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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Veterans Affairs Medical Center, Long Beach; and 2Departments of Medicine (Nephrology) and Physiology/Biophysics and 3Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, California
Submitted 4 October 2006 ; accepted in final form 27 April 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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integrative transport physiology; folate transport; transport regulation
The nematode Caenorhabditis elegans has been used as an animal model in which to delineate molecular mechanisms of complicated functions (31, 32). This animal model has a host of unique features that include simple anatomy (it has a total of 959 highly differentiated cells), transparency, defined genomic and tractable genetics, ease of maintenance and growth, a defined life cycle, and a short lifespan (2–3 wk). In addition, many human physiological functions appear to have analogs in this animal model, and many human genes have orthologs in the genome of this nematode, i.e., 28.4% of the worm genome has one or more human orthologs, and also 83% of worm proteins have domains with significant similarity to human genes (24, 34). Furthermore, this animal model allows great flexibility in manipulating physiological events and in performing certain experiments that are otherwise difficult to perform at the whole animal level in vivo in more complicated organisms (e.g., quantitative imaging and promoter analysis both can be performed at the whole animal level) (31, 32). Using this animal model, we have undertaken a series of investigations into the integrative aspects of the folate uptake process.
In this report, we present our findings on the cloning of a C. elegans folate uptake system, folt-1, the functional characterization both in vitro and in vivo, and the effect of folt-1 on development and the prevailing substrate level on different parameters of the folate uptake process at the level of whole animal. Our results show for the first time the existence of a specialized folate uptake system ( folt-1) in this species. This system appears to be similar in many ways to the folate uptake process that operates in the human intestine, being more active at acidic compared with alkaline buffer pHs, having similar affinity to reduced and substituted folate derivatives, being sensitive to the effects of the anti-inflammatory agent sulfasalazine, being inhibited by the anion transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and having a similar apparent Km. In addition, the folt-1 system was found to be expressed in different tissues of the nematode and appears to be developmentally and adaptively regulated.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Nematode growth. The wild-type nematode strain is C. elegans N2. For routine experiments, the animals were maintained at 15–20°C on nematode growth medium (NGM) agar plates, and Escherichia coli strain OP50 was used as the food source (4). Total RNA were prepared from worms by freezing the pelleted worms in liquid nitrogen and grinding them in the presence of TRIzol, as described by the manufacturer's protocol (Life Technologies, Rockville, MD).
Cloning of RFC-like transporters from C. elegans.
Three genes have been reported in the worm genome (http://www.wormbase.org) as being hRFC-like genes: C06H2.4, C50E3.11, and F37B4.7. These genes were named folt-1, folt-2, and folt-3, respectively. Our own search of the C. elegans genome has confirmed that the C. elegans genes 5L621 and 5D352 are indeed putative hRFC-like transporters sharing 40 and 31% identity, respectively, with the hRFC, but C50E3.11 did not show significant sequence homology with the hRFC gene. Thus we have renamed the putative folt-1 and folt-3 as folt-1 and folt-2, respectively, to avoid confusion. To date, no other gene with similarity to the hRFC gene was found. We also searched for orthologs to the human folate receptor and the proton-coupled folate transporter/heme carrier protein (PCFT/HCP1) in the worm genome but found none. We focused our investigations on the putative folt-1 and folt-2. To clone these C. elegans RFC-like transporters, we obtained a RT-PCR product using C. elegans poly(A)+ RNA and primers designed on the basis of the predicted exonic sequences of these genes. Briefly, total RNA was isolated from adult C. elegans using TRIzol reagent. A pair of PCR primers specific for the putative folt-1 and folt-2 genes was designed based on the sequences of the cosmids C06H2.4 and F37B4.7 (http://www.wormbase.org). Poly(A)+ RNA was used as template to perform the RT-PCR, employing the Superscript RT-PCR kit (Invitrogen) to synthesize first-strand cDNA. To amplify the open reading frame (ORF) of the putative folt-1 and folt-2, we used the following two gene-specific primers: for folt-1, the forward primer was 5'-CCGCTC GAGATGAGCTGGCGTACCAC-3' and the reverse primer was 5'-CGGGATCCTCAATTTT GGTCTAGAAAGACTG-3'; for folt-2, the forward primer was 5'-ATGGAGCAATGGAA AGTGATG-3' and the reverse primer was 5'-TCAATTAGTACTCGTTTTGAAAAACCG-3'. The PCR conditions used were as follows: 95°C/10 min for 1 cycle; 95°C/30 s, 54°C/1 min, and 72°C/3 min for 40 cycles. A single PCR product was obtained for each ORF with an estimated size of 
1.2 and 1.6 kb for the folt-1 and the folt-2 genes, respectively, as predicted by the distance between these primers in each pair. The PCR products were gel purified and subcloned into pGEM-T Easy Vector (Promega, Madison, WI). The molecular identity of the inserts was established by sequencing (Laragen). The identified ORF were subcloned into the mammalian expression vector pLenti6/V5-Dest, again verified by sequencing, and then expressed in the human retinal pigment epithelial cells (ARPE-19) to determine functionality.
