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Biochemical and Molecular Action of Nutrients

Folate Receptor Function Is Regulated in Response to Different Cellular Growth Rates in Cultured Mammalian Cells^{1 ,2}

Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30335

³To whom correspondence should be addressed. E-mail: vlsteve{at}emory.edu .

ABSTRACT

The folate receptor (FR) binds physiologic folates with nmol/L affinities and is expected to play an important role in transporting serum folates into cells that express this receptor. Although it has been shown that FR expression increases when extracellular levels of folate are low, whether this receptor is regulated in response to altered cellular requirements for folates or by intracellular levels of this vitamin has not been investigated. In this study, FR levels, FR function and cellular folate levels were measured in cells with different growth rates to investigate FR regulation of this receptor under conditions in which cellular requirements for folate are altered. These experiments used cells that endogenously express FR (JAR, Caco-2 and MA-104) and cells stably transfected with this receptor (FRGPI-16 and FRTM-8). FR function decreased as cellular growth slowed in four of the five cell lines examined. Although cellular folate levels also decreased as cells reached confluence, the total amount of cellular folate in the culture remained constant, suggesting the depleted cellular folate was because of the cell partitioning its pool throughout cell division, not because of decreased FR function. Conversely, there was an inverse association with FR levels and cell growth (r = -0.998 to -0.999, P < 0.05) in cells endogenously expressing FR, with a significant increase in the percentage of total FR located in an intracellular compartment as growth slowed. These results suggest FR function is regulated by cellular requirements for folates but not in response to changing FR levels or cellular levels of this vitamin.

KEY WORDS: • humans • folate receptor • folate uptake • cell growth

Folates are essential cofactors for many biochemical reactions involving one-carbon metabolism, including purine and thymidine synthesis, remethylation of homocysteine to methionine and conversion of serine to glycine. The role of this vitamin in the production of precursors for DNA synthesis and repair makes it essential for proliferating cells. Cells can acquire the folate needed for these processes by two separate mechanisms. One involves a transmembrane (TM)⁴ transporter known as the reduced folate carrier (RFC). This ubiquitously expressed protein has a high affinity for 5-methyltetrahydrofolate (5-MTHF) (K_d = 0.3–4.0 µmol/L), the predominant folate found in serum, and a much lower affinity for folic acid (K_d=100–200 µmol/L) (1 ).

The other folate uptake system uses a glycosylphosphatidylinositol (GPI)-anchored protein known as the folate receptor (FR) that transports folates into the cytosol via fluid-phase endocytosis (2 –6 ). This receptor binds its ligands with very high affinity, having a K_d of 0.4 nmol/L for folic acid and 3 nmol/L for 5-MTHF. There are three isoforms of FR that vary somewhat in sequence, ligand preference and tissue distribution, such that only one isoform, designated FR-, is thought to be physiologically relevant (7 –10 ). The expression of FR- is limited to normal differentiated epithelial cells, including those of the choroid plexus, placenta, kidney and lung (11 ). FR- has also been found to be overexpressed in malignant tissues of epithelial origin, including the uterus, ovary, breast, colon and testis (12 ).

The difference in affinities for folic acid and 5-MTHF between FR and RFC (10⁵-fold for folic acid and 100-fold for 5-MTHF) suggests that the GPI-anchored receptor may play an important role in transporting folates when extracellular concentrations are in the nmol/L range. This hypothesis is supported by the finding that introduction of FR by transfection into cells that normally do not express this receptor allows the cells to grow in low (nmol/L) folate concentrations (13 , 14 ). Several investigators have found that FR mRNA and protein levels increase when extracellular levels of folate are low, suggesting that cells upregulate FR when the amount of folate available for transport falls to levels at which it can only be effectively transported by this receptor (15 –17 ). However, whether increased levels of FR actually reflect increased function of this receptor under these circumstances has not been investigated. The possibility that FR levels and function are not coordinately regulated was suggested by the findings of Lark et al. (18 ). These investigators found that FR function and levels are discordantly altered in the monkey kidney epithelial cell line MA-104 as rates of cell growth change. As cell growth slowed, FR function decreased, but FR levels increased. Because FR expression was lowest when the rate of cell replication was greatest, these investigators concluded that this receptor does not play an important role in the acquisition of folates required for rapid growth of these cells.

In this study, FR function and levels were measured in cells with different growth rates to investigate the regulation of this receptor under conditions in which cellular requirements for folates are altered. These experiments were done with three different cell lines that normally express high levels of FR to determine if the results found represent common regulatory mechanisms or if there is variability between cells from different species and tissues (3 , 8 , 18 –22 ). These three cell lines were JAR, a human placental choriocarcinoma cell line, Caco-2, a human adenocarinoma cell line, and MA-104, a monkey kidney epithelial cell line. Because some studies of FR function and endocytosis have utilized cells in which FR is not normally expressed but has been introduced by transfection (2 , 13 , 23 ), a Chinese hamster ovary (CHO) cell line stably transfected with FR- (designated FRGPI-16) has also been analyzed. Finally, to assess the role of the GPI anchor in the behavior of FR in response to different cellular requirements for folate, a CHO cell line that stably expresses a form of FR in which the GPI anchor was replaced with a TM protein span (designated FRTM-8) was also studied. Cellular folate levels were also measured to determine the influence of this parameter on FR regulation. The findings of this study indicate that FR function may be regulated by cellular requirements for folates but not in response to cellular levels of this vitamin.

MATERIALS AND METHODS

Materials.

Fetal bovine serum was purchased from Atlanta Biologicals, (Norcross, GA). RPMI 1640 medium, folate-free RPMI 1640 medium and Ham’s F12 medium were purchased from Life Technologies, (Rockville, MD). Folate-free Ham’s F12 medium was purchased from Specialty Media (Phillipsburg, NJ). [3,5,7,9-³H]Folic acid (25–30 Ci/mmol, 99% pure) and [methyl-³H]thymidine (25 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA) and Amersham Pharmacia Biotech (Piscataway, NJ), respectively. Folic acid casei assay medium, lactobacilli MRS broth and chicken pancreas were purchased from Becton Dickinson (Sparks, MD). Zeocin was purchased from Invitrogen (Carlsbad, CA). Eco-lite scintillation fluid was purchased from ICN (Costa Mesa, CA). Folic acid, folinic acid, leupeptin, aprotinin, charcoal and all other chemicals were purchased from Sigma (St. Louis, MO). The reagents for the bicinchoninic acid (BCA) protein assay were purchased from Pierce (Rockford, IL).

Cell culture.

MA-104 cells (BioWhittaker, Walkersville, MD), JAR and Caco-2 cells (American Type Culture Collection, Manassas, VA) were routinely grown in monolayer cultures in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 10⁵ units/L penicillin and 100 mg/L streptomycin in an atmosphere of 5% CO₂ at 37°C. Cells were routinely passaged with either 0.5 mmol/L EDTA in phosphate-buffered saline (PBS) (137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L Na₂HPO₄ and 1.4 mmol/L KH₂PO₄) (JAR cells) or 0.5 mmol/L EDTA in PBS containing 1.25 g/L trypsin (Caco-2 and MA-104 cells). Cell numbers were determined by counting with a hemocytometer.

FRGPI-16 cells were generated by stable transfection of CHO-K1 cells with a cDNA encoding the FR- protein [obtained from Dr. Manohar Ratnam (Medical College of Ohio, Toledo, OH)] and put into the mammalian expression vector pZeoSV2(+) using standard cloning methods. FRTM-8 cells, which express a TM form of FR-, were generated by stable transfection of CHO-K1 cells with a cDNA in which the COOH-terminal GPI anchor signal of FR- (19 amino acids) had been replaced with the sequence for the COOH-terminal TM domain (encoding 85 amino acids) of the complement membrane cofactor protein (MCP or CD46). Both cell lines were cloned by limiting dilution after surviving selection in Zeocin. The mechanism of membrane attachment of FR- in these cell lines was tested by treatment with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC), followed by partitioning in the detergent Triton X-114 and Western blotting. FR- was susceptible to cleavage by PI-PLC in the FRGPI-16 cells and not in the FRTM-8 cells, indicating that it was attached to the membrane via a GPI anchor in the former and a TM span in the latter (data not shown). Both cell lines were routinely grown in monolayer cultures in Ham’s F12 medium supplemented with 10% (v/v) fetal bovine serum, 10⁵ units/L penicillin, 100 mg/L streptomycin and 175 mg/L Zeocin in an atmosphere of 5% CO₂ at 37°C. Cells were passaged by treatment with 0.5 mmol/L EDTA in PBS.

For all experiments, cells were plated at 2.5 x 10⁵ cells/25 cm² flasks in folate-free medium (RPMI 1640 for JAR, MA-104 and Caco-2; Ham’s F12 for FRGPI-16 and FRTM-8) and grown to the indicated confluency. The medium was changed every 2 d (to prevent depletion of nutrients other than folate). Cultures were judged to be subconfluent or confluent when the cells covered 75 or 100%, respectively, of the flask’s growth surface. Cells were defined as postconfluent when they had grown for 2 d after reaching confluence.

[³H]folic acid purification.

[3,5,7,9-³H]folic acid was spotted on cellulose 300, F254 plastic-backed plates (Selecto Scientific, Suwanee, GA) and chromatographed in the dark in 50 mmol/L HEPES, pH 7.5, containing 286 mmol/L ß-mercaptoethanol as the developing agent. [³H]folic acid was identified by comparison to unlabeled folic acid spotted on the thin-layer chromatography plate in an adjacent lane and visualized with UV light. The [³H]folic acid was scraped from the plate and eluted three times with 50 mmol/L HEPES, pH 7.5, containing 341 mmol/L ethanol and 286 µmol/L ß-mercaptoethanol. The specific activity (dpm/pmol) of the [³H]folic acid was calculated using the extinction coefficient, _mM = 27.6 (282 nm, pH 7.0). The purified [³H]folic acid was either used immediately or stored at -80°C and used within 7 d.

Folic acid uptake.

The internalization of folic acid into the cytosol was quantitated as previously described with a few minor variations (24 ). Briefly, cells were rinsed twice with PBS followed by the addition of folate-free growth medium containing 200 nmol/L [³H]folic acid to each flask. After incubating the cells at 37°C for the specified times (depending on cell line as detailed in the results), the cells were placed on ice for 5 min, incubation medium was removed, and the flasks were rinsed four times with 3 mL of ice-cold PBS. One milliliter of ice-cold lysis buffer (10 mmol/L Tris-HCl, pH 8.0, 20 mg/L leupeptin, 20 mg/L aprotinin and 5 mmol/L unlabeled folic acid) was added to each flask. The cells were then lysed by placing the flasks at -80°C for 15 min and thawed on ice. Each lysed sample was then transferred to a centrifuge tube, and the flask was rinsed with an additional 1 mL of ice-cold lysis buffer. The samples were ultracentrifuged for 35 min at 100,000 x g at 4°C to separate the membrane (pellet) from the cytosolic (supernatant) fractions. Aliquots of the supernatant were counted by scintillation counting and normalized to protein content determined by the BCA assay (25 ). Nonspecific uptake was determined in each experiment by measuring [³H]folic acid uptake in the presence of 750-fold excess unlabeled folic acid. Specific uptake values were determined by subtracting the nonspecific value from the respective total radioactivity.

Folic acid binding.

Folic acid binding was used to estimate the total amount of FR in the cell as well as the amount of receptor involved in endocytosis. To determine the total amount of FR in the cell, cellular membranes were solubilized with 1% (v/v) Triton X-100 before the binding assay to allow the [³H]folic acid access to all pools of receptor in the cell. The pool of FR with access to the cell surface, which includes receptor on the plasma membrane and in the endocytic pathway, was quantified by performing the binding reaction in viable cells maintained in culture conditions at 37°C. We have defined this FR pool measured in intact cells as the "cycling" receptor.

For solubilized folate binding, cells were washed twice with PBS, scraped and centrifuged for 5 min at 1,000g; the supernatant was removed; and the pellet was solubilized in 1 mL ice-cold solubilization buffer (25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA and 1% (v/v) Triton X-100) for 20 min on ice. [³H]folic acid (20–175 nmol/L, depending on the cell line) was added and the samples were incubated at 37°C for 1 h to allow ligand binding to the receptor. The samples were then placed on ice for 5 min, and 1 mL dextran-coated charcoal solution (25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 8 mol/L dextran and 80 mol/L activated charcoal) was added to each tube to absorb unbound radiolabel. The samples were vortexed and incubated on ice for 15 min, followed by centrifugation for 30 min at 35,000 x g at 4°C. Aliquots of the supernatant were counted by scintillation counting. Nonspecific binding was determined in each experiment by measuring [³H]folic acid binding in the presence of 750-fold excess unlabeled folic acid. Specific binding values were determined by subtracting the nonspecific value from the respective total radioactivity.

Intact binding was measured using the same methodology, except the incubation with [³H]folic acid was done with living cells at 37°C for the amount of time necessary to allow the FR to completely cycle through the endocytic pathway (1–3 h, depending on cell line). The optimal conditions for the intact binding assay (ligand concentration and incubation time) were defined for each cell line by increasing the [³H]folic acid concentration and incubation time (independently) until maximal binding was obtained (data not shown).

Cellular folate quantitation.

Cellular folate was quantitatively measured using a 96-well plate adaptation of the Lactobacillus casei (L. casei) microbiological assay (26 ). Briefly, cells were washed twice with PBS, scraped from the dish and pelleted by centrifugation (1,000 x g for 5 min at 4°C). The cell pellet was resuspended in sodium-phosphate buffer, pH 8.6 (473.5 g/L Na₂HPO₄ and 26.5 g/L NaH₂PO₄), 572 µmol/L ß-mercaptoethanol and freshly prepared 1 g/L ascorbic acid. The samples were immediately vortexed, placed in a boiling water bath for 5 min and put on ice to cool. The cooled samples were centrifuged at 6,000g for 10 min, a 100-µL aliquot of the supernatant was treated with conjugase buffer (850 µL sodium-phosphate buffer, pH 8.6, freshly prepared 10 g/L ascorbic acid and 200 µL partially-purified chicken pancreas conjugase) to convert polyglutamates to their corresponding mono- and di-glutamate derivatives, and the samples were incubated at 37°C for 24 h and placed at -20°C for 30 min. The chicken pancreas was partially purified as previously described (27 ).

The folate content of the conjugase-treated samples was quantitatively measured by the L. casei microbiological assay. Briefly, the thawed samples were diluted 1:500 in sodium-phosphate buffer, pH 6.3 (112.5 g/L Na₂HPO₄ and 387.5 g/L NaH₂PO₄) and 1 g/L freshly prepared ascorbic acid. L. casei bacteria cryopreserved in 5.4 mol/L glycerol was thawed and diluted with sterile 9 g/L NaCl and added to freshly prepared folic acid casei assay medium containing 0.5 g/L ascorbic acid. Inoculated medium (150 µL) was pipetted into 96-well plates and allowed to grow for 24 h at 37°C. Each plate also contained a standard curve in which L. casei growth in concentrations of folinic acid, ranging from 0.005–0.15 ng/0.3 mL, was assessed in the same way. Bacterial growth was assessed by measuring absorbance at 595 nm in a Bio-Rad reader (Bio-Rad Laboratories, Hercules, CA). Sample concentrations were determined by comparison to the growth of the standards and were normalized to protein content.

[³H]thymidine incorporation.

Cells were rinsed twice with PBS and folate-free medium containing 0.03 µCi [³H]thymidine (total volume of 1 mL) was added to each flask. After 2 h of incubation at 37°C, the cells were placed on ice for 5 min, the incubation medium was removed, and the flasks were rinsed four times with ice-cold PBS. The rinsed cells were scraped and transferred to a filtration device (25-mm stainless steel filter holder with 125 mL Erlenmeyer filter flask; VWR, Willard, OH) containing a glass microfiber filter (25-mm diameter; Whatman GF/C, Maidstone, England). The filter was then washed with three applications of 100 g/L trichloroacetic acid (2 mL each) to precipitate the DNA to the glass microfibers and to remove free [³H]thymidine. The filter was then rinsed three times with 2 mL of 14.3 mol/L ethanol to remove residual acid and unincorporated [³H]thymidine. Radioactivity on the filter was measured by scintillation counting.

Statistics.

The values are reported as means ± SEM of measurements performed in triplicate. Statistical analysis of the data was performed using the Minitab statistical software program (Minitab, Release 12; Minitab, State College, PA). Statistical comparisons between two means were conducted by Student’s t test using P < 0.05 as the level of significance. For comparison of the relationship between cell growth and FR number, a Pearson’s correlation coefficient was used with P < 0.05 as the level of significance. Uptake and binding data were transformed by regression analysis and plotted using Microsoft Excel 2000 software (Redmond, WA).

RESULTS

Cellular growth rates vary with confluence.

For most of the cell lines, the rate of [³H]thymidine incorporation was highest before the cells reached confluence and then decreased as the cells grew to confluence and beyond (Fig. 1 ). Thus, the rate of growth was relatively rapid at subconfluence, somewhat slower at confluence and more reduced at postconfluence. The specific points on the growth curve corresponding to the various confluencies (subconfluence, confluence and postconfluence) at which FR function and levels were measured in each cell line are indicated with arrows in Figure 1 .

View larger version (13K): [in this window] [in a new window]   Figure 1. Cellular growth rates as measured by [3H]thymidine incorporation in JAR, Caco-2, MA-104, FRGPI-16 and FRTM-8 cells. Cells were incubated in folate-free medium containing 0.03 µCi [3H]thymidine for 2 h at 37°C. [3H]thymidine incorporation into DNA was measured as described in MATERIALS AND METHODS and shown in the indicated panels. The arrows () indicate the number of days after plating that corresponded to the various confluencies (SC, subconfluency; C, confluency; PC, postconfluency) for each of the cell lines. Values are means ± SEM (n = 3).

As shown in Figure 2 A, the rate of cellular folate uptake decreased as the cells grew to confluence and beyond. FR levels at each growth rate were also determined. Total cellular FR was determined by measuring specific [³H]folic acid binding in detergent solubilized cells, while the amount of receptor involved in endocytosis (referred to as cycling FR) was quantified as the specific [³H]folic acid binding in intact cells maintained at 37°C. The level of cycling FR in these cells stayed relatively constant (4–8 pmol/mg protein) as growth slowed, while the total amount of FR increased dramatically (from 4 to 26 pmol/mg protein) (Fig. 2 B). Comparison of the amount of cycling and total FR indicates that almost all of the receptor in the cell participated in endocytosis when the cells were growing rapidly at subconfluence. However, as growth slowed, there was a significant increase in the total amount of receptor in the cell. Because this increase was not reflected in the cycling pool of FR, this result suggests that the additional receptor is located in an intracellular location. The finding that cellular levels of FR do not decrease as the function of this receptor does indicates that FR function is not regulated by FR levels in JAR cells.

View larger version (20K): [in this window] [in a new window]   Figure 2. FR function and levels in human placental choriocarcinoma (JAR) cells. [3H]folic acid uptake (A) was measured at each of the indicated confluencies (SC, subconfluency; C, confluency; PC, postconfluency) by incubating cells in folate-free medium containing 200 nmol/L [3H]folic acid for the indicated time. Solubilized [3H]folic acid binding (Total; B) was conducted in detergent-solubilized cells incubated with 175 nmol/L [3H]folic acid for 1 h at 37°C. Intact binding (Cycling; B) was performed in live cells maintained at 37°C for 3 h in folate-free medium containing 175 nmol/L [3H]folic acid. Data are expressed as pmol [3H]folic acid transported (A) or bound (B) per milligram cellular protein. Uptake and binding values are means ± SEM (n = 3). Asterisks (B) indicate significant difference between total and cycling data for the indicated confluency (P < 0.05).

As in JAR cells, the rate of cellular folate uptake decreased as growth rate decreased (Fig. 3 A). Receptor levels also changed in a manner similar to that observed in JAR cells with the amount of cycling FR remaining fairly constant (0.2 pmol/mg protein) and total FR levels dramatically increasing as growth slowed (from 0.2 to 1.5 pmol/mg protein) (Fig. 3 B). All of the FR in the cell appeared to be involved in endocytosis in subconfluent cells, whereas only 20% of the receptor was cycling in postconfluent cells. As in the JAR cells, the inverse relationship between FR function and levels in response to different growth rates suggests that these parameters are differentially regulated in Caco-2 cells.

View larger version (21K): [in this window] [in a new window]   Figure 3. FR function and levels in human adenocarinoma (Caco-2) cells. [3H]folic acid uptake (A) was measured at each of the indicated confluencies (SC, subconfluency; C, confluency; PC, postconfluency) by incubating cells in folate-free medium containing 200 nmol/L [3H]folic acid for the indicated time. Solubilized [3H]folic acid binding (Total; B) was conducted in detergent-solubilized cells incubated with 20 nmol/L [3H]folic acid for 1 h at 37°C. Intact binding (Cycling; B) was performed in live cells maintained at 37°C for 3 h in folate-free medium containing 20 nmol/L [3H]folic acid. Data are expressed as pmol [3H]folic acid transported (A) or bound (B) per milligram cellular protein. Uptake and binding values are means ± SEM (n = 3). Asterisks (B) indicate significant difference between total and cycling data for the indicated confluency (P < 0.05).

The rate of folic acid uptake in MA-104 cells slightly increased as growth rate decreased (Fig. 4 A). Both cycling and total FR levels were significantly increased as growth slowed in the MA-104 cells (0.3–8 and 0.5–17 pmol/mg protein, respectively) (Fig. 4 B). There was a greater increase in total FR as compared with cycling receptors, with approximately one-half of the total FR functioning in the cycling pool in postconfluent cells.

View larger version (21K): [in this window] [in a new window]   Figure 4. FR function and levels in monkey kidney epithelial (MA-104) cells. [3H]folic acid uptake (A) was measured at each of the indicated confluencies (SC, subconfluency; C, confluency; PC, postconfluency) by incubating cells in folate-free medium containing 200 nmol/L [3H]folic acid for the indicated time. Solubilized [3H]folic acid binding (Total; B) was conducted in detergent-solubilized cells incubated with 100 nmol/L [3H]folic acid for 1 h at 37°C. Intact binding (Cycling; B) was performed in live cells maintained at 37°C for 1 h in folate-free medium containing 100 nmol/L [3H]folic acid. Data are expressed as pmol [3H]folic acid transported (A) or bound (B) per milligram cellular protein. Uptake and binding values are means ± SEM (n = 3). Asterisks (B) indicate significant difference between total and cycling data for the indicated confluency (P < 0.05).

As shown in Figure 5 , FR function (Fig. 5 A) and levels (Fig. 5 B) both decreased as rate of growth decreased. The FRGPI-16 cells express a high level of FR that does not appear to be active, as only approximately one-fourth of the total FR was in the cycling pool (from 25 to 5 pmol/mg protein from subconfluence to postconfluence). The total amount of FR in the cell decreased more than the cycling pool (from 100 to 48 pmol/mg protein), indicating that FR was lost from both the endocytic pathway and the inactive pool as the growth of the FRGPI-16 cells slowed.

View larger version (21K): [in this window] [in a new window]   Figure 5. FR function and levels in Chinese hamster ovary (CHO) cells stably transfected with GPI-anchored FR (FRGPI-16 cells). [3H]folic acid uptake (A) was measured at each of the indicated confluencies (SC, subconfluency; C, confluency; PC, postconfluency) by incubating cells in folate-free medium containing 200 nmol/L [3H]folic acid for the indicated time. Solubilized [3H]folic acid binding (Total; B) was conducted in detergent-solubilized cells incubated with 50 nmol/L [3H]folic acid for 1 h at 37°C. Intact binding (Cycling; B) was performed in live cells maintained at 37°C for 2 h in folate-free medium containing 50 nmol/L [3H]folic acid. Data are expressed as pmol [3H]folic acid transported (A) or bound (B) per milligram cellular protein. Uptake and binding values are means ± SEM (n = 3). Asterisks (B) indicate significant difference between total and cycling data for the indicated confluency (P < 0.05).

Increased confluence in FRTM-8 cells resulted in a reduction in both receptor function and levels (Fig. 6 A and 6B). The total amount of FR decreased from 70 to 27 pmol/mg protein while the cycling pool decreased from 47 to 23 pmol/mg protein as cells grew from subconfluence to postconfluence. Although FRTM-8 cells expressed similar FR levels as FRGPI-16 cells, a greater proportion of the receptors (approximately two-thirds of the total FR pool) was cycling in the endocytic pathway.

View larger version (21K): [in this window] [in a new window]   Figure 6. FR function and levels in Chinese hamster ovary (CHO) cells stably transfected with TM-attached FR (FRTM cells). [3H]folic acid uptake (A) was measured at each of the indicated confluencies (SC, subconfluency; C, confluency; PC, postconfluency) by incubating cells in folate-free medium containing 200 nmol/L [3H]folic acid for the indicated time period. Solubilized [3H]folic acid binding (Total; B) was conducted in detergent-solubilized cells incubated with 50 nmol/L [3H]folic acid for 1 h at 37°C. Intact binding (Cycling; B) was performed in live cells maintained at 37°C for 2 h in folate-free medium containing 50 nmol/L [3H]folic acid. Data are expressed as pmol [3H]folic acid transported (A) or bound (B) per milligram cellular protein. Uptake and binding values are means ± SEM (n = 3). Asterisks (B) indicate significant difference between total and cycling data for the indicated confluency (P < 0.05).

The amount of folate per cell decreased in each of the cell lines as the confluence of the culture increased (Fig. 7 ). However, the total level of folate in the culture remained constant as the cells grew to confluency and beyond (data not shown). Comparison of the amount of folate transported by FR over a 24-h period in the five cell lines analyzed (estimated using the rates of uptake from Figs. 2 3 4 5 6 ) to the folate pools reveals that the former is significantly smaller than the latter, even at subconfluence, when transport rates are the greatest (P < 0.05). Therefore, gain or loss of FR-mediated transport is not expected to have dramatic effects on cellular folate pools over a short time period (i.e., several days).

View larger version (18K): [in this window] [in a new window]   Figure 7. Cellular folate levels for each of the five cell lines as measured by the L. casei assay. Cellular folate levels were measured by conducting the L. casei assay at each of the indicated confluencies as described in MATERIALS AND METHODS. The data, expressed as pmoL folate per milligram cellular protein, are the means ± SEM (n = 3).

DISCUSSION

Rapidly growing cells are expected to have higher folate requirements than slowly dividing cells. To assess the effect of differing requirements for folates on the regulation of this receptor, FR function and levels were determined in cells growing at different rates. Measurement of folic acid uptake demonstrated that FR function was downregulated as growth slowed in four of the five cell lines tested. This relationship was observed in both cells that endogenously express FR (JAR and Caco-2) and cells stably transfected with this receptor (FRGPI-16 and FRTM-8). The concomitant decrease in FR function and cellular growth rate suggests that the downregulation of FR function was in response to a decreased need for this vitamin. Furthermore, the fact that FR function is downregulated as cellular folate pools are decreasing indicates that transport is not stimulated by cellular losses of this vitamin.

The one cell line in which FR function was not downregulated as growth slowed was MA-104. In these cells, uptake