1Veterans Affairs Medical Center, Long Beach 90822; 2University of California, Irvine, California 92697; and 3University of New Mexico and Albuquerque Veterans Affairs Medical Center, Albuquerque, New Mexico 87131-0001
Submitted 8 January 2004 ; accepted in final form 1 March 2004
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ABSTRACT |
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human reduced folate carrier; small interfering RNA; transport regulation
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MATERIALS AND METHODS |
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Cell Culture and Uptake Studies
Human-derived pancreatic carcinoma epithelial MIA PaCa-2 cells (passage 125; American Type Culture Collection, Manassas, VA; these cells were derived from tumor tissue of the pancreas obtained from a 65-year-old Caucasian male) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum in 75-cm2 plastic flasks at 37°C in 5% CO2-95% air atmosphere, with the medium changed every 23 days. MIA PaCa-2 cells were subcultured by trypsinization (subcultivation ratio 1:5) and plated onto 24-well plates. Uptake studies were performed on confluent cell monolayers (between passages 127 and 140) 23 days after confluence.
[3H]folic acid uptake was examined in cells incubated in Krebs-Ringer buffer containing (in mM) 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 5.0 (unless otherwise stated). [3H]folic acid was added to the incubation medium at the outset of the uptake experiment, and the reaction was terminated after 3 min (unless otherwise stated) by the addition of 1 ml of ice-cold buffer followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer and lysed with 1 ml of 1 N NaOH. Lysates were neutralized with HCl, and then radioactivity was measured in a scintillation counter. The protein content of cell digests were measured in parallel wells using a Bio-Rad Dc protein assay kit (Bio-Rad, Richmond, VA).
Western Blot Analysis
Western blot analysis was performed as described previously (4) with the use of anti-hRFC polyclonal antibodies. Briefly, MIA PaCa-2 cells were lysed with 20 mM Tris·HCl, pH 7.4, containing 100 mM KCl, 0.9% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 0.5 µg/ml leupeptin. Postnuclear extracts (100 µg protein) were subjected to SDS-8% PAGE and electroblotted on Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). After blocking with 5% powdered defatted milk in PBS-Tween 20, blots were incubated with rabbit anti-hRFC polyclonal antibodies. Immunodetection was performed with goat anti-rabbit IgG conjugated to horseradish peroxidase, using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Specific bands were quantitated with the use of the Eagle Eye II system (StrataGene, La Jolla, CA).
Pretreatment with Gene-Specific Small Interfering RNA
Pretreatment of MIA PaCa-2 cells with hRFC gene-specific RNA was performed as described previously (3). The targeted region for silencing of hRFC (GenBank accession no. NM_003056) was selected from the cDNA sequence beginning 612 nt downstream of the start codon ATG. Custom-made hRFC gene-specific small interfering RNA (siRNA; double-stranded RNA of 21 nucleotides: 5'-aa gcgccccaagcgcagcctc dTdT-3') was chemically synthesized by Qiagen-Xeragon (Germantown, MD). Both the sense and antisense strands were modified at their 3' ends to increase stability (5). Before the experiments were performed, siRNA duplexes were dissolved in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, pH 7.4) and reheated to 90°C for 1 min followed by 1 h at 37°C. MIA PaCa-2 cells (4050% confluent) were transiently transfected with 1 µg siRNA duplex/well of a 24-well plate with the use of Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Two types of controls were used to estimate the specificity of the silencing effect: 1) cells transfected with 1 µg/well scrambled siRNA (5'-aa gcgtgggcatgtctacctt dTdT-3') and 2) cells pretreated with Oligofectamine reagent. Assays for silencing were performed on confluent monolayers 23 days after transfection.
RT-PCR Analysis
Oligo(dT) primers and 5 µg of total RNA isolated from MIA PaCa-2 cells were used with a SuperScript RT-PCR kit (Life Technologies, Rockville, MD) to synthesize first-strand cDNA. To amplify the open reading frame of hRFC, we used two gene-specific primers (5'- GCGCGGGTCTACAACGG-3' and 5'-CAGCATGGCCCCCAAGAAGTAG-3') corresponding to the sequence in the open reading frame of hRFC to produce a 453-bp product. To determine the level of endogenous hRFC in siRNA-pretreated and control cells, we performed PCR within the linear range of amplification. The conditions for semiquantitative PCR were 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min (24 cycles). The products were analyzed on 2% agarose gels, the images were captured using an Eagle Eye II system, and the amplified RT-PCR products were normalized to amplified -actin controls. To confirm the specificity of the siRNA effect on hRFC, we measured the mRNA level of the human thiamin transporter SLC19A2 in siRNA-pretreated and control cells.
