Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

https://doi.org/10.1016/j.taap.2016.02.018Get rights and content

Highlights

  • FB1 treatment results in accumulation of Sa1P primarily in the nucleus of MEFs.

  • FB1 treatment and elevated nuclear Sa1P are associated with HDAC inhibition.

  • Sphk2 inhibition alone significantly decreases nuclear Sa1P in response to FB1.

  • Sphk1 and Sphk2 inhibitors prevent nuclear Sa1P accumulation in response to FB1.

Abstract

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.

Introduction

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (previously F. moniliforme), a common fungal contaminant of maize (Gelderblom et al., 1988, Marasas et al., 2004). FB1 has been found in maize and maize-based foods and feed worldwide, including in the United States (Marasas, 1995, Marasas, 2001). Exposure to FB1 has been shown to cause equine leukoencephalomalacia (ELEM) (Marasas et al., 1988, Wilson et al., 1990), porcine pulmonary edema (PPE) (Harrison et al., 1990), and hepatotoxicity, nephrotoxicity, and liver and kidney carcinomas in laboratory rodents (International Agency for Research on Cancer, 2002, International Programme on Chemical Safety, 2000). Human populations that consume large amounts of FB1-contaminated foods (maize-based foods) have demonstrated a higher incidence of esophageal (Chu and Li, 1994) and liver cancer (Sun et al., 2007), and ingestion during early pregnancy is associated with increased risk of having a child with a neural tube defect (NTD) (Hendricks, 1999, Marasas et al., 2004).

NTDs (i.e. spina bifida and anencephaly) are one of the most common types of birth defect, and occur within the first month of gestation when the developing embryonic neural tube fails to close properly. The worldwide NTD average is approximately 1 in 1000 live births; however, in regions of the world where maize is a primary food source (parts of China, Guatemala, Mexico, South Africa), the incidence of NTDs is often 6–11 times higher than the global average (Botto et al., 1999, Imhoff-Kunsch et al., 2007, Li et al., 2006, Marasas et al., 2004, Ncayiyana, 1986). The causes for NTDs vary greatly and are thought to be multifactorial, resulting from complex interactions between genetics, maternal nutrition, and environmental factors (Green et al., 2009). The ability of FB1 to interfere with folate transport and sphingolipid metabolism have been suggested as possible mechanisms linking FB1 exposure to human NTDs (Bulder et al., 2012, Marasas et al., 2004, Wilde et al., 2014).

FB1 has a chemical structure that is similar to that of the sphingoid bases deoxysphinganine (Zitomer et al., 2009), resulting in inhibition of ceramide synthases (CerS1–6), key enzymes involved in sphingolipid biosynthesis (Wang et al., 1991). This dysregulation of both the de novo and recycling pathways of sphingolipid biosynthesis (Fig. 1), causes an accumulation of the sphingoid bases, sphinganine (Sa) and sphingosine (So), and their phosphorylated metabolites, sphinganine-1-phosphate (Sa1P) (also known as dihydro-S1P) and sphingosine-1-phosphate (S1P) (Gelineau-van Waes et al., 2009, Merrill et al., 2001, Zitomer et al., 2009). Sphinganine and sphingosine are phosphorylated by sphingosine kinase (Sphk) enzymes to form Sa1P and S1P, respectively. Sa1P and S1P are bioactive signaling molecules that act as ligands for a group of five G protein-coupled receptors (GPCRs), known as sphingosine-1-phosphate (S1P1–5) receptors (Brinkmann, 2007, Callihan et al., 2012, Spiegel and Milstien, 2002). S1P receptors are found throughout the body and are involved in regulating a wide range of biological processes (Brinkmann, 2007, Rosen et al., 2009), and play a crucial role in embryonic development as regulators and mediators of neurogenesis and angiogenesis (Kono et al., 2004, Mizugishi et al., 2005).

