RESUMEN
Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Fibroblastos/efectos de los fármacos , Fumonisinas/toxicidad , Histona Desacetilasas/metabolismo , Defectos del Tubo Neural/metabolismo , Esfingosina/análogos & derivados , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/patología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Embrión de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/patología , Ratones , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/patología , Cultivo Primario de Células , Espectrometría de Masa por Ionización de Electrospray , Esfingosina/metabolismo , Espectrometría de Masas en TándemRESUMEN
FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 in vivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Defectos del Tubo Neural/inducido químicamente , Organofosfatos/toxicidad , Esfingosina/análogos & derivados , Animales , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoplasma/metabolismo , Fibroblastos/metabolismo , Histona Desacetilasas/metabolismo , Histonas/efectos de los fármacos , Ratones Endogámicos , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/metabolismo , Organofosfatos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/toxicidadRESUMEN
BACKGROUND: Fumonisin B(1) (FB(1)) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB(1)-contaminated food is associated with increased risk for neural tube defects (NTDs). FB(1) induces NTDs in inbred LM/Bc mice. FB(1) inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. METHODS: Pregnant SWV and LM/Bc mice were treated with FB(1) (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5-8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5-8.5). LC/MS was used to detect sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB(1) (40 µM for 24 hr) and LC/MS was used to detect sphingoid base-1-phosphates. RESULTS: FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB(1) or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB(1), and Sa1P was higher in MEFs generated from the FB(1)-NTD-susceptible LM/Bc strain. CONCLUSIONS: Elevated sphingoid base-1-P after FB(1) or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB(1) or FTY720. Sa1P may represent a biomarker for FB(1)-NTD risk assessment.