Functional expression of the cloned folt-1 and folt-2 in ARPE-19 cells using a lentiviral expression system. The ARPE-19 cells have been successfully used to functionally characterize cloned C. elegans transporters (6, 7, 41). Using these cells, we expressed the putative folt-1 and folt-2 using the lentivirus expression system as described previously (17, 22). Viral stocks were prepared using folt-1 and folt-2-cDNA and the pLenti6/V5-Dest kit (Invitrogen) as per the manufacturer's protocols. ARPE-19 cells (60–70% confluent) were transiently transfected with 10 µl of pLenti6/V5-DEST folt-1 or folt-2 cDNA virus per well of a 12-well plate in the presence of polybrene (Fisher Scientific, Tustin, CA), i.e., infected with a lentivirus at a multiplicity of 5–10 plaque-forming units/cell. The cells were incubated at 37°C for 72 h and used for determination of transport activity. Cells transfected with vector alone without the cDNA insert were used as the control to determine endogenous transport activity in these cells. [3H]folate uptake was determined at 37°C in Krebs-Ringer buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 5.5; unless otherwise stated). The 3H-radioactivity taken up by the cells was determined by means of scintillation counting. Protein content of cell digests was measured in parallel wells using a Bio-Rad protein assay kit (Bio-Rad, Richmond, VA). Transport activity attributable to the expressed folt was determined by subtracting folate uptake by vector-transfected ARPE-19 cells from total uptake by folt-1- and folt-2-expressing cells.
Folate uptake by the whole C. elegans.
The simplicity of the C. elegans body and its demonstrated ability to take up large and small molecules from exogenous sources (e.g., large dsRNAs; Refs. 8, 15) have led us to test the [3H]folic acid uptake at the whole animal level. To obtain synchronized populations, we isolated eggs from gravid adult animals that were treated with a hypochlorite-NaOH solution (14) to isolate eggs. Eggs were incubated in M9 buffer and allowed to hatch overnight (12 h) at room temperature. The resulting synchronized L1-stage worms were put on standard NGM plates with feeding bacteria (25°C) to develop to different stages. Worms at different developmental stages were collected, washed with M9 buffer (containing 0.01% Triton X-100, Sigma), gently pelleted by centrifugation, and washed several times to remove residual OP50 bacteria. They were then used in functional uptake assays at the whole animal level to determine mRNA levels and green fluorescent protein (GFP) expression patterns. To examine the effect of folt-1 gene-specific RNA interference (RNAi) on folate uptake, we followed the protocol described by Timmons and Fire (35). The commercially available folt-1 gene-specific RNAi feeder clone, obtained from Open Biosystems (Huntsville, AL), was transformed into an E. coli strain, HT115 (DE3). The transformed bacteria culture was grown overnight and then induced with 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG) for 3 h; it was then seeded on NGM plates containing 1 mM IPTG. To test the effect of folt-1RNAi, synchronized young adult C. elegans were transferred to the plates, and the functional uptake assay was performed after 16 h of pretreatment. For the whole animal uptake studies, five age-synchronized individual young adult nematodes were placed in a test tube and preincubated in Krebs-Ringer buffer for 20 min at room temperature. [3H]folic acid was then added, and the reaction was terminated after 5 min (initial rate; data not shown) by the addition of 1 ml of ice-cold Krebs-Ringer buffer followed by immediate placing of the animals on Millipore filters (0.22 micron) under negative pressure, followed by 3x washing with ice-cold buffer. The filters with the animals on them (verified by microscope) were then transferred into vials containing scintillation fluid and counted for radioactivity.
