Altered signal transduction in Folr1-/- mouse embryo fibroblasts
Dennis R Warner1, Cynthia L Webb, Robert M Greene
1University of Louisville Birth Defects Center, Department of Molecular, Cellular and Craniofacial Biology, School of Dentistry, KY 40292, U.S.A.
Abstract:
Mice lacking the gene for Folr1 (folic acid receptor 1) have an NTD (neural tube defect) that is rescued by maternal folate supplementation. Primary cultures of MEFs (mouse embryonic fibroblasts) were established from these embryos and the effect on various signalling pathways examined. TGFβ1 (transforming growth factor β1) inhibited the proliferation of wild-type and Folr1-/- MEFs, and folate restriction, either in growth medium or through folate uptake, led to further inhibition of growth. This effect may be Smad-independent because reporter assays using the Smad-dependent reporter, p3TP-lux, revealed attenuation of TGFβ1/Smad signalling in Folr1-/- MEFs. Signalling through the canonical Wnt pathway, measured by Wnt-3a stimulated expression of the target gene, Axin2, demonstrated increased activity in Folr1-/- MEFs. Only minor changes in the expression of a panel of TGFβ (transforming growth factor β) and Wnt pathway-associated genes were revealed when Folr1-/- MEFs were compared with wild-type cells. These results demonstrate that under conditions of reduced folate (Folr-/-) signalling, pathways crucial for proper development of the neural tube are significantly altered.
Insights
Mice lacking folic acid receptor 1 (Folr1) show neural tube defects. Folate restriction alters TGFβ1 and Wnt signaling pathways, impacting embryonic development.
Area of Science:
- Developmental biology
- Molecular signaling
- Nutritional science
Background:
- Folic acid is essential for embryonic development, particularly neural tube formation.
- Folr1 plays a critical role in folate transport and metabolism.
- Folr1 deficiency in mice leads to neural tube defects (NTDs) that can be rescued by maternal folate supplementation.
Purpose of the Study:
- To investigate the impact of Folr1 deficiency on cellular signaling pathways in mouse embryonic fibroblasts (MEFs).
- To determine how folate restriction affects TGFβ1 and Wnt signaling in the context of Folr1 deficiency.
Main Methods:
- Establishment of primary MEF cultures from wild-type and Folr1-/- embryos.
- Assessment of cell proliferation under varying folate conditions.
- Analysis of TGFβ1/Smad signaling using reporter assays (p3TP-lux).
- Measurement of Wnt pathway activity via Wnt-3a stimulated Axin2 expression.
Main Results:
- TGFβ1 inhibited proliferation in both wild-type and Folr1-/- MEFs, with enhanced inhibition under folate restriction.
- TGFβ1/Smad signaling was attenuated in Folr1-/- MEFs, suggesting a Smad-independent effect.
- Canonical Wnt pathway activity was increased in Folr1-/- MEFs.
- Gene expression analysis revealed only minor changes in TGFβ and Wnt pathway-associated genes.
Conclusions:
- Folr1 deficiency significantly alters TGFβ1 and Wnt signaling pathways.
- These pathway alterations are implicated in the neural tube defects observed in Folr1-/- mice.
- Folate availability is crucial for maintaining proper signaling balance during embryonic development.
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