Antisense modulation of the coding or regulatory sequence of the folate receptor (folate binding protein‐1) in mouse embryos leads to neural tube defects

Abstract

BACKGROUND: Although folic acid decreases the incidence of neural tube defects (NTDs) in humans, the mechanism for this protection is unknown. We have employed antisense technology to alter expression of the gene for the folate receptor (folate binding protein-1 [Folbp1]) in mouse embryos cultured in vitro. METHODS: Embryos were explanted on day 8 of gestation and cultured for 44 hr. Several oligodeoxyribonucleotides designed to modulate the coding region or a regulatory sequence in the 5Ј-untranslated region of Folbp1 were microinjected into the amniotic sac of embryos at the beginning of the culture period. RESULTS: Two different antisense sequences to the 5Ј and 3Ј coding region in Folbp1 produced concentration-dependent increases in the number of embryos with NTDs. Coinjection of 5-methyltetrahydrofolate with these sequences decreased the frequency of abnormal embryos. A semi-quantitative RT-PCR technique used to measure the amount of Folbp1 mRNA in treated and control embryos confirmed that the mRNA level was decreased by treatment with the antisense sequences. An antisense oligodeoxyribonucleotide to a 17 base cis regulatory element also generated a concentrationdependent increase in the frequency of embryos with NTDs, and a decrease in the level of Folbp1 mRNA. CONCLUSIONS: These results demonstrate that alterations in expression of Folbp1 by perturbing either the coding sequence or a critical regulatory cis-element can play a role in NTDs. Birth Defects Research

Maternal folate deficiency results in selective upregulation of folate receptors and heterogeneous nuclear ribonucleoprotein-E1 associated with multiple subtle aberrations in fetal tissues

“…Knockout of Folbp2 had no observed adverse effects (Piedrahita et al, 1999). Folbp1 is expressed in the cells of the neural tube during early organogenesis (Saitsu et al, 2003), and the induction of neural tube defects and neurocristopathies by "knockdown" of FRs in murine fetuses with antisense oligonucleotides on GD8.5 (Hansen et al, 2003) and by exposure of pregnant rats to anti-FR antisera (daCosta et al, 2003), collectively indicate that murine FRs play primary roles in the early development of the embryo. The recent identification of blocking autoantibodies against FRs in women with pregnancy complicated by a neural tube defect (Rothenberg et al, 2004) is an interesting human correlate to the experimental studies.…”

“…Our "knockdown" studies using antisense oligonucleotides to the 17-base cis-element of murine FR (Hansen et al, 2003) have also highlighted the potential for perturbation of the FR translational regulatory unit in the genesis of neural tube defects and neurocristopathies (Antony and Hansen, 2000). This regulatory unit involves an interaction between the FR mRNA cis-element and a trans-factor (Sun and Antony, 1996) that we identified as heterogeneous nuclear ribonucleoprotein-E1 (hnRNP-E1) (Xiao et al, 2001).…”

“…In particular, we looked for evidence of upregulation of FR and for the possibility that limiting folate could result in disturbed fetal cell proliferation and organ development. And because of the importance of hnRNP-E1 to the translational regulatory unit for FR in the fetus (Hansen et al, 2003), we determined if the expression of the murine homolog of hnRNP-E1 was altered in some tissues in folate deficiency and whether there was evidence for increased RNA-protein interaction in fetal tissues during hyperhomocysteinemia, as observed in cultured human cells (Antony et al, 2004).…”

Incrimination of Heterogeneous Nuclear Ribonucleoprotein E1 (hnRNP-E1) as a Candidate Sensor of Physiological Folate Deficiency

“…High affinity folate receptors (FR), 2 which were first introduced to the biomedical literature 30 years ago (2), are critical for the cellular uptake of 5-methyltetrahydrofolate in several human (normal and cancer) cells (3). Among three human FR (FR-␣, FR-␤, and FR-␥), the FR-␣ are ubiquitous and physiologically relevant for proper development of the neural tube and neural crest (4,5), for hematopoiesis (6), for transplacental maternal-to-fetal folate transport (7), for cerebral folate transport (8), and for renal conservation of folates (3,9,10).…”

“…For example, the mouse homologue of FR-␣ is folate-binding protein-1 (Folbp1) (4). FR also functions in murine cells (4,5,60,61), and mice have a similar regulatory unit involving the interaction between a 17-base cis-element in the 5Ј-UTR of FR mRNA and a transfactor called mouse poly(rC)-binding protein (␣CP1), which is homologous with human hnRNP-E1 and found in several murine tissues, including the brain (12,52,62,63). Based on our data on the interaction of homocysteinylated hnRNP-E1 with mRNA cis-elements from TH (45) and NF-M (31, 64 -67), it is plausible that homocysteinylation of hnRNP-E1 within murine tissues can also trigger the binding of several diverse mRNAs with common signature poly(rC)-rich or poly(U)-rich sequences (18,20,31,38,40,43,47,48,58,68,69) that are part of a nutrition-sensitive (homocysteine-responsive), posttranscriptional RNA operon in mice.…”