Characterizing the effects of in utero valproic acid exposure on NF-κB signaling in CD-1 mouse embryos during neural tube closure

Highlights

• Gestational valproic acid exposure perturbates NF-kB signaling in a mouse model.

• Valproic acid exposure downregulates p65, Pim-1, p105/p50 mRNA in GD9 mouse embryos.

• Downregulated p50 protein levels are observed in valproic acid induced exencephaly.

Abstract

Nuclear factor kappa B (NF-κB) is a heterodimer of protein subunits p65 and p50, that regulates the expression of a large number of genes related to cell growth and proliferation. The p65 subunit is activated after phosphorylation by Pim-1, while the p50 subunit is the cleaved product of its precursor molecule p105. Valproic acid (VPA), an antiepileptic drug, is a known teratogen and its exposure during pregnancy is associated with 1–2% of neural tube defects in the offspring. The current study aimed at investigating the effects of in utero VPA exposure on the key components of the NF-κB signaling pathway including p65, p50, and Pim-1 in CD-1 mouse embryos during the critical period of neural tube closure. Here we report that p65, Pim-1 and p105/p50 mRNA were significantly (p < 0.05) downregulated at 1 and 3 h following in utero exposure to a teratogenic dose (400 mg/kg) of VPA in gestational day (GD)9 exposed embryos. At GD13 heads of control, non-exencephalic and exencephalic embryos were used for analysis and we found significant upregulation of p65 protein expression in non-exencephalic GD13 heads while p50 protein levels were significantly downregulated in both non-exencephalic and exencephalic groups. On the other hand, p65 and p50 protein levels remained unchanged in the nuclear extracts of the VPA-exposed non-exencephalic and exencephalic GD13 embryo heads. The reported results suggest that VPA exposure perturbates p65, p105/p50, Pim-1 transcript and p65/p50 protein levels in mouse embryos.

Introduction

Embryogenesis involves processes including cell division, growth and differentiation, tightly controlled by specific molecular signaling pathways (Ufer, 2011). The brain and spinal cord develop from cranial and caudal ends of the neural tube (NT), respectively (Padmanabhan, 2006). The NT arises from the neural plate which is formed on the dorsal surface of the embryo by ectodermal thickening and differentiation under the influence of the notochord (Greene and Copp, 2014). The neural groove forms in the neural plate as a midline depression which develops into raised tissue margins that fuse in the midline forming the NT (Padmanabhan, 2006). In the mouse, NT closure begins on embryonic day 8.5, spreads bidirectionally from the sites of closure and completes on embryonic day 10 (Greene and Copp, 2014). NT development and successful closure depends on rapid growth and proliferation of neuroepithelial cells (Copp and Greene, 2013). Failed fusion of NT margins within either the cranial or caudal regions can result in NT defects (NTDs), which are multifactorial in causation (Copp and Greene, 2013). Disruptions in neural cell growth, proliferation and differentiation following exposure to exogenous agents, such as valproic acid (VPA) can result in NTDs (Wells et al., 2010).

VPA is a well-recognised treatment for certain types of seizures, mood disorders and migraines (Adedinsewo et al., 2013; Bowden, 2009) and is being increasingly prescribed as an effective treatment for psychiatric illnesses in women within the reproductive age group (Gotlib et al., 2016). VPA is a known teratogen and maternal exposure to VPA during pregnancy significantly increases the risk of major malformations (Wyszynski et al., 2005). Birth defects caused by VPA include NTDs, congenital heart defects and musculoskeletal abnormalities (Koren et al., 2006; Moore et al., 2000). The overall Incidence of NTDs is 1–2% in VPA-exposed pregnancies, while the rate of spina bifida specifically is 10–20 times the rate in the general population (Ornoy, 2009). Despite the known potential harmful effects associated with in utero VPA exposure, therapeutic use of VPA during pregnancy is often continued to prevent the adverse outcomes associated with loss of seizure control and disturbances in mood disorders (Genton et al., 2006; Tomson et al., 2016b). Perturbation in gene expression and signaling pathways involved in cell growth and proliferation are a proposed mechanism of VPA-induced NTDs, with the nuclear factor kappa B (NF-κB) being one such pathway (Dawson et al., 2006).

Members of the NF-κB family of transcription factors regulate the expression of a large number of genes related to immune responses, inflammation, cell survival and proliferation (Häcker and Karin, 2006). The NF-κB family consist of five proteins in the Rel family: RelA (p65), RelB, c-Rel, p50, and p52, which form homo- and hetero-dimeric complexes to function (Hayden and Ghosh, 2004). The p65 and p50 subunits are bound to the inactive inhibitory IκB complex and are predominantly located in the cytoplasm (Häcker and Karin, 2006). Following activation of the bound complex, the p65 and p50 heterodimer (NF-κB) is released and localizes to the nucleus (Wan and Lenardo, 2009). Recruitment of the active NF-κB heterodimer to specific DNA sites within the nucleus is fundamental for targeted gene transcription (Wan and Lenardo, 2009). To become transcriptionally active, the subunit p65 undergoes several post-translational modifications including phosphorylation by Pim-1 kinase at Ser276 (Nihira et al., 2010). The NF-κB1 (p105) gene encodes for the p105 protein which is cleaved by proteasomal degradation to produce the subunit p50 (Lin et al., 2000; Moorthy et al., 2006; Yu et al., 2009). p50 has a DNA binding domain but lacks a transactivation domain, therefore p65 and p50 need to combine as a heterodimer to regulate DNA transcription (Schwertheim et al., 2014; Yu et al., 2009).

NF-κB transcriptional activity is present in a broad range of tissues during development including multiple regions of the developing central nervous system (Dickson et al., 2004). p65 knockout mice show lethality at 15–16 days of gestation with extensive apoptosis and liver damage (Beg et al., 1995). Exposure to teratogens, such as cyclophosphamide, result in decreased NF-κB DNA-binding in the brain and liver of gestational day (GD) 12 mouse embryos (Torchinsky et al., 2002). We have previously shown that VPA exposure decreases mRNA and protein levels of p65 and nuclear p50 protein levels in P19 cells (Lamparter et al., 2017). P19 cells are derived from a pluripotent embryonal carcinoma and are used as an in vitro model to study the cellular and molecular aspects of early embryonic differentiation (McBurney, 1993).

Therefore, here we hypothesis that in utero VPA exposure results in alterations in key components of NF-κB signaling including p65, p105, Pim-1 and p50, during the critical period of NT closure in mice on GD9, GD10 and in exencephalic embryos on GD13.