Data Presentation and Statistical Analysis
Transport data presented in this article are means ± SE of multiple separate uptake determinations and are expressed in picomoles or femtomoles per milligram of protein per time unit. Data were analyzed by performing the Student's t-test or ANOVA, with statistical significance set at 0.05. Kinetic parameters of saturable folic acid uptake, i.e., maximal velocity (Vmax), and the apparent Michaelis-Menten constant (Km) were calculated by using a computerized model of the Michaelis-Menten equation as described by Wilkinson (18). All semiquantitative RT-PCR and Western blot analyses were performed on at least three separate occasions with comparable results. Representative data are presented in this report.
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RESULTS |
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Time course, possible metabolism during transport, and temperature and energy dependency of the uptake process. Uptake of folic acid (11.6 nM) by MIA PaCa-2 cells was examined as a function of time at pH 5.0, was found to be linear for up to 10 min of incubation, and occurred at a rate of 52 fmol·mg protein1·min1 (Fig. 1). Thus we chose a 3-min period as the standard incubation time.
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We also examined the effect of incubation temperature on the initial rate of folic acid (11.6 nM) uptake at pH 5.0 and determined the Q10 value (i.e., the ratio of transport rate at 37°C to transport rate at 27°C). The Q10 value was found to be 2.04, which suggests the involvement of a mediated process. In other studies, we examined the effect of pretreating the cells for 30 min with the metabolic inhibitors 2,4-dinitrophenol (DNP) and iodoacetate (both at 1 mM) on the initial rate of folic acid (11.6 nM) uptake at pH 5.0. The results show significant (P < 0.01) inhibition in the substrate uptake by both pretreatments (136.09 ± 0.9, 60.88 ± 1.5, and 82.79 ± 2.2 fmol·mg protein1·3 min1 for control and in cells pretreated with DNP and iodoacetate, respectively).
Effect of Na+ and incubation buffer pH. We sought to determine whether the uptake of folic acid into MIA PaCa-2 cells depends on the availability of Na+ in the incubation medium. We examined the effect of isosmotically replacing Na+ (123 mM) in the incubation medium with the monovalent cations K+ and Li+ (123 mM) on the initial rate of folic acid (11.6 nM) uptake at pH 5.0. The results show that replacing Na+ with either K+ or Li+ had no significant effect on vitamin uptake [148.3 ± 7.4, 148.7 ± 2.9, and 146.4 ± 2.5 fmol·mg protein1·3 min1 for control (Na+) and in the presence of K+ and Li+, respectively]. We also examined the effect of pretreating MIA PaCa-2 cells for 30 min with the Na+-K+-ATPase inhibitor ouabain (1 mM) on the initial rate of folic acid (11.6 nM) uptake at pH 5.0. The results show similar uptake in ouabain-pretreated and control cells (131.00 ± 3.3 and 136.09 ± 0.9 fmol·mg protein1·3 min1, respectively).
In another study, we examined the effect of varying incubation buffer pH over the range 5.0 to 8.0 on the initial rate of folic acid (11.6 nM) uptake by MIA PaCa-2 cells. The results (Fig. 2) show that decreasing the incubation buffer pH from 8.0 to 5.0 caused a sharp increase in folic acid uptake with maximum uptake at pH 5.0. In fact, folic acid uptake at pH 7.4 was only 2% of that at pH 5.0. For this reason, we performed all other experiments at buffer pH 5.0. In a related study, we tested the effect of pretreating for 30 min MIA PaCa-2 cells with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 50 µM) on the initial rate of folic acid (11.6 nM) uptake at pH 5.0 and 7.4. FCCP caused significant (P < 0.01) inhibition in folic acid uptake in cells incubated at buffer pH 5.0 (137.42 ± 6.4 and 77.4 ± 1.5 fmol·mg protein1·3 min1 for control and FCCP-pretreated cells, respectively) but not in those incubated at buffer pH 7.4 (3.08 ± 0.5 and 3.24 ± 0.5 fmol·mg protein1·3 min1 for control and FCCP-pretreated cells, respectively).
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Uptake as a function of concentration. In this study, we examined the initial rate of folic acid uptake (3 min) as a function of increasing the substrate concentration in the incubation medium (0.0210 µM). The results (Fig. 3) show that the uptake exhibited saturation with respect to increasing the folic acid concentration in the medium. Uptake by the saturable component was determined by subtracting the uptake by simple diffusion from the total folic acid uptake at each substrate concentration examined. Uptake by simple diffusion was calculated as the slope of the line between uptake at high pharmacological concentration of folic acid (1 mM) and the point of origin. The apparent Km and Vmax of the saturable uptake component were then calculated as described in MATERIALS AND METHODS and found to be 0.762 ± 0.10 µM and 6.115 ± 0.29 fmol·mg protein1·3 min1, respectively.
