The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

The RFC (reduced folate carrier) is the principal mechanism by which folates and clinically used antifolates are delivered to mammalian cells. hRFC (human RFC) is subject to complex transcriptional controls and exists as homo-oligomer. To explore the post-transcriptional regulation of hRFC by exogenous folates, hRFC-null HeLa cells were stably transfected with hRFC under control of a constitutive promoter. hRFC transcripts and the total membrane protein increased with increasing LCV [(6R,S)5-formyl tetrahydrofolate (leucovorin)] with a maximum at 20 nM LCV, attributable to reduced turnover of hRFC transcripts. hRFC homo-oligomerization was unaffected by increasing LCV. Cell surface hRFC paralleled [3H]methotrexate transport and increased from 0.5 to 2 nM LCV, and then decreased (~2-fold) with increasing LCV up to 20 nM. hRFC was localized to the cell surface at low LCV concentrations (0.5-1.5 nM). However, at higher LCV concentrations, significant intracellular hRFC was localized to the ER (endoplasmic reticulum), such that at 20 nM LCV, intracellular hRFC was predominated. Our results demonstrate a novel post-transcriptional regulation of hRFC involving: (i) increased hRFC transcripts and proteins, accompanying increased extracellular folates, attributable to differences in hRFC transcript stabilities; and (ii) increased retention of hRFC in the ER under conditions of folate excess, because of impaired intracellular trafficking and plasma membrane targeting.

The functional consequences of the G80A RFC SNP on the expressed reduced folate carrier protein were evaluated by looking at the relationship between intake of folate, plasma folate and cellular stores of the vitamin. The effect on homocysteine was also examined. Homocysteine is a thiol that is known to be inversely associated with folate, and which is considered to be both thrombo- and athrogenic. At high levels, homocysteine may also interfere with nitric oxide mediated vasodilation, cause oxidative injury to, and proliferation of the vascular endothelium, and alter the elastic properties of the vascular wall, contributing to increased blood pressure. Participants (119; 52 male, 67 female) from a NSW retirement village were assessed. Independent of gender, the assimilation of folate from dietary sources into red cells showed a significant association for GG (r=0.399; p=0.022) and GA (r=0.564; p<0.0001) subjects, but not homozygous recessive (AA) individuals (r=0.223; p=0.236). The same genotype based pattern of significance was shown for the association between dietary folate and plasma folate (GG: r=0.524; p=0.002, GA: r=0.408; p=0.002). No genotype-related pattern of significance was shown for the association between dietary folate and homocysteine. When examined by gender, some differences were apparent; one-way ANOVA showed that genotype influenced diastolic blood pressure in males (p=0.019), while only females showed a significant correlation between dietary folate and blood pressure within specific genotypes (Systolic pressure GA: r=-0.372; p=0.025, carriage of A: r=0.-0.357; p=0.011. Diastolic pressure GA: r=-0.355; p=0.034, carriage of A: r=0.-0.310; p=0.029). The G80A RFC SNP had an impact on the absorption and cellular translocation of dietary folate and its association with blood pressure in an elderly population.

Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known.

RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) alpha helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311-314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1-6 (N6) and TMD7-12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6-C6 and N6-N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.

The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.

Reduced accumulation of cisplatin is the most consistent feature seen in cisplatin-resistant (CP-r) cells that are cross-resistant to other cytotoxic compounds, such as methotrexate. In this report, defective uptake of a broad range of compounds, including [(14)C]-carboplatin, [(3)H]MTX, [(3)H]folic acid (FA), [(125)I]epidermal growth factor, (59)Fe, [(3)H]glucose, and [(3)H]proline, as well as (73)As(5+) and (73)As(3+), was detected in CP-r human hepatoma and epidermal carcinoma cells that we have previously shown are defective in fluid-phase endocytosis. Downregulation of several small GTPases, such as rab5, rac1, and rhoA, which regulate endocytosis, was found in CP-r cells. However, expression of an early endosomal protein and clathrin heavy chain was not changed, suggesting that the defective endocytic pathway is clathrin independent. Reduced expression of the cell surface protein, folate-binding protein (FBP), which is a carrier for the uptake of MTX, was also observed in the CP-r cells by confocal immunofluorescence microscopy and Real-Time PCR. Reactivation of the silenced FBP gene in the CP-r cells by a DNA demethylation agent, 2-deoxy-5-aza-cytidine (DAC) demonstrates that hypermethylation occurred in the CP-r cells. The uptake of [(14)C]carboplatin, [(3)H]FA, and [(3)H]MTX increased in an early stage CP-r cell line (KB-CP1) after treatment with DAC. Both a defective endocytic pathway and DNA hypermethylation resulting in the downregulation of small regulatory GTPases and cell surface receptors contribute to the reduced accumulation of a broad range of compounds in CP-r cells.