Semiquantitative RT-PCR.
An RT-PCR assay (using folt-1-specific primers) was used to study the level of expression of the endogenous folt-1 mRNA under different conditions [different developmental stages: early larvae (L1–L3), young adult, and adult; different level of exogenous folate]. Poly(A+) RNA was used as a template to perform reverse transcription using an RT-PCR kit. The reverse transcription was initiated with random oligonucleotides and carried out in a DNA thermal cycler (Light Cycler PCR System, Bio-Rad Laboratories, Hercules, CA) as per the manufacturer's procedures. Reverse transcription was followed by real-time PCR in a single-well format in which the gene-specific primers and the primers for the housekeeping gene (-actin) with their PCR mix (SYBR Green kit, Qiagen, Valencia, CA) were combined separately at a predefined ratio. The PCR cycle number was titrated according to the manufacturer's protocol to ensure that the reaction was within the linear range. The resultant PCR products were monitored during real time and then resolved in a 3.0% agarose gel for further confirmation. The steady-state levels of folt-1 mRNA were assessed from the cycle threshold (Ct) values during real-time PCR of the folt-1-specific RT-PCR product relative to the Ct values of the -actin at each developmental stage/condition and were calculated using a relative relationship method supplied by the iCycler manufacturer (Bio-Rad).
Analyses of folt-1 knockout.
To further our understanding of the role of folt-1 in folate uptake in C. elegans, we obtained a deletion strain, VC959 {WormBase deletion strain: VC959;tag-330(ok1460)V/nT1 [qIs51] (IV;V); C. elegans gene knockout (KO) consortium, Oklahoma Medical Research Foundation, Oklahoma City, OK}, which has a deletion of 1,330 bp that covers part of the first exon to the 3'-untranslated region of folt-1 (see Fig. 7C), resulting in complete removal of the coding sequences that code for most of the TM domains of folt-1. As per the KO consortium report, the homozygous ok1460 animals are sterile, but the basis of the sterility and at what stage the sterility occurs are not clearly known. These animals do not produce offspring, so the ok1460 mutation has been balanced by nT1[qIs51]. The heterozygote strains can be maintained and produce offspring. The nT1[qIs51] animals are also not viable. So, the only animals that were obtained on the plate were fertile heterozygotes and sterile ok1460 homozygotes. We furthered our studies with the sterile ok1460 homozygotes. We have selected the homozygote worms on the basis of their phenotype (slow movement on the plate) and non-GFP expression for uptake studies. The additional confirmations were performed by single-worm PCR (39), using the following nested primers: forward primers, 5'-TCTGCAACCGCAACGTATAA-3' and 5'-TTTCACCGGTCCATGAAAGT-3'; reverse primers, 5'-TAACC-TTACTTCGACTT-3' and 5'-TCTT-GGCTCGGAGAA-3'. They produced a PCR product of 2,250 and 1,040 bp for wild-type and folt-1 KO animals, respectively.
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50 ng of C. elegans genomic DNA were used to clone the entire (1.4 kb) 5'-regulatory region of the folt-1 gene (including its ATG). Then, we generated a transcriptional fusion construct that contains a 1.4-kb 5'-regulatory region of the folt-1 gene with the GFP reporter gene obtained from the pPD95.75 vector (gift from A. Fire, Carnegie Institution of Washington, Baltimore, MD) by PCR (11). The fusion construct was verified by sequencing and was microinjected into the syncytial gonad of adult wild-type C. elegans. The plasmid pRF4 containing the dominant injection marker Rol-6 (20) was coinjected as a transgenic marker. Transformants were scored on the basis of roller phenotype behavior, and GFP expression was observed under a fluorescence microscope (Zeiss Axio Plan II equipped with a fluorescence light source; Oberkochen, Germany). Four independent lines carrying extra chromosomal arrays were obtained, and all gave similar patterns of GFP expression. The measurement of the GFP fluorescence was done at the anterior, central, and posterior intestine of these transgenic lines. The transgenic animals were recorded for their GFP fluorescence with fixed exposed time, and the intensities were measured easily using Adobe Photoshop. The area of selection from each animal was identical for all of our measurements. Effect of exogenous folate level on expression of folt-1 and on folate uptake by the whole living C. elegans. The effects of oversupplementation of C. elegans with folate on the levels of expression of folt-1 and on the uptake of folate by the whole living C. elegans were examined. In these studies, wild-type and transgenic C. elegans expressing the transcriptional construct folt-1::GFP were incubated for 24 h on nematode culture medium petri dishes supplemented with a larger dose of folate (1 mM) along with an E. coli OP50 bacteria lawn. Findings with these animals were compared with findings in wild-type and transgenic nematodes maintained in the absence of supplemented folate and fed bacteria lacking the ability to synthesize folate [E.coli K12 MH828 and MH829 strains, which are folA null mutants (10); these strains were kindly provided by Dr. Muriel B. Herrington of Concordia University, Montreal, Canada]. The level of expression of the endogenous folt-1 mRNA, and the level of expression of folt-1::GFP in transgenic C. elegans were then determined.