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Effect of membrane transport inhibitors. We examined the effect of inhibitors of anion exchangers, i.e., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (both at 0.5 mM), on the initial rate of folic acid (11.6 nM) uptake by MIA PaCa-2 cells. Both membrane transport inhibitors significantly (P < 0.01) inhibited the folic acid uptake (150.2 ± 3.7, 3.6 ± 0.6, and 15.0 ± 0.8 fmol·mg protein1·3 min1 for control and in cells pretreated with DIDS and SITS, respectively).
Expression of hRFC at RNA and Protein Levels in MIA PaCa-2 Cells and Evidence for Functional Contribution of hRFC
The findings that the folic acid uptake process by MIA PaCa-2 cells is markedly higher at acidic pH compared with alkaline pH, that the process has a similar affinity for oxidized, reduced, and substituted folate derivatives, and that it has an apparent Km in the micromolar range strongly suggest that the process is mediated via hRFC (1114). Thus our next aim was to establish the expression of the hRFC at the mRNA and protein levels in MIA PaCa-2 cells and to further demonstrate its functional contribution to carrier-mediated folate uptake. We performed RT-PCR with the use of gene-specific primers corresponding to a sequence in the open reading frame of hRFC (see MATERIALS AND METHODS) and obtained the fragment of expected size (Fig. 4A). Sequencing of the fragment showed that it was identical to that of hRFC. Expression of hRFC in these cells at the protein level was confirmed by Western blot analysis with the use of specific anti-hRFC polyclonal antibodies and postnuclear extracts of MIA PaCa-2 cells (Fig. 4B).
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After establishing the mechanism of folic acid uptake by MIA PaCa-2 cells, we examined possible regulation of the uptake process by specific intracellular regulatory pathways. We focused on pathways for which there is a consensus sequence in the hRFC protein (i.e., PKC and PKA) (9, 10) and on those that have been shown to play a role in the regulation of uptake of other nutrients in other cellular systems (PTK- and Ca2+/calmodulin-mediated pathways; see Ref. 7).
Involvement of a PKC-mediated pathway in the regulation of folic acid uptake by MIA PaCa-2 cells was examined by pretreating (for 1 h) confluent monolayers with modulators of PKC activity before uptake measurements. We tested the effect of the PKC activator phorbol 12-myristate 13-acetate (PMA) and the PKC inhibitors chelerythrine and bisindolylmaleimide (all at 10 µM) on the initial rate of folic acid (11.6 nM) uptake. None of these modulators were found to significantly affect folic acid uptake (106.7 ± 2.8, 98.2 ± 7.9, 108.3 ± 2.2, and 98.1 ± 6.0 fmol·mg protein1·3 min1 for control cells and in cells pretreated with PMA, chelerythrine, and bisindolylmaleimide, respectively).
The role of a cAMP (or PKA)-mediated pathway in the regulation of folic acid uptake by MIA PaCa-2 cells was investigated by examining the effect of pretreating the cells for 1 h with compounds that are known to increase intracellular cAMP level, namely, dibutyryl cAMP (DBcAMP) and isobutylmethylxanthine (IBMX), on the initial rate of folic acid (11.6 nM) uptake. DBcAMP and IBMX were found to cause a significant and concentration-dependent inhibition in folic acid uptake (Table 1). Pretreating the cells with the PKA inhibitor H-89 failed to cause any significant effect on folic acid uptake. In addition, pretreating cells with H-89 (100 µM) first for 30 min, followed by the addition of 1 mM DBcAMP and subsequent incubation for 30 min, failed to prevent the inhibition in folic acid uptake caused by DBcAMP alone (112.2 ± 2.6, 67.6 ± 2.0, and 64.0 ± 0.7 fmol·mg protein1·3 min1 for the control, DBcAMP-treated, and both DBcAMP- and H-89-treated cells, respectively). We also examined the effect of DBcAMP on the kinetic parameters of the folic acid uptake process by MIA PaCa-2 cells. This was accomplished by studying the effect of DBcAMP (2 mM) on the initial rate of folic acid uptake as a function of concentration and comparing the results with that of untreated control. The results show carrier-mediated folic acid uptake to be saturable in both the absence and the presence of DBcAMP but that uptake in the presence of DBcAMP was lower than that of control (Fig. 6). The kinetic parameters of saturable uptake showed a decrease in the Vmax of folic acid uptake in DBcAMP-pretreated cells compared with control cells (2.418 ± 0.07 and 6.115 ± 0.29 fmol·mg protein1·3 min1, respectively), as well as a decrease in the apparent Km (0.358 ± 0.03 and 0.762 ± 0.10 µM, respectively).