(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O6-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2'-deoxycytidine similar values were obtained. RX-3117 also increased PCFT gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.

Acute or chronic kidney disease can cause micronutrient deficiency. Patients with end-stage renal disease, kidney transplantation or on dialysis have reduced circulating levels of folate, an essential B vitamin. However, the molecular mechanism is not well understood. Reabsorption of folate in renal proximal tubules through folate transporters is an important process to prevent urinary loss of folate. The present study investigated the impact of acute kidney injury (AKI) on folate transporter expression and the underlying mechanism. AKI was induced in Sprague-Dawley rats that were subjected to kidney ischemia (45 min)-reperfusion (24 h). Both male and female rats displayed kidney injury and low plasma folate levels compared with sham-operated rats. The plasma folate levels were inversely correlated to plasma creatinine levels. There was a significant increase in neutrophil gelatinase-associated lipocalin (NGAL) and IL-6 mRNA expression in the kidneys of rats with ischemia-reperfusion, indicating kidney injury and increased inflammatory cytokine expression. Ischemia-reperfusion decreased mRNA and protein expression of folate transporters including folate receptor 1 (FOLR1) and reduced folate carrier (RFC); and inhibited transcription factor Sp1/DNA binding activity in the kidneys. Simulated ischemia-reperfusion through hypoxia-reoxygenation or Sp1 siRNA transfection in human proximal tubular cells inhibited folate transporter expression and reduced intracellular folate levels. These results suggest that ischemia-reperfusion injury downregulates renal folate transporter expression and decreases folate uptake by tubular cells, which may contribute to low folate status in AKI. In conclusion, ischemia-reperfusion injury can downregulate Sp1 mediated-folate transporter expression in tubular cells, which may reduce folate reabsorption and lead to low folate status.

Folic acid is an essential micronutrient, deficiency of which can lead to disturbance in various metabolic processes of cell. Folate transport across intestine occurs via the involvement of specialized folate transporters viz. proton coupled folate transporter (PCFT) and reduced folate carrier (RFC), which express at the membrane surfaces. The current study was designed to identify the regulatory mechanisms underlying the effects of folate deficiency (FD) on folate transport in human intestinal cell line as well as in rats and to check the reversibility of such effects. Caco-2 cells were grown for five generations in control and FD medium. Following treatment, one subgroup of cells was shifted on folate sufficient medium and grown for three more generations. Similarly, rats were fed an FD diet for 3 and 5 months, and after 3 months of FD treatment, one group of rats were shifted on normal folate-containing diet. Increase in folate transport and expression of folate transporters were observed on FD treatment. However, when cells and rats were shifted to control conditions after treatment, transport and expression of these genes restored to the control level. FD was found to have no impact on promoter methylation of PCFT and RFC; however, messenger RNA stability of transporters was found to be decreased, suggesting some adaptive response. Overall, increased expression of transporters under FD conditions can be attributed to enhanced rate of transcription of folate transporters and also to the increased binding of specificity protein 1 transcription factor to the RFC promoter only.

