Citation:

Moorefield, S., D. Belair, AND B. Abbott. Disruption of epithelial cell migration as a potential mechanism of cleft palate induction. NCSOT, RTP, NC, October 30, 2017.

Impact/Purpose:

The purpose of this product was to establish an assay of epithelial cell migration, which is a key event during palate fusion. The data generated for the product suggest key cellular events and pathways needed to validate an adverse outcome pathway for cleft palate. The method for assessing epithelial cell migration behavior in vitro will enable future chemical testing to predict and prioritize which chemicals would likely interfere with palate fusion during development. This product demonstrates some validation of the in vitro model and identified that perturbation of either epidermal growth factor signaling or bone morphogenetic protein signaling interferes with epithelial cell migration and would be candidate pathways for identifying a set of chemicals from ToxCast that would likely interfere with one or more key events of palate fusion.

Description:

Cleft palate occurs in about one in seven hundred births per year, making it the most prevalent craniofacial birth defect in the world. During embryonic development, tissue fusion is a critical step in the formation of the palate, cornea, urethra, and neural tube. Epithelial cell migration, epithelial-mesenchymal transition, and apoptosis are important mechanisms of tissue fusion. Chemicals such as valproic acid and tretinoin are known to lead to cleft palate in mice, with a few incidences reported in humans. However, the mechanisms by which many environmental chemicals induce cleft palate are still unknown. Disruption of epithelial cell migration is one proposed mechanism whereby chemicals may elicit cleft palate, but few studies have robustly studied the influence of cleft palate teratogens on migration. We adapted the scratch assay to measure human epidermal keratinocyte progenitor cell migration and coupled the assay with another that measures cellular ATP content. We tested the application of the assay by examining the effects of nine different pharmacological inhibitors at sub-toxic and toxic concentrations against known epithelial cell migration pathways. We have shown preliminarily that disruption of the Hedgehog, BMP, EGF, IGF, and HGF pathways decreased epithelial cell migration, disruption of the FGF and Wnt/β-catenin pathways decreased cell viability and migration, and disruption of the Notch pathway decreased epithelial cell viability. Specifically, Erlotinib (EGF inhibitor) and K02288 (BMP inhibitor) at sub-toxic concentrations were the most potent inhibitors and elicited an 85% and 89% reduction in epithelial cell migration at 48 hours relative to the DMSO control, respectively. Both BMP signaling and EGF signaling are known to be important for palatogenesis, which suggests that the epithelial cell migration assay here can be used to identify chemical effects that are related to cleft palate. Ongoing studies address the hypothesis that cleft palate teratogens (e.g. valproic acid, tretinoin, TCDD) have the potential to disrupt epithelial cell migration. The assay format used here is designed to recapitulate an important key event of palate fusion and will enable mechanistic studies of the effects of chemicals on epithelial cell migration and viability.

Record Details: