RFC-1 Gene Expression Regulates Folate Absorption in Mouse Small Intestine*

(Received for publication, December 3, 1996, and in revised form, February 18, 1997)

Judy H. Chiao , Krishnendu Roy , Berend Tolner , Ching-Hsiung Yang and F. M. Sirotnak Dagger §

From the Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center and Dagger  Graduate School of Medical Sciences, Cornell University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l,L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (Km or Ki = 2 to 4 µM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 µM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l,L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.


INTRODUCTION

Transport of folate compounds into mammalian cells can occur via carrier-mediated (1-5) as well as receptor-mediated (3-7) mechanisms depending on the relative level at which each is expressed in the plasma membrane of these cells. Carrier-mediated mechanisms are involved (1-4) in the internalization of both natural folates and their cytotoxic analogues in tumor and normal proliferative tissues. Thus, their level of expression in these tissues has pharmacological significance. Carrier-mediated transport of folate compounds among tumor cells from various tissues in different mammalian species appears to share (1-4) similar properties. They exhibit (1-4) similar diversity in saturability for various folate compounds and their analogues with extremely low saturability for folic acid compared with folate coenzymes. The properties of folate compound transport in normal tissues appear to be much more diverse (reviewed in Ref. 2) and often differ substantially from those characteristic of mechanisms found in tumor cells. Carrier-mediated transport of folates associated (8-11) with absorption in the small intestine of rodents appears to be uniquely different from that in tumor cells (1-4, 12), particularly in terms of its pH dependence and saturability. For instance, in absorptive luminal epithelium of the rat small intestine, a mechanism in the brush-border membrane has been described (11, 13) which exhibits an optimum at pH 5.5-6 and relatively high saturability (Km < 5 µM) for internalization of all folate compounds and their analogues examined, including folic acid. Another carrier-mediated mechanism transporting folate compounds has been described (14) in the luminal epithelium of mouse small intestine. This moderately saturable mechanism mediates high capacity influx and does not appear (14) to be associated with folate absorption. Since this mechanism is operable (14) at the same level in both absorptive and nonabsorptive proliferative tissue in the luminal crypt, it would appear to be localized in the plasma membrane at the basolateral surface.

Recently, a murine cDNA clone (RFC-1) was isolated (15) by expression cloning from an L1210 cell cDNA library. This cDNA appears to encode a protein with many of the properties of the tumor cell, 1-carbon reduced folate transporter. However, expression of the RFC-1 gene has also been shown (16) to occur in the small intestine of rodents and appears to be under developmental regulation at least in rat intestine. This was based on the finding in the rat of higher levels of RFC-1 mRNA in mature absorptive cells compared with that found in immature proliferative cells in the crypt. However, direct evidence showing that this gene does, in fact, regulate folate absorption in this tissue was not provided.

The present studies are an extension of these recently reported (16) and of our own earlier (14) studies of folate compound transport in luminal epithelial cells isolated from mouse small intestine. In these new studies, we were able to delineate by kinetic and other means acid pH-dependent influx of l,L-5-CH3-H4folate1 from non-pH-dependent influx mediated (14) by the mechanism that appears to operate in the plasma (basolateral) membrane of these cells. We also provide evidence showing that acid pH-dependent folate compound transport in brush-border membrane in mature absorptive cells is inhibited by a folate-based affinity label and by antibodies against RFC-1-specific peptides. Folate transport in absorptive epithelial cells from the mouse also appears to be dependent on the expression of the RFC-1 gene (15) in the form of a 2.5-kilobase transcript and a 58-kDa transporter encoded by this transcript and detected by the same folate-based affinity label and by immunoblotting in the brush-border membrane of these cells.


EXPERIMENTAL PROCEDURES

Isolation of Luminal Epithelial Cell Fractions

The isolation of luminal epithelial cells from the small intestine of C57BL/6 × DBA/2 F1 (BD2F1) mice was carried out at 0-4 °C by a previously published (14) method. These cells were obtained in the form of four individual fractions varying in their stage of maturation using marker enzyme analysis (14) to monitor fractionation. The isolation of purified brush-border membrane from Fraction I luminal epithelial cells was the same as that described (13, 17) for rat luminal epithelial cells.

Transport Methodology and Affinity Labeling

Freshly isolated cells in Fractions I and IV were utilized in experiments measuring the mediated internalization at 37 °C of l,L-5-CH3-H4folate using published procedures (14, 18). The transport buffer (Hepes-sucrose-MgO) used during these experiments contained 20 mM Hepes and 227 mM sucrose adjusted to the appropriate pH with MgO. A few experiments were carried out using a physiological salts solution plus 7 mM D-glucose (14) as the transport buffer. Cells in the Hepes-sucrose-MgO buffer were affinity labeled with 50 nM N-hydroxysuccinimide ester of [3H]aminopterin as described by Henderson and Zevely (19). The alkaline-washed purified membranes (20) were fractionated (20) by SDS-polyacrylamide gel electrophoresis and gel slicing, and the radioactivity in each fraction was determined by scintillation counting.

Preparation of Polyclonal Antibodies and Western Blotting

Antibodies against two murine RFC-1-specific peptides were prepared in rabbits according to standard protocols (21). Before injection, the peptides were either linked to keyhole limpet hemocyanin (22) or converted to macromolecular size with multiple antigenic peptide technology (23). The peptides prepared consist of 19 amino acids encoded at the NH2-terminal (Met1 through Asp19) or COOH-terminal (Ser495 through Ala512) regions of the murine RFC-1 cDNA. Before use, the antibodies in the immune serum were purified by immunoaffinity fractionation with the NH2- and COOH-terminal peptides linked to cyanogen bromide-activated Sepharose (24). Western blotting with enhanced chemiluminescence (Amersham) detection was carried out by a standard procedure (25) following SDS-polyacrylamide gel electrophoresis of purified plasma membrane (20).

Northern Blotting of RFC-1 mRNA

Standard procedures (26) were used for detection by blotting of RFC-1 mRNA in luminal intestinal epithelial cell fractions using a murine RFC-1 cDNA probe (15), a generous gift of Dr. K. H. Cowan. Total RNA was obtained from these cells by means of RNA STAT-60 (TEL-TEST, Inc.). Poly(A)+ RNA was subsequently obtained by fractionation on an oligo(dT) column (27). All blots were normalized with a gamma -actin probe (28). Labeling of each probe was by random priming (Boehringer Mannheim) using [alpha -32P]dCTP (3000 Ci/mmol) and 10 ng of insert.

Quantitative Reverse Transcription-PCR of RFC-1 cDNA

Total RNA was prepared by two treatments with RNA STAT-60 according to the manufacturer's (TEL-TEST, Inc.) instructions. A 5-µg aliquot of methylmercuric hydroxide-treated RNA was used to synthesize cDNA by a standard procedure (29) and an oligo(dT) primer (Life Technologies, Inc.). Quantitative PCR was carried out with Amplitag DNA polymerase (Perkin-Elmer) in the recommended buffer, 400 pmol of RFC-1 sense (5-GAGCCTTTAGGTTAGG-3') and antisense (5'-CAAGCACCTCCGATAG-3') primers, 200 µM dNTPs, and 10 µCi [32P]dCTP in a total volume of 50 µl. In a parallel reaction, sense (5'-TATCAGCACTGGATCG-3') and antisense (5'-TTACAGGTGTCGATGC-3') gamma -actin primers were used. After the initial denaturation step of 5 min at 95 °C, 30 cycles of 1.5 min at 95 °C, 1 min at 60 °C, and 1.5 min at 72 °C, respectively, were carried out. After this, the reaction was extended for 10 min at 72 °C. The relative amount of product formation was determined by transblotting (26) to Nytran-Plus membrane (Schleicher and Schuell) and radioautography. The amount of RFC-1 and gamma -actin cDNA included in the reaction sustained amplification in the linear range of product formation at the time the amount of each product was compared.

Other Materials and Methodology

l,L-[3H]-5-CH3-H4folate (specific activity, 30 Ci/mmol) and [3H]aminopterin (specific activity, 10-12 Ci/mmol) were purchased from Moravek Biochemicals (City of Industry, CA) l,L-5-CH3-H4folate was purchased from Sigma Biochemical (St. Louis, MO). All other reagents were reagent grade. Radiolabeled folate compounds were analyzed for purity (>95%) by HPLC (30) before use or repurified by HPLC (30).


RESULTS AND DISCUSSION

Kinetic Delineation between Mechanisms Mediating l,L-[3H]-5-CH3-H4folate Influx in Luminal Epithelial Cells

The velocity of initial influx of l,L-[3H]-5-CH3-H4folate was determined with Fraction I cells suspended in transport buffer with 150 µM DIDS. At this concentration of the stilbene, influx by the moderately saturable system previously described (14) for these cells was almost completely inhibited. Also, the concentration of the folate utilized (2.5 µM) was >50-fold lower than the apparent Ki determined (14) for inhibition by this folate of folate compound influx by this moderately saturable system. Data shown in Fig. 1A document rapid internalization of l,L-[3H]-5-CH3-H4folate under these conditions in a pH-dependent manner. A steady-state level of internalization was achieved within 5-10 min at 37 °C at pH 6.2 or 7.4. However, initial influx and steady-state level achieved were both 4-fold greater at pH 6.2. Under the same conditions at pH 6.2, the initial influx and steady-state level of this folate achieved in Fraction IV cells (Fig. 1B) were severalfold lower. Influx mediated by the highly saturable, pH-dependent system in Fraction IV cells is probably even lower than would appear from these data in view of the residual influx mediated by the moderately saturable system in contaminating Fraction I cells that would be expected to be present in the Fraction IV preparation.


Fig. 1. Time course for mediated accumulation at 37 °C of l,L-[3H]-5-CH3-H4folate by luminal epithelial cells from mouse small intestine. A, influx by Fraction I cells at pH 6.2 and pH 7.4. B, influx by Fraction I and Fraction IV cells at pH 6.2; ext, external. Data are averages from three experiments with S.E. <±14%. Other experimental details are provided in the text.
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Data on the concentration response for initial influx at 37 °C of l,L-[3H]-5-CH3-H4folate in Fraction I and IV cells is shown in Fig. 2A. At pH 6.2, initial influx exhibits complex saturation kinetics in the form of highly saturable and moderately saturable components. The highly saturable component was decreased at pH 7.4 in Fraction I cells and in Fraction IV cells at pH 6.2 or pH 7.4 (data not shown). Moreover, in the presence of 150 µM DIDS, only a highly saturable component of influx is observed at pH 6.2 in Fraction I cells. A double-reciprocal plot of some of the data obtained in this type of experiment carried out over a wider concentration range in the presence an absence of 150 µM DIDS is shown in Fig. 2B. It can be seen that the two saturable components of influx observed in the absence of DIDS (Fig. 2B) are converted to a single highly saturable component in the presence of 150 µM DIDS. From the data presented in Fig. 2B, an apparent Km and Vmax for each saturable component at pH 6.2 or at pH 7.4 (not shown) in Fraction I cells could be obtained. In Fraction I cells, DIDS-insensitive influx of l,L-[3H]-5-CH3-H4folate exhibits (Table I) an apparent Km in the low micromolar range and an extremely low Vmax compared with DIDS-sensitive influx of this folate compound. Only influx Vmax exhibited pH dependence, with the higher value for Vmax occurring in the acidic range. In contrast, DIDS-sensitive influx of this folate compound exhibits both a high Km and a high Vmax, neither of which are pH dependent. Therefore, at the low micromolar range of concentration or below, influx of l,L-[3H]-5-CH3-H4folate by the DIDS-insensitive system would predominate in Fraction I cells, but only at pH 6.2. While at pH 7.4 and at the high micromolar range, DIDS-sensitive influx would predominate. The data in Table I also document a much lower Vmax for l,L-[3H]-5-CH3-H4folate influx at pH 6.2 in Fraction IV cells in the presence of DIDS.


Fig. 2. Concentration response for mediated accumulation at 37 °C of l,L-[3H]-5-CH3-H4folate by Fraction I and Fraction IV cells. A, influx following 1 min of incubation at different concentrations of l,L-[3H]-5-CH3-H4folate in Fraction I cells at pH 6.2 ± 150 µM DIDS or pH 7.4 and in Fraction IV cells at pH 6.2. B, double-reciprocal plot of data on influx at pH 6.2 ± 150 µM DIDS; ext, external. Other experimental details are provided in the text. Data are averages from three experiments with S.E. <±16%.
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Table I.

Kinetic properties of mediated ell ,L-[3H]-5-CH3-H4 folate influx in luminal epithelial cells of mouse small intestine

The values shown are averages ± S.E. for influx by Fraction I or Fraction IV cells in 3-5 separate experiments. Influx by the high and low affinity systems was delineated by measurements made in the presence and absence of 150 µM DIDS. Other experimental details are provided in the text.


Fraction DIDS (150 µM) pH Km Vmax

µM pmol/min/mg protein
I + 6.2 1.8  ± 0.4 0.8  ± 0.2
7.4 2.3  ± 0.2 0.2  ± 0.05
 - 6.2 128  ± 23 18  ± 5
7.4 115  ± 20 16  ± 4
IV + 6.2 1.9  ± 0.3 0.13  ± 0.03
 - 6.2 119  ± 18 19  ± 4

The two systems delineated above on the basis of inhibition by DIDS and pH dependence were also distinguishable by their interaction with various folate compounds as inhibitors of l,L-[3H]-5-CH3-H4folate influx. In the presence of 150 µM DIDS, influx of this folate by the DIDS-insensitive system was inhibited (Table II) by 5-methyl- or 5-formyl-substituted H4folate as well as folic acid in the low micromolar range, yielding values for Ki of 2-4 µM. Similar values for Ki were obtained with the 4-aminofolate analogues, aminopterin and methotrexate. These values are consistent with other values for Ki obtained when most of these same compounds were used as inhibitors of folic acid influx by brush-border membrane vesicles in studies by others (13). In contrast, inhibition by these same compounds of l,L-[3H]-5-CH3-H4folate influx by the DIDS-sensitive system (see Table II) was more variable and much less in extent.

Table II.

Relative effects of various folate compounds as competitive inhibitors of influx of ell ,L-[3H]-5-CH3-H4 folate by Fraction I cells

Influx was measured at pH 6.2 in the presence of 150 µM DIDS or at pH 7.4. Data are averages of three separate experiments ± S.E. Additional experimental details are provided in the text.


Permeant Transport system
Brush-border specifica Nonspecificb

 ell ,L-5-CH3-H4 folate 2.1  ± 0.3 128  ± 26
 ell ,L-5-CHO-H4 folate 1.7  ± 0.2 111  ± 20
Folic acid 4.1  ± 0.9 >2000
Aminopterin 3.4  ± 0.7 16  ± 3
Methotrexate 2.9  ± 0.4 107  ± 18

a Influx of ell ,L-[3H]-5-CH3-H4 folate (2 µM) was measured at pH 6.2 in the presence of 150 µM DIDS.
b Influx of ell ,L-[3H]-5-CH3-H4 folate (40 µM) was measured at pH 7.4.

Affinity Labeling of Fraction I Luminal Epithelial Cells with NHS-[3H]Aminopterin

Fraction I cells were affinity labeled with 50 nM NHS-[3H]aminopterin, and purified plasma membrane (20) from these cells was electrophoretically fractionated by SDS-polyacrylamide gel electrophoresis using a 15% polyacrylamide disc gel. The electrophoretic profile for radioactivity shown in Fig. 3 delineates the major peak at about 58 kDa with some minor peaks discernible at a lower range of kDa. A similar fractionation of NHS-[3H]aminopterin-labeled plasma membrane from Fraction IV cells (not shown) did not delineate a predominant peak within the same overall range of kDA. To associate the differential in affinity labeling of Fraction I and Fraction IV cells in the form of a 58-kDA protein with influx of l,L-[3H]-5-CH3-H4folate by Fraction I cells, we determined the effect of affinity labeling with NHS-aminopterin on the influx of this folate compound. The data in Fig. 4 show that within the range of 0-10 µM, affinity labeling with NHS-aminopterin effectively inhibited (IC50 = 0.65 ± 0.05 µM) influx of l,L-[3H]-5-CH3-H4folate. It was also shown (data not given) that l,L-5-CH3-H4folate and folic acid were also effective inhibitors of NHS-[3H]aminopterin labeling of Fraction I cells.


Fig. 3. SDS-polyacrylamide gel electrophoresis (PAGE) of plasma membrane proteins from Fraction I cells after affinity labeling with NHS-[3H]aminopterin. The cells were labeled by 10 min of exposure to 50 nM NHS ester at room temperature and washed in buffer before plasma membrane preparation. The experimental details are given in the text. Data are for one of two representative fractionations monitored by radioactive counting of various fractions prepared with a gel slicer.
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Fig. 4. Effect of NHS-aminopterin (AM) affinity labeling on l,L-[3H]-5-CH3-H4folate influx by Fraction I cells. The cells were exposed to different concentrations of affinity label for 10 min at room temperature and washed in Hepes-NaCl before measurement of l,L-[3H]-5-CH3-H4folate influx; ext, external. Additional experimental details are given in the text. Data are the average of three experiments with S.E. <±13%.
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Detection of RFC-1 mRNA in Luminal Epithelium from Mouse Small Intestine

The data from a Northern blot in Fig. 5 show that a substantial level of RFC-1 RNA transcript was found with an RFC-1 cDNA probe in this tissue compared with liver when both blots were normalized to the level of gamma -actin RNA transcripts in each tissue. The direct detection of RFC-1 RNA in isolated Fraction I versus Fraction IV cells by Northern blotting was not possible because of the large number of Fraction IV cells that was required for the preparation of adequate amounts of poly(A)+ RNA. Because of the size of these preparations (usually 20-30 mice each), the quality of the RNA was consistently poor. Therefore, quantitative reverse transcription-PCR was used to determine the relative amount of RFC-1 mRNA in preparations of much smaller numbers of Fraction I and Fraction IV cells that were obtained from only a few mice. The results of these reverse transcription-PCR reactions in the form of a radioautograph are given in Fig. 6. The data shown are for the amount of RFC-1 cDNA product obtained normalized to the amount of gamma -actin cDNA product obtained when run in a parallel PCR reaction. From Fig. 6, it can be seen that RFC-1 mRNA is present in Fraction IV cells at a much lower level than in Fraction I cells.


Fig. 5. Detection of RFC-1 mRNA in mouse tissues by Northern blotting. Northern blot of RFC-1 mRNA in total small intestine and liver from BD2F1 mice. The total amount of poly(A)+ RNA from each tissue added to the gel was 5 µg. The RFC-1 mRNA blot was normalized with respect to a blot of gamma -actin mRNA. Additional experimental details are given in the text. Data for each tissue are the results of one of two representative blots. kb, kilobases.
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Fig. 6. Detection of RFC-1 mRNA in Fraction I and Fraction IV cells by quantitative reverse transcription-PCR. The PCR product in each case was radioactively labeled by incorporation of [alpha -32P]dCTP in the PCR reaction. Following blotting, radioautography was performed to detect the PCR products. See text for additional details. Data are the results of two representative experiments.
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Western Blotting

Further evidence for an association between l,L-[3H]-5-CH3-H4folate influx in Fraction I cells and RFC-1 gene expression was obtained in the form of immunoblotting with antibodies raised in rabbits against anti-RFC-1-specific peptides. The blots in Fig. 7 reveal a prominent protein band at 58 kDa and a faint band at 46 kDa in plasma membrane from Fraction I cells. In contrast, blotting of plasma membrane from Fraction IV cells revealed neither of these protein bands but occasionally detected a weakly reacting protein band in the vicinity of 35 kDa. Western blotting with anti-RFC-1 peptide antibody was also carried out with purified brush-border membrane protein from Fraction I cells. From data shown in Fig. 6B, it can be seen that this antibody detected a 58-kDa protein band in this specialized membrane in addition to another band at ~35 kDa. A plot of plasma membrane protein from Fraction IV cells run in parallel is also shown in Fig. 6.


Fig. 7. Western blotting with anti-RFC-1 peptide antibody. A, blotting of plasma membrane protein from Fraction I and Fraction IV cells. The amount of solubilized plasma membrane protein in each lane was 10 µg. B, blotting of brush-border membrane protein from Fraction I cells. Sample size was 10 µg of protein. Additional experimental details are given in the text. Data are representative results from one of several blots.
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Further Evidence for a Role of the Transporter Encoded by the RFC-1 Gene in Folate Absorption

The results of both NHS-[3H]aminopterin affinity labeling and immunoblotting with anti-RFC-1 peptide antibody presented above clearly suggest that there is an association between a 58-kDa protein and folate transport in absorptive luminal epithelium (Fraction I cells) from mouse small intestine. More direct evidence was obtained by determining the effect of the anti-RFC-1 peptide antibody on l,L-[3H]-5-CH3-H4folate influx by Fraction I cells at pH 6.2 in the presence of 150 µM DIDS. The results presented in Fig. 8 show that influx of this folate compound under these conditions by Fraction I cells was inhibited substantially by the prior incubation of Fraction I cells with varying amounts of anti-RFC-1 peptide antibody and that inhibition occurred in a concentration-dependent manner. No effect was obtained with an irrelevant antibody of the same IgG class against a mouse folylpolyglutamate synthetase peptide purified in the same manner from rabbit serum.


Fig. 8. The inhibition by anti-RFC-1 peptide antibody of l,L-[3H]-5-CH3-H4folate influx in Fraction I cells. The cells in Hepes-NaCl were exposed to different amounts of antibody (ab) for 30 min at room temperature. After the cells were washed in Hepes-NaCl, influx of l,L-[3H]-5-CH3-H4folate was measured for an interval of 1 min, and the cells were processed for scintillation counting. Additional experimental details are given in the text. Data are an average of two experiments differing by <±13%.
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The studies described here on folate compound influx by isolated luminal epithelial cells from mouse small intestine are in close agreement with those published earlier (11, 13) by Selhub and Rosenberg using either everted sacs or brush-border membrane vesicles from rat small intestine. Under conditions that suppress non-pH-dependent influx (14), the role of a highly saturable, pH-dependent system in mediating influx of folate compounds that operates only in absorptive epithelial cells has been documented with characteristics very similar to that found (13) in the brush-border membrane of rat small intestine. These studies were further extended to show that the dependence of influx of l,L-[3H]-5-CH3-H4folate on pH reflected a difference at each pH in values for Vmax but not for apparent Km for this system. The basis for this effect on maximum capacity rather than saturability is not understood but could reflect a difference in the rate of translocation of the transporter in response to a difference in the proton gradient (inside versus outside) at each pH.

In further support of the notion that the acid pH-dependent system in the luminal epithelium of mouse small intestine is involved in folate absorption, we have also provided data that show that acid pH-dependent transport in luminal epithelium of this organ is developmentally regulated. Physiologically significant levels of acid pH-dependent influx of l,L-[3H]-5-CH3-H4folate and of the 58-kDa putative transporter occurs only in mature absorptive cells apparently within the brush-border membrane. The relevance of this protein to acid pH-dependent transport in these cells was strongly suggested by the results of folate-based affinity labeling and immunoblotting. Also, the murine RFC-1 gene, which encodes a 58-kDa membrane protein (15), appears primarily to be transcriptionally active in mature absorptive cells. Finally, it was found that the folate-based affinity label and antibodies against murine RFC-1-specific peptides will inhibit influx of l,L-[3H]-5-CH3-H4folate in mature absorptive cells.

In the report by Said et al. (16), the RFC-1 cDNA derived from poly(A)+ RNA of mouse small intestine incorporated a different 5' end from that initially reported for an RFC-1 cDNA derived from L1210 cell poly(A)+ RNA. We have now obtained data to show (31) that the RFC-1 cDNA derived from mouse small intestine is a splice variant incorporating an alternative to exon 1 of the RFC-1 gene, which encodes this different 5' end. Although supporting data are still needed, it is likely that transcription of this splice variant, which is initiated at the alternate to exon 1 in small intestine, is under the control of a second promoter. Transcription of genes that encode alternate 5' ends in the form of splice variants have frequently been shown (32-35) to be under the control of multiple promoters. Additional work will be required to establish this point.

Lastly, it is of interest to compare data obtained in the current studies on mediated folate compound transport in luminal epithelium of small intestine to that documented (1, 2, 12, 20, 36) in detail for L1210 cells. If we assume that transport of these compounds in both of these tissues is a direct result of RFC-1 gene expression alone, a paradox emerges pertaining to the properties of the transport system in each case. Although the murine RFC-1 gene encodes a 58-kDa protein, the mass of this protein actually detected is 58 kDa in luminal intestinal epithelium but only 46 kDa in L1210 cells (20, 36). Moreover, discrepancies exit with regard to both pH dependence and structural preferences among folate compounds as permeants. The transport system in L1210 cells exhibits (12) a moderately alkaline pH optimum and marked differences (1, 2, 12, 18) in preferences among folate compounds as permeants. In contrast, the system in mouse luminal intestinal epithelium exhibits (see above) an acid pH optimum and close similarity in preferences among the same folate compounds as permeants. There are a number of possible explanations for these apparent discrepancies that are consistent with the role of the RFC-1 gene in each case. These could pertain to alternate splicing, post-transcriptional modification and (or) the role of accessory proteins. However, since there is currently a lack of relevant data, further work will be required that addresses each aspect of this question.


FOOTNOTES

*   Supported in part by Grants CA 08748 and CA 55617 from the National Cancer Institute.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.
§   To whom correspondence and requests for reprints should be addressed: Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7952; Fax: 212-794-4342; E-mail: sirotnaf{at}mskcc.org.
1   The abbreviations used are: l,L-5-CH3-H4folate, the natural diastereomer of 5-methyltetrahydrofolate; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; NHS, N-hydroxysuccinimide.

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