Microbial metabolites, including B complex vitamins contribute to diverse aspects of human health. Folate, or vitamin B9, refers to a broad category of biomolecules that include pterin, para-aminobenzoic acid (pABA), and glutamate subunits. Folates are required for DNA synthesis and epigenetic regulation. In addition to dietary nutrients, the gut microbiota has been recognized as a source of B complex vitamins, including folate. This study evaluated the predicted folate synthesis capabilities in the genomes of human commensal microbes identified in the Human Microbiome Project and folate production by representative strains of six human intestinal bacterial phyla. Bacterial folate synthesis genes were ubiquitous across 512 gastrointestinal reference genomes with 13% of the genomes containing all genes required for complete de novo folate synthesis. An additional 39% of the genomes had the genetic capacity to synthesize folates in the presence of pABA, an upstream intermediate that can be obtained through diet or from other intestinal microbes. Bacterial folate synthesis was assessed during exponential and stationary phase growth through the evaluation of expression of select folate synthesis genes, quantification of total folate production, and analysis of folate polyglutamylation. Increased expression of key folate synthesis genes was apparent in exponential phase, and increased folate polyglutamylation occurred during late stationary phase. Of the folate producers, we focused on the commensal Lactobacillus reuteri to examine host-microbe interactions in relation to folate and examined folate receptors in the physiologically relevant human enteroid model. RNAseq data revealed segment-specific folate receptor distribution. Treatment of human colonoid monolayers with conditioned media (CM) from wild-type L. reuteri did not influence the expression of key folate transporters proton-coupled folate transporter (PCFT) or reduced folate carrier (RFC). However, CM from L. reuteri containing a site-specific inactivation of the folC gene, which prevents the bacteria from synthesizing a polyglutamate tail on folate, significantly upregulated RFC expression. No effects were observed using L. reuteri with a site inactivation of folC2, which results in no folate production. This work sheds light on the contributions of microbial folate to overall folate status and mammalian host metabolism.

Folate is involved in metabolic processes and it has been implicated in both aggravation and amelioration of seizures. The aim of the current work was to study the effect of chronic temporal lobe epilepsy (TLE) on the plasma and brain concentrations of folate and on its uptake carriers in the brain - the reduced folate carrier (RFC), folate receptor α (FRα) and proton coupled folate transporter (PCFT). We utilized the rat lithium pilocarpine model for TLE. Approximately two months following status epilepticus, rats with spontaneous recurrent seizures (SRS) were sacrificed for brain and plasma folate concentration analyses and folate uptake carrier expression studies. RT-PCR and western blot analyses were utilized for quantification of folate carriers' mRNAs and proteins, respectively. The distribution of folate carriers in the brain was studied using immunohistochemistry. In the SRS rats we found lower plasma concentrations (10 ± 0.9 in control vs. 6.6 ± 1.6 ng/ml in SRS, P < 0.05), but preserved cortical and increased hippocampal levels of folate (0.5 ± 0.1 in control vs. 0.9 ± 0.2 ng/mg in SRS, P = 0.055). Hippocampus - to - plasma ratio of folate concentration was 3-fold higher in the SRS group, compared with the controls (0.13 ± 0.03 vs. 0.04 ± 0.02, respectively; P < 0.01). mRNA and protein levels of the folate uptake carriers did not differ between SRS rats and controls. However, immunofluorescent staining quantification revealed that the emission intensity of both RFC and FRα was elevated 8-fold and 4-fold, respectively, in hippocampal CA1 neurons of SRS rats, compared to controls (P < 0.01). PCFT was unquantifiable. If corroborated by complementary research in humans, the findings of this study may be utilized clinically for supplemental therapy planning, in imaging the epileptic focus, and for drug delivery into the epileptic brain. Further studies are required for better elucidating the clinical and mechanistic significance of altered folate balances in the epileptic brain.

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis.

We investigated the molecular response to folate metabolism inhibition by exposing human lymphoblast cell lines to the methionine adenosyltransferase inhibitor cycloleucine. We carried out microarray analysis on replicate control and exposed cells by examining 47,000 transcripts on the Affymetrix HG U133 plus 2.0 arrays. We identified 13 genes that we considered reliable responders to cycloleucine treatment: chemokine receptor 3 (CXCR3), prostaglandin-endoperoxide synthase 2, growth arrest-specific 7, reduced folate carrier, klotho beta, early growth response 1, diaphanous homolog 3, prostaglandin D2 synthase (PGDS), butyrophilin-like 9, low-density lipoprotein receptor-related protein 11, chromosome 21 orf15, G-protein-coupled receptor 98 (GPR98) and cystathionine-beta-synthase (CBS). We further demonstrated that four of these genes, CXCR3, PGDS, GPR98 and CBS, consistently responded to cycloleucine treatment in additional experiments over a range of concentrations. We carried out gene-specific DNA methylation analysis on five genes, including CBS, and found no evidence that DNA methylation changes were mediating the gene expression changes observed. Pathway analysis of the microarray data identified four pathways of relevance for response to cycloleucine; the immune response NF-AT signaling pathway was the most statistically significant. Comparison with other gene expression studies focusing on folate deficiency revealed that gene products related to immune cells or the immune response is a common theme. This indicates that apart from their role in the immune response, it is likely that these gene products may also have a role to play in the cellular response to folate status.

MYCN amplification occurs in 20% of neuroblastomas and is strongly related to poor clinical outcome. We have identified folate-mediated one-carbon metabolism as highly upregulated in neuroblastoma tumors with MYCN amplification and have validated this finding experimentally by showing that MYCN amplified neuroblastoma cell lines have a higher requirement for folate and are significantly more sensitive to the antifolate methotrexate than cell lines without MYCN amplification. We have demonstrated that methotrexate uptake in neuroblastoma cells is mediated principally by the reduced folate carrier (RFC; SLC19A1), that SLC19A1 and MYCN expression are highly correlated in both patient tumors and cell lines, and that SLC19A1 is a direct transcriptional target of N-Myc. Finally, we assessed the relationship between SLC19A1 expression and patient survival in two independent primary tumor cohorts and found that SLC19A1 expression was associated with increased risk of relapse or death, and that SLC19A1 expression retained prognostic significance independent of age, disease stage and MYCN amplification. This study adds upregulation of folate-mediated one-carbon metabolism to the known consequences of MYCN amplification, and suggests that this pathway might be targeted in poor outcome tumors with MYCN amplification and high SLC19A1 expression.

Folates are a family of B9 vitamins that serve as one-carbon donors critical to biosynthetic processes required for the development and function of the central nervous system (CNS) in mammals. Folate transport is mediated by three highly specific systems: (1) folate receptor alpha (FRα; FOLR1/Folr1), (2) the reduced folate-carrier (RFC; SLC19A1/Slc19a1) and (3) the proton-coupled folate transporter (PCFT; SLC46A1/Slc46a1). Folate transport into and out of the CNS occurs at the blood-cerebrospinal fluid barrier (BCSFB), mediated by FRα and PCFT. Impairment of folate transport at the BCSFB results in cerebral folate deficiency in infants characterized by severe neurological deficiencies and seizures. In contrast to the BCSFB, CNS folate transport at other brain barriers and brain parenchymal cells has not been extensively investigated. The aim of this study is to characterize folate transport systems in the murine CNS at several known barriers encompassing the BCSFB, arachnoid barrier (AB), blood-brain barrier (BBB) and parenchymal cells (astrocytes, microglia, neurons).

Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta.

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells. We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR. The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

It is well understood that maternal folate deficiency can cause abnormal fetal development. However, the extent to which placental development and function are also dependent upon folate uptake and metabolism remains unclear. To understand which trophoblast cell types may be affected by folate deficiency or abnormal folate metabolism, we completed a comprehensive spatial and temporal protein expression analysis of folate receptor (Folr), folate transporters (proton-coupled folate receptor [Slc46a1 or PCFT] and reduced folate carrier-1 [Rfc1]) and folate metabolic enzymes (5,10-methylenetetrahydrofolate reductase [Mthfr] and methionine synthase [Mtr]) in histological sections of mouse placentas from early development (E8.5) until term (E18.5). We observed that the highest level of protein expression was during early development (E8.5-E10.5), prior to the formation of the three main layers of the mature placenta suggesting that folate uptake and metabolism may be required for placental development, itself. As expected, the labyrinth trophoblast cells, which are responsible for nutrient transport, expressed these proteins throughout pregnancy, including robust expression in the sinusoidal trophoblast giant cells that line the maternal blood spaces. Other trophoblast giant cell (TGC) subtypes (parietal-TGCs and canal-TGCs), whose function does not include nutrient transport, expressed folate transporters and enzymes from E8.5 onwards. Remarkably, these proteins were also detected in glycogen trophoblast cells from E12.5-E18.5 suggesting a new role in folate uptake and metabolism for these cells. Together, these data provide evidence that folate may be necessary for normal placental development and function, and perturbations in its availability or metabolism may lead to secondary effects on fetal development.