Statistical analysis. All uptake studies were performed at least in triplicate on different occasions using different batches of cells/nematodes, and the data were expressed as means ± SE in moles per milligram of protein per unit of time. Statistical analysis was performed using the Student's t-test or one-way ANOVA followed by Tukey's honestly significant difference (HSD) test, with statistical significance being set at 0.01 (P < 0.01). Kinetic parameters of the saturable folic acid uptake process were calculated using a computerized model of the Michaelis-Menten equation as described previously by Wilkinson (38). Uptake by the saturable process was determined by subtracting the diffusing component (determined from the slope of the uptake line between a high pharmacological concentration of folic acid of 1 mM and the point of origin) from the total uptake. Studies involving quantitative PCR, analysis of promoter activity, and distribution of expression of folt-1 under different conditions were performed on at least 20–30 different nematodes. For GFP analysis, 15–20 age-synchronized nematodes were transferred to slides and were scored for fluorescence intensity under identical times for comparison.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1.23 and 1.6 kb for folt-1 and folt-2, respectively, and their identities were confirmed by sequencing. Of the two cloned sequences, only folt-1 was found to have active folate transporter activity (see below), and thus we focused our characterization of this transporter. We also did both 5'-RACE using Ambion's FirstChoice RLM RACE kit (Austin, TX) and 3'-RACE using an Invitrogen kit to confirm the initiation and stop codons of folt-1. The results confirmed the start and stop codons and showed a lack of existence of any variance.
The deduced amino acid sequence of folt-1compared with hRFC is given in Fig. 1A. The cDNA of the folt-1 gene consisted of 1,566 bp, of which 1,233 bp represent the ORF. This encodes for a protein of 410 amino acids with a predicted molecular mass of 46.5 kDa. Hydropathy analysis (HMMTOP; http://www.enzim.hu/hmmtop1.1/server/hmmtop.cgi) predicted the protein to have 10 TM domains with a long intracellular loop of 58 amino acids between TM domains 5 and 6 (Fig. 1B). This resembles the situation with the hRFC where a large intracellular loop also exists. When the membrane topology was modeled, both the NH2- and COOH-terminal ends were found to be directed toward the intracellular side (Fig. 1B). Direct studies, however, are needed to establish the orientation of these terminals and the topology of the folt-1 transporter. The folt-1 polypeptide was predicted to carry two potential NH2-glycosylation sites (Asn-36, Asn-260; NetNGlyc 1.0 server), five potential PKC phosphorylation sites (Ser-2, Ser-186, Ser-192, Ser-212, and Ser-304), and two potential cAMP- and cGMP-dependent protein kinase phosphorylation sites (Lys-128 and Lys-226; NetPhos 2.0 server).
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6.5-fold) higher in cells transfected with the full-length folt-1 cDNA compared with control cells (8.96 ± 0.56 and 1.4 ± 0.15 fmol·mg protein–1·7 min–1 for folt-1-transfected and control cells, respectively). Similar results were obtained when a slightly shorter folt-1 protein (that lacks the last 53 AA that represents the COOH-terminal tail and the last TM domain) was used, in that folic acid (16 nM) uptake was significantly (P < 0.01) (8-fold) induced in cDNA-transfected cells compared with control (Fig. 2B). The latter finding suggests that the COOH-terminal and last TM domain of folt-1 do not play a role in the transport function of the carrier protein. Unless otherwise stated below, the functional characterization studies were done using the slightly shorter form of folt-1.