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DISCUSSION |
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Folic acid uptake by pancreatic MIA PaCa-2 cells was found to be carrier mediated in nature. This conclusion is based on the observation that the initial rate of folic acid uptake was saturable as a function of substrate concentration, with an apparent Km value of 0.762 ± 0.10 µM. The ability of the folate structural analogs 5-FTHF and MTX to inhibit the initial rate of [3H]folic acid uptake further confirms this conclusion. It is interesting to note that the Ki of [3H]folic acid uptake by 5-FTHF and MTX is competitive in nature, with apparent Ki of 1.0 and 0.83 µM, respectively. The similarity of the Ki values for 5-FTHF and MTX and the value of the apparent Km for folic acid uptake by MIA PaCa-2 cells strongly suggests that these three compounds have the same affinity for the involved uptake system and thus clearly justifies the use of folic acid as a representative folate compound in this study. It is interesting that the pH profile and the similar affinity for oxidized, reduced, and substituted folate derivatives are similar to those observed in human intestinal and colonic epithelial cells (7, 1113). These features of the folate uptake process are in contrast to the features observed in cells such as L1210 cells, however, in which uptake is higher at neutral and alkaline than at acidic buffer pH and in which the system has higher affinity for reduced over oxidized folate derivatives (for review, see Ref. 15). Uptake of folic acid by MIA PaCa-2 cells also was found to be sensitive to the effect of the anion exchange inhibitors DIDS and SITS.
The finding of greater folate uptake by MIA PaCa-2 cells at acidic pH than at alkaline pH, the similar affinity for oxidized, reduced, and substituted folate derivatives, and the fact that the apparent Km of the uptake system is in the micromolar range strongly indicate that this system is the hRFC (1114). RT-PCR and Western blot analysis results confirmed the expression of hRFC at the RNA and protein levels, respectively. To further confirm the functional involvement of the hRFC in folate uptake by MIA PaCa-2 cells, we examined the effect of silencing the hRFC gene on the ability of the cells to take up folic acid by the carrier-mediated process. This experiment was performed with the use of gene-specific siRNA. First, we validated that such an approach does indeed lead to specific depletion in the level of hRFC mRNA and the protein level in the pretreated cells compared with controls. The results show that pretreating the MIA PaCa-2 cells with hRFC gene-specific siRNA leads to a specific and marked decrease in hRFC mRNA and protein levels. We then examined the effect of such siRNA pretreatment on the ability of the cells to transport folic acid. The results showed significantly less folic acid uptake in siRNA-pretreated cells than in control cells. This effect was found to be specific for folic acid because uptake of the unrelated biotin was similar in hRFC siRNA-pretreated and control cells. These findings clearly confirm the functional role of hRFC in folic acid uptake by MIA PaCa-2 cells.
After the determination of the cellular mechanism of folate uptake by MIA PaCa-2 cells, we investigated the possible involvement of certain intracellular protein kinase-mediated regulatory pathways in the regulation of folic acid uptake by these pancreas-derived cells. We focused on those pathways for which there is consensus sequence in the hRFC polypeptide (PKC and PKA; see Refs. 9, 10) and on those that have been shown to regulate the uptake of other nutrients in other cell systems. Our results show that although PKC- and Ca2+/calmodulin-mediated pathways have no role in the regulation of folic acid uptake by MIA PaCa-2 cells (as indicated by lack of effect on vitamin uptake by modulators of these pathways), evidence was obtained that suggests the involvement of cAMP- and PTK-mediated pathways in the regulation of the uptake process. The effect of cAMP was found to be mediated via changes in the Vmax and the apparent Km of the folic acid uptake process, suggesting an effect on the activity and affinity of the folate carrier system, respectively. The effect of increasing the level of intracellular cAMP on folic acid uptake did not appear to be mediated by the activation of PKA. This conclusion is based on the observation that the addition of the PKA-specific inhibitor H-89 failed to prevent the inhibitory effect on folic acid uptake caused by subsequent addition of DBcAMP. The inhibitory effect of genistein was mediated via a decrease in the Vmax and an increase in the apparent Km of the folic acid uptake process, suggesting that the effect is mediated via alterations in the activity (and/or number) and the affinity of the folic acid uptake carriers. These results regarding the possible role of cAMP and PTK in the regulation of folic acid uptake by MIA PaCa-2 cells are similar to those observed for substrate uptake by other cells of the digestive system (namely, the human colonocytes) (7).
In summary, the results of our study demonstrate for the first time that folate uptake by human-derived pancreatic MIA PaCa-2 cells is mediated via a specialized, acidic pH-dependent, carrier-mediated mechanism that involves the hRFC. In addition, the study shows the folate uptake process of these cells to be under the possible regulation of intracellular cAMP- and PTK-mediated pathways.
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GRANTS |
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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.
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