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Title: Elevated nuclear and cytoplasmic FTY720-phosphate in mouse embryonic fibroblasts suggests the potential for multiple mechanisms in FTY720-induced neural tube defects

item GARDNER, NICOLE - Creighton University
item Riley, Ronald
item Showker, Adele
item Voss, Kenneth
item SACHS, ANDREW - Creighton University
item MADDOX, JOYCE - Creighton University
item GELINEAU-VAN WAES, JANEE - Creighton University

Submitted to: Toxicological Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2015
Publication Date: 12/29/2015
Citation: Gardner, N., Riley, R.T., Showker, A.J., Voss, K.A., Sachs, A., Maddox, J., Gelineau-Van Waes, J. 2015. Elevated nuclear and cytoplasmic FTY720-phosphate in mouse embryonic fibroblasts suggests the potential for multiple mechanisms in FTY720-induced neural tube defects. Toxicological Sciences. 150(1):161-168.

Interpretive Summary: Fumonisins are fungal toxins found in corn and in corn-based foods. They are toxic to animals. Fumonisin B1 (FB1), the most common fumonisin, causes a birth defect in mice known as a neural tube defect and is a suspected risk factor for these serious birth defects in humans. FB1 exposure leads to the accumulation of a novel type of fat called sphinganine and sphingosine and their metabolites sphinganine-1-phosphate and sphingosine-1-phosphate in cells. The 1-phosphate metabolites are involved in multiple physiologically important cell signaling pathways; however, their role in neural tube development and FB1-induced neural tube defects in mice is poorly understood. FTY720 is an FDA-approved drug that is structurally similar to sphinganine and sphingosine. Like FB1, it causes neural tube defects in mice. The drug is metabolized to FTY720-1-phosphate which, because of its structural similarity to the sphinganine- and sphingosine-1-phosphates, mimics some of the latter metabolites’ physiological functions. FTY720 is therefore a useful research tool to study physiological mechanisms that are involved in neural tube defect induction by FB1. Mouse embryonic fibroblast cells were exposed to FTY720 in culture. Significant amounts of the drug and its FTY720-1-phosphate metabolite accumulated in both the cytoplasmic and nuclear cell fractions. FTY-1-phosphate accumulation in the nuclear fractions was significantly reduced by exposing the cells to an inhibitor of the enzyme that metabolizes FTY720 in the nucleus. Furthermore, the amount of FTY720-1-phosphate found in the nucleus was associated with increased acetylation of selected proteins known as histones. Acetylation status of histones influences gene expression. These findings are important in that they suggest inappropriately high levels of sphinganine- and sphingosine-1-phosphates resulting from FB1 exposure might influence the expression of genes that are critical for proper neural tube closure. Studies comparing the biomolecular effects of FB1 and FTY720 will be useful for elucidating the mechanisms of FB1 induced neural tube defects.

Technical Abstract: FTY720 (fingolimod) is an FDA-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 four of the five 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 dual mechanisms: (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.