Fumonisin B(1) induces apoptosis in LLC-PK(1) renal epithelial cells via a sphinganine- and calmodulin-dependent pathway.
Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B(1) kills LLC-PK(1) renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B(1)-induced cell death. Fumonisin B(1) produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B(1) blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B(1)-induced apoptosis, beta-fluoroalanine (betaFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. betaFA blocked sphinganine accumulation and prevented fumonisin B(1)-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B(1) is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B(1). Differential expression in response to fumonisin B(1) of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because betaFA reduces induction of calmodulin mRNA by fumonisin B(1). Fumonisin B(1) also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B(1)-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B(1)-induced apoptosis. In contrast, fumonisin B(1) had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B(1) kills LLC-PK(1) kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B(1)-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.
Kim
M S
MS
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan, 48824-1224, USA.
Lee
D Y
DY
Wang
T
T
Schroeder
J J
JJ
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism; D002264:Carboxylic Acids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D003720:Densitometry; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D037341:Fumonisins; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018374:LLC-PK1 Cells; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013552:Swine; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000963:Antimetabolites; D002147:Calmodulin; D002264:Carboxylic Acids; D037341:Fumonisins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019938:Cyclin D1; C056933:fumonisin B1; C007742:3-fluoroalanine; D013110:Sphingosine; D000409:Alanine; C005682:safingol