lesser extent sphingosine or its metabolite in LLC-PK sub(1) cells. These findings suggest that the direct or indirect modulation of PKC by these sphingolipids is involved at least in part in the action of fumonisin B sub(1).