There are two isoforms of Sphks, Sphk1 and Sphk2. These enzymes are largely homologous, sharing 80% sequence similarity, and are conserved across multiple species (Hait et al., 2006, Taha et al., 2006). Sphk1 and Sphk2 appear to have some redundant physiological functions as Sphk1- or Sphk2-null mice alone demonstrate reduced Sphk activity but appear to be viable, fertile, and lacking any abnormalities or malformations (Mizugishi et al., 2005). However, combined loss of both kinases results in embryos with severe abnormalities, including exencephaly (Mizugishi et al., 2005). Although similar, the subcellular localization of Sphk1 and Sphk2, as well as tissue distribution (Blondeau et al., 2007, Fukuda et al., 2003, Liu et al., 2000), are different. Sphk1 is predominantly cytoplasmic, moving to the plasma membrane upon activation, and stimulating DNA synthesis (Hengst et al., 2009, Igarashi et al., 2003, Inagaki et al., 2003, Olivera et al., 1999). Sphk2, however, is predominantly associated with the nucleus and causes inhibition of DNA synthesis and cell cycle arrest (Igarashi et al., 2003, Maceyka et al., 2005). Sphk1 is thought to be responsible for regulating levels of cytoplasmic and extracellular sphingoid base-1-phosphates (Sa1P and S1P) (Spiegel and Milstien, 2003), whereas Sphk2 is thought to be responsible for generating nuclear Sa1P and S1P (Hait et al., 2009, Riccio, 2010, Spiegel et al., 2012). Although it is widely thought that Sphk1 and Sphk2 have distinct subcellular localizations, these appear to be dependent on cell type and density (Hait et al., 2005, Igarashi et al., 2003).

FB1-induced NTDs have previously been studied using cultured mouse embryos (Sadler et al., 2002) and, more recently, using an in vivo mouse model (Gelineau-van Waes et al., 2005). Mouse neurulation begins at E7.5 and is usually complete by E9.5, with these two days representing the crucial window of neural tube closure. In the inbred LM/Bc mouse strain, maternal FB1 exposure during early gestation (E7.5–E8.5) results in a dose-dependent increase in the number of embryos with exencephaly (Gelineau-van Waes et al., 2005). At the highest dose of FB1 administered (20 mg/kg body weight/day), 79% of the LM/Bc embryos were affected with an NTD. In contrast, this dose induced < 1% NTDs and a significant increase in the number of resorptions in the inbred SWV mouse strain (Gelineau-van Waes et al., 2005, Gelineau-van Waes et al., 2009); suggesting that genetic background plays a role in susceptibility to NTDs and/or embryonic lethality following maternal exposure to FB1. Blood spots and plasma collected from LM/Bc and SWV mice treated with FB1 demonstrate a significant elevation in sphinganine and Sa1P, suggesting that FB1 effectively inhibits CerS in the de novo pathway of sphingolipid biosynthesis (Gelineau-van Waes et al., 2012). Significantly higher accumulation of Sa, So and their corresponding 1-phosphates in the LM/Bc strain, compared to the SWV strain, may play a role in their susceptibility to FB1-induced NTDs (Gelineau-van Waes et al., 2012). Similar strain-specific alterations in sphingolipid metabolism have also been observed in LM/Bc and SWV strain-specific mouse embryonic fibroblasts (MEFs) exposed to FB1 (Gelineau-van Waes et al., 2012). However, in that previous study, the analysis in MEFs was done using a whole cell lysis buffer and therefore did not differentiate between Sa1P accumulation in the nuclear or cytoplasmic fractions in response to FB1 (Gelineau-van Waes et al., 2012).

We previously demonstrated that administration of the S1P receptor agonist FTY720 to pregnant LM/Bc and SWV mice results in significant accumulation of the active ligand FTY720-P in embryonic tissue and the induction of NTDs in offspring from both strains (Gelineau-van Waes et al., 2012). These results demonstrate ‘proof-of-concept’ that elevated levels of the bioactive ligand FTY720-P, a known S1P receptor agonist and/or functional antagonist, is associated with NTDs in mice. Aberrant and/or sustained activation of S1P receptors by FTY720-P during early embryonic development may play a role in the failure of neural tube closure, and these findings could implicate a potential (similar) role for elevated Sa1P and altered S1P receptor-mediated signaling in FB1-induced NTDs. Most research regarding sphingoid base-1-phosphates has focused on their role as S1P receptor ligands; however, recent studies have shown that Sphk2-generated S1P/Sa1P and FTY720-P can bind to the active sites of histone deacetylases 1 and 2 (HDAC1, HDAC2), and act as endogenous inhibitors of these HDAC enzymes, leading to increased histone acetylation (Hait et al., 2009, Hait et al., 2014). Gestational exposure to the known HDAC inhibitors valproic acid (VPA) and trichostatin A (TSA) have been shown to cause NTDs in mice and/or humans (Svensson et al., 1998, Wiltse, 2005).

