Inhibition of sphingolipid biosynthesis decreases phosphorylated ERK2 in LLC-PK 1 cells

Abstract

Fumonisin B 1 (FB 1) is a fungal toxin produced by Fusarium verticillioides that inhibits ceramide synthase (CS), a key enzyme in the de novo sphingolipid biosynthesis pathway. In LLC-PK 1 cells, FB 1 inhibits cell proliferation and induces apoptosis, which can be prevented by inhibitors of serine palmitoyltransferase (SPT). Inhibition of SPT prevents the FB 1-induced accumulation of free sphinganine, a precursor of ceramide biosynthesis. However, not all of the effects of FB 1 in LLC-PK 1 cells can be explained solely by the increase in free sphingoid bases. The downstream signaling pathways that are affected by FB 1-induced disruption of sphingolipid biosynthesis are not well understood. This study determined, in LLC-PK 1 cells, changes in p42 MAP kinase (phosphorylated ERK2 [pERK2]) phosphorylation in response to various inhibitors of key enzymes of the de novo sphingolipid biosynthesis pathway (CS, SPT, and glucosylceramide synthase [GlcCer synthase]). The results show that inhibition of any of the three enzymes caused a similar decrease in the extent of phosphorylation of ERK2 with no reduction in total ERK2. The co-treatment of FB 1 (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB 1-induced reduction in pERK2 phosphorylation, indicating that FB 1-mediated changes in phosphorylation of pERK2 was independent of increases in free sphinganine or its metabolites or a reduction in ceramide. Nonetheless, the decrease in pERK2 phosphorylation was dependent on inhibition of de novo sphingolipid biosynthesis. Decreased pERK2 activity could contribute to the physiological effects of FB 1 in LLC-PK 1 cells that are not due to alteration in pathways modulated by free sphingoid bases and their metabolites but are sensitive to inhibition of glycosphingolipid biosynthesis.