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In contrast to folt-1, transfection of ARPE-19 cells with a cDNA for folt-2 failed to show any increase in the initial rate of folic acid (16 nM) uptake compared with controls, both at buffer pH 5.5 (2.19 ± 0.17 and 2.18 ± 0.05 fmol·mg protein–1·7 min–1, respectively) and buffer pH 7.4 (1.71 ± 0.07 and 1.69 ± 0.20 fmol·mg protein–1·7 min–1, respectively).
Effect of buffer pH and role of Na+ in folic acid uptake by the folt-1 system. The effect of variation in incubation buffer pH on the initial rate of folic acid (16 nM) uptake by the induced carrier following transfection of ARPE-19 cells with cDNA of the shortened folt-1 was examined. The results showed an increase in folic acid uptake by the induced carrier as a function of decreasing the incubation buffer pH; uptake was significantly (P < 0.01) higher at buffer pH 5.5 compared with pH 7.4 (Fig. 3A). Similarly, uptake of folic acid (16 nM) by ARPE-19 cells transfected with full-length folt-1 was found to be significantly (P < 0.01) higher at pH 5.5 compared with pH 7.4 (8.96 ± 0.56 and 0.19 ± 0.03 fmol·mg protein–1·7 min–1 at pH 5.5 and 7.4, respectively). Incubation buffer pH 5.5 was used in all subsequent investigations.
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) on the initial rate of folic acid (16 nM) uptake by the induced system in shortened folt-1-expressing cells. The results showed the induced folic acid uptake to be similar in the presence and absence of Na+ both at pH 5.5 (Fig. 3B) and at pH 7.4 (uptake of 1.04 ± 0.02, 1.07 ± 0.03, 1.11 ± 0.01, 1.08 ± 0.01, 1.18 ± 0.1, and 1.13 ± 0.2 fmol·mg protein–1·7 min–1 for the incubation medium containing Na+, K+, Li+, Tris, choline, and NH, respectively). We also examined the effect of pretreatment of (for 30 min) the shortened folt-1-expressing ARPE-19 cells with the Na+-K+-ATPase inhibitor ouabain (0.5 mM) on the initial rate of folic acid (16 nM) uptake. The results show induced folic acid uptake to be similar in ouabain-pretreated and control cells (10.86 ± 1.0 and 11.61 ± 0.562 fmol·mg protein–1·7 min–1, respectively). Kinetic parameters of the induced folic acid uptake by folt-1-expressing ARPE-19 cells. In this study, we examined the initial rate of folic acid uptake by the induced system in shortened folt-1-expressing ARPE-19 cells as a function of increasing substrate concentration in the incubation medium (0.01–10 µM). Uptake by the induced folic acid transport system includes a saturable component (Fig. 4). The apparent Km and Vmax of the saturable uptake component were then calculated as described in MATERIALS AND METHODS and found to be 1.23 ± 0.18 µM and 7.28 ± 1.1 pmol·mg protein–1·7 min–1, respectively.
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Confirmation of functionality of folt-1 as a folate transporter in vivo: effect of folt-1 knockdown (silencing) and KO.
To establish the functionality of folt-1 as a folate transporter in vivo in C. elegans, we used two approaches. In the first, we examined the effect of specific folt-1 gene silencing on whole animal folate uptake, with the use of a gene-specific RNAi feeder clone. In these studies, wild-type animals were fed with E. coli expressing the folt-1RNAi plasmids for 16 h; control C. elegans were fed bacteria without the RNAi. Real-time PCR analysis was then performed on samples from these animals, with the results showing a significant (P < 0.01) reduction in mRNA levels of folt-1 in the RNAi-fed animals compared with controls (Fig. 7A). This reduction appears to be specific for folt-1, as no change in the level of expression of the housekeeping gene -actin was observed. Next, we examined the functional consequences of folt-1 silencing on the whole animal folate uptake [5 min; uptake by the whole animal was linear for up to 20 min (data not shown)]. The results showed a significant (P < 0.01) reduction in folate (16 nM) uptake in RNAi-fed compared with control animals (Fig. 7B). Uptake of the unrelated ascorbic acid, on the other hand, was similar in the two animal groups (Fig. 7B).