The mechanism(s) underlying FB1 exposure and failure of neural tube closure are undoubtedly complex, as numerous changes occur in the balance of sphingolipid metabolites (both up- and downstream of ceramide synthase) (Gelineau-van Waes et al., 2012, Marasas et al., 2004, Merrill et al., 1993) and disruptions in folate uptake and transport (Gelineau-van Waes et al., 2005, Marasas et al., 2004, Stevens and Tang, 1997). However, in order to determine whether key mechanisms for consideration could include cytoplasmic/extracellular Sa1P accumulation and activation of S1P receptors, and/or nuclear accumulation of Sa1P and HDAC inhibition, it is necessary to identify the specific subcellular compartment in which Sa1P accumulates in response to FB1. The purpose of this study was to determine the levels of Sa1P (and S1P) that accumulate in nuclear and cytoplasmic extracts after FB1 treatment in LM/Bc and SWV MEFs and to evaluate the effects of FB1 treatment on HDAC activity and histone acetylation. Further studies utilized specific Sphk1 and/or Sphk2 inhibitors in an attempt to selectively reduce cytoplasmic vs. nuclear Sa1P accumulation in response to FB1. Histone post-translational modifications (PTMs) are epigenetic modifications that affect chromatin remodeling, and have the potential to alter gene regulation. Altered gene expression during the critical window of neurulation can lead to disruption of crucial, time-sensitive signaling pathways important for proper neural tube closure. Establishing a role for cytoplasmic and/or nuclear accumulation of Sa1P in response to FB1 could provide significant insight into the potential mechanism of FB1-induced NTDs.

Section snippets

Mouse embryonic fibroblast (MEF) cell lines and treatments

Strain-specific primary MEF cell lines were generated from gestational day 12.5 embryos harvested from untreated SWV and LM/Bc dams and cultured using standard conditions as previously described (Gelineau-van Waes et al., 2012). Primary fibroblasts were routinely passaged every 3 days through initial growth and senescence. Spontaneous immortalization was achieved after more than 20 passages (Gelineau-van Waes et al., 2012). For experiments, spontaneously immortalized MEFs were cultured in 100-mm

Elevated sphingoid bases and 1-phosphates in the nucleus of LM/Bc and SWV MEFs after FB1 treatment

LC-ESI-MS/MS analysis of the cytoplasmic and nuclear extracts was performed to determine the subcellular localization of free sphingoid bases (Sa and So) and sphingoid base-1-phosphates (Sa1P and S1P) after FB1 treatment (Fig. 2A–B). Sa and Sa1P were significantly elevated in the cytoplasm and nucleus of both SWV and LM/Bc MEFs 24 h after FB1 treatment (Fig. 3A–B). Although the FB1-induced increase in Sa and Sa1P was observed in both fractions, the amount found in the nuclear fraction was

Discussion

The present study demonstrates that FB1 treatment of LM/Bc MEFs results in a significant accumulation of Sa1P, primarily in the nuclear protein fraction. The total amount of Sa1P detected in both the nuclear and cytoplasmic fractions of LM/Bc MEFs is more than 25-times the amount detected in SWV MEFs. These results parallel the strain-specific differences in accumulation of sphingolipid metabolites in blood and tissues when pregnant LM/Bc and SWV mice are treated with FB1. The amount of nuclear

Conflict of interest

All authors state no conflicts of interest.

Transparency document

Transparency document.

Acknowledgments

We would like to thank Mark Rainey for the initial development of the LM/Bc and SWV MEFs.

This work was supported by USDA-ARS NP108 in house project 6612-42000-012-00D; RC4HD067971 Eunice Kennedy Shriver National Institute of Child Health & Human Development & NIH Office of the Director; and LB692 Nebraska Tobacco Settlement Biomedical Research Development Fund.

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      Of note is the increased accumulation of SA compared to SO and its rapid conversion to SA1P that is considered to be the mediator of fumonisin induced toxic effects [169,172,173]. In LM/Bc mouse embryonic fibroblasts treated with FB1, SA1P was already shown to be able to decrease histone deacetylase activity and increased histone acetylation that could be related with neural tube defects [169]. SA1P was also shown to be related with the kidney and liver toxicity with more severe consequences for the kidney cells [166].

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