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Title: INHIBITION OF SPHINGOLIPID BIOSYNTHESIS DECREASES PHOSPHORYLATED ERK2 IN LLC-PK1 CELLS.

Author
item RENTZ, SARAH - PHARMACOL/UGA/ATHENS,GA
item Showker, Adele
item MEREDITH, FILMORE - RETIRED - ARS
item Riley, Ronald

Submitted to: Food and Chemical Toxicology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2004
Publication Date: 1/1/2005
Citation: Rentz, S.S., Showker, A.J., Meredith, F.I., Riley, R.T. 2005. Inhibition of sphingolipid biosynthesis decreases phosphorylated ERK2 in LLC-PK1 cells. Food and Chemical Toxicology. 43:123-131.

Interpretive Summary: Fumonisin B1 (FB1) is a fungal toxin produced by the fungus Fusarium verticillioides. It is an inhibitor of an enzyme known as ceramide synthase, a key enzyme necessary for making a unique type of fat known as sphingolipids. In kidney cells, FB1 slows cell growth and causes cells to die and also causes changes in the way that cells maintain contact with each other. The effects of FB1 on cell growth and cell death, but not the changes in cell contact can be prevented by inhibitors of another enzyme known as serine palmitoyltransferase. The way that inhibition of sphingolipid biosynthesis causes changes in cell behavior is not entirely understood. However, one protein that is suspected of being involved in the changes in cell behavior is p42 Map kinase. This study determined, in kidney cells, changes in phosphorylated p42 MAP kinase in response to various inhibitors of key enzymes of sphingolipid biosynthesis. The results show that inhibition of any of three key enzymes caused a similar decrease in phosphorylation of p42 Map kinase. This indicates that p42 Map kinase phosphorylation in kidney cells is modulated by sphingolipid biosynthesis. More specifically, decreased phosphorylation of p42 Map kinase could contribute to the changes in cell contact caused by FB1 in kidney cells.

Technical Abstract: Fumonisin B1 (FB1) 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-PK1 cells, FB1 inhibits cell proliferation and induces apoptosis, which can be prevented by inhibitors of serine palmitoyltransferase (SPT). Inhibition of SPT prevents the FB1-induced accumulation of free sphinganine, a precursor of ceramide biosynthesis. However, not all of the effects of FB1 in LLC-PK1 cells can be explained solely by the increase in free sphingoid bases. The downstream signaling pathways that are affected by FB1-induced disruption of sphingolipid biosynthesis are not well understood. This study determined, in LLC-PK1 cells, changes in phosphorylation status of p42 MAP kinase (phosphorylated ERK2 [pERK2]) 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 phosphorylation of ERK2 with no reduction in total ERK2. The co-treatment of FB1 (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB1-induced reduction in phosphorylation of ERK2, indicating that FB1-mediated changes in phosphorylation of ERK2 were independent of the FB1-induced increases in free sphinganine or reduction in ceramide. Nonetheless, the decrease in pERK2 content was dependent on inhibition of de novo sphingolipid biosynthesis. Decreased phosphorylated ERK2 could contribute to the physiological effects of FB1 in LLC-PK1 cells that are not due to alteration in pathways modulated by free sphingoid bases and their metabolites.