In the second approach, we used a folt-1 deletion strain (Fig. 7C) of the C. elegans and performed similar functional folate uptake studies. Consistent with the KO consortium report, we found the homozygotes of this mutant to be defective in reproduction (sterile) and displaying very slow (sluggish) movement. The identity of the strain was confirmed by PCR (39) and by the phenotypic characteristics. Wild-type animals showed a PCR product of 2,250 bp, whereas the folt-1–/– KO animals showed a deletion product of 1,040 bp (Fig. 7D).
Using the homozygote folt-1–/– deletion strains, we examined folate uptake by these animals and compared the findings with those in wild-type animals of identical stages (synchronized). The results showed a significantly (P < 0.01) lower folic acid (16 nM) uptake in the homozygote folt-1–/– KO animals compared with wild type (Fig. 7E). Uptake of the unrelated biotin, on the other hand, by the homozygous folt-1–/– KO animals was similar to that of wild-type animals (Fig. 7E).
Analyses of the expression pattern of folt-1. To study the pattern of expression of the folt-1 gene in the whole living nematode, a transcriptional folt-1::GFP fusion was constructed (see MATERIALS AND METHODS) and then used to generate transgenic nematodes expressing this transgenic construct. In this transcriptional construct, the expression of the GFP would be indicative of the expression pattern of the folt-1 gene. The results showed expression of the GFP in different C. elegans tissues, thus establishing promoter activity of the cloned genomic fragment. Expression was consistently higher in the pharynx and the posterior part of the intestine; it was also observed in the body wall muscles, head muscles, and vulva muscles of these transgenic animals (Fig. 8).
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-actin transcripts from the same samples served as an internal control in these experiments. There was a gradual decrease in the expression levels of folt-1 mRNA with development, with the decrease reaching a significant (P < 0.01) level when the animal reached the adult stage.
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Effect of exogenous folate level on parameters of folate uptake and on the level of folt-1 expression. The effect of maintaining (for 72 h) C. elegans in a culture medium containing high concentrations of folic acid (1 mM) on the level of expression of folt-1 mRNA and other parameters of folate uptake was examined. Comparison was made with data from C. elegans maintained in regular culture medium (no folate supplementation) and fed E. coli that lacks the ability to synthesize folate (we used this type of E. coli to further minimize the level of exogenous folate that is available to the animals so that a clearer comparison can be made). The latter animals were considered controls in this study. Results of the quantitative PCR assay showed the level of folt-1 mRNA to be significantly (P < 0.01) lower in the C. elegans maintained in folate-oversupplemented culture medium compared with the controls (Fig. 10A).
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To link the above observed changes in the level of expression of folt-1 mRNA and in folic acid uptake on folate oversupplementation with possible transcriptional regulatory events, transgenic nematodes carrying the folt-1::GFP transcriptional construct were maintained under folate-oversupplemented and control conditions followed by determination of GFP fluorescence intensity in the intestine. There was a significantly (P < 0.01) lower level of GFP expression in the intestine of the folate-oversupplemented transgenic animals compared with controls (Fig. 11, A–C).
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DISCUSSION |
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An interesting observation was the ability of the anti-inflammatory agent sulfasalazine to competitively inhibit the induced folic acid uptake in folt-1-expressing ARPE-19 cells. Sulfasalazine is an anti-inflammatory agent that is widely used in the treatment of IBD and has been shown to competitively inhibit folate uptake in the human intestine (42). In addition and as seen with the human intestinal folate uptake process (26, 29), the anion transport inhibitors DIDS and SITS were both found to be strong inhibitors of folic acid uptake by the induced system in the folt-1-expressing ARPE-19 cells. The latter two findings further indicate the similarity between the functionality of the folate uptake process mediated by folt-1 in C. elegans and that of the human intestinal folate uptake process.
To confirm the functionality of the folt-1 system in vivo, we utilized two different approaches. In the first approach, we examined the effect of knocking down (silencing) the folt-1 gene with the use of gene-specific RNAi on folate uptake. In C. elegans, the RNAi-mediated gene silencing process is so robust that exposure of the animals to RNAi in their environment is sufficient to induce genetic interference (35). The results showed that silencing the folt-1 gene leads to a substantial reduction in folate uptake compared with the control (Fig. 7B). In the second approach, we used folt-1 KO C. elegans and examined folate uptake; results were compared with those of identical-stage wild-type animals. The folt-1 KO worms were obtained from the deletion strain VC959. First, we selected the homozygote animals based on phenotype and PCR data (genotyping). We then used these animals in folate uptake studies and compared the findings with those of wild-type worms of identical age. The results showed folate uptake to be severely inhibited in the KO worms compared with controls. These findings collectively suggest a critical role for folt-1 in folate uptake in C. elegans in vivo.
To gain insight into the transcriptional regulation of the folt-1 gene, we cloned the 5'-regulatory region of the gene and fused the cloned genomic fragment to the GFP reporter gene. Promoter activity of the cloned folt-1 genomic fragment was demonstrated in vivo by generating transgenic worms expressing folt-1::GFP, which showed expression of GFP in the living animals. This study, in addition to demonstrating promoter activity of our cloned genomic fragment, also provided important information on the pattern of expression of folt-1 in different tissues of the intact whole C. elegans in vivo, since expression of the GFP was driven by the folt-1 promoter. The results showed the highest level of expression to be in two organs of the digestive system, namely the pharynx and the (posterior portion of) intestine of the transgenic animals. While the intestinal area is composed of highly differentiated epithelial cells, the pharynx area contains different cell types, including epithelial cells, muscle cells, and secretory glands (1). The latter cell type is believed to be involved in the secretion of digestive enzymes (1). The high level of expression of folt-1 in the cells of the digestive system raises the possibility of its involvement in micronutrient absorption in this organism. Further studies are, however, needed to confirm this suggestion.
We also investigated possible developmental regulation of folt-1 mRNA expression in wild-type and in transgenic C. elegans expressing the transcriptional folt-1::GFP construct. The results showed the highest level of expression of folt-1 mRNA to be in the larva 1 stage, but the expression declined with maturation. This pattern of decline was also observed in the intestine of transgenic animals expressing the transcriptional construct folt-1::GFP, thus confirming the in vitro observations. The observation that folt-1 is developmentally regulated is similar to the observations reported for mammalian RFC, whose expression in the gut was shown to be developmentally regulated and in a similar manner (2, 28).
Possible adaptive regulation of folt-1 expression and function was investigated using both wild-type and transgenic animals carrying the folt-1::GFP construct. Maintaining C. elegans in culture medium oversupplemented with high pharmacological doses of folic acid was found to lead to a significant decrease in the level of mRNA expression of folt-1. This decrease was associated with a specific decrease in the level of folic acid uptake by wild-type C. elegans. However, such a regulation by external folate level was not observed with KO worms, further supporting the present data on the important role played by the folt-1gene in the folate uptake process in C. elegans. These changes indicate that uptake of folate by folt-1 is adaptively regulated by exogenous substrate level. The observation of a decreased expression of folt-1::GFP in the intestine of the C. elegans maintained in folate-oversupplemented medium compared with control suggests that a transcriptional regulatory mechanism(s) may (at least in part) be involved in mediating the observed adaptive response. Again, these observations are similar to those reported with mammalian RFC in the intestine on changing extracellular folate levels (Refs. 27 and 33 and unpublished observations).
In summary, results of these investigations have identified for the first time the existence of a functional and specialized folate uptake system in the nematode C. elegans and showed the system to be similar to that of the human intestinal folate uptake process in that it is acidic pH dependent; has similar affinity to oxidized, reduced, and substituted folate derivatives; is sensitive to the inhibitory effects of the anti-inflammatory agent sulfasalazine; and is inhibited by the anion transport inhibitors DIDS and SITS. In addition, functionality of this system was confirmed in vivo by gene knockdown and KO approaches. Furthermore, folt-1 appears to be expressed in different tissues of C. elegans (including the intestine), and its expression is regulated during development and by substrate level in the culture medium. These studies establish the suitability of C. elegans as a model for detailed investigations into integrative aspects of the folate uptake process at the whole animal level.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. Balamurugan and B. Ashokkumar contributed equally to this work. 
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) and 1 (
) µM] and different concentrations of 5-formyltetrahydrofolic acid (folinic acid) and MTX were added at the onset of incubation. Data are means ± SE of at least 3 separate uptake determinations.
