Involvement of sphingoid bases and their metabolites in fumonisin B1-induced alterations in protein kinase C-mediated signaling in LLC-PK1 cells

Abstract

Fumonisin B1 (FB1), produced by the mycotoxin of Fusarium verticillioides, is a carcinogen and causes various species-specific toxicoses. FB1 inhibits ceramide synthase and induces tumor necrosis factor a (TNFa) expression. Modulation of protein kinase C (PKC) by FB1 may be involved in its cytotoxicity in porcine renal epithelial (LLC-PK1) cells. Temporal effects of 1 mM FB1 revealed selective and transient increased cytosol to membrane translocation of PKCa exclusively at 5 min, which was correlated with an increase in PKC activity. FB1-induced increased cytosol PKCa protein concentration at 15 min was not associated with increased activity and independent of protein biosynthesis. A concentration-dependent increase in membrane PKCa was observed on exposure to FB1 concentrations of 0.1-1 mM. Intracellular sphinganine and sphingosine concentrations were unaffected by FB1 up to 120 min. However, myriocin, the serine palmitoyltransferase inhibitor, did not prevent the short-term FB1 effects on PKCa. PKCa activation by 1 mM FB1 was directly correlated with activation of the transcription factor, nuclear factor-kappa B (NF-kB) at 5 min. Sequential activation of TNFa mRNA expression at 15 min and caspase 3 at 60 min by 1 mM FB1 via a PKC-dependent pathway was also observed in LLC-PK1 cells. A concentration-dependent inhibition of PKC-a, -d, -e and -z isoforms, NF-kB, and TNFa was observed on exposure to FB1 concentrations of 1-50 mM at 24, 48, and 72 h in LLC-PK1 cells. FB1-induced apoptosis (³ 10 mM) at 48 h was associated with an increase in caspase-3 activity. Intracellular sphinganine and sphingosine concentrations were increased in a concentration-dependent manner. Exogenous sphinganine 1-phosphate stimulated cytosolic to membrane translocation of PKCa comparative to 10 mM FB1 at 5 min. Inhibition of sphinganine kinase by N,N-dimethylsphingosine, prevented the FB1-induction of PKCa at 5 min, suggesting that the selective and transient activation of PKCa is due to the FB1-induced accumulation of sphinganine 1-phosphate. FB1, sphinganine, sphingosine and ceramide repressed all PKC isoforms at 48 h. Co-exposure of myriocin prevented the inhibitory effects of FB1 on PKC isoforms in LLC-PK1 cells, suggesting that accumulation of sphinganine and its metabolite may be predominantly involved in the repression of PKC in LLC-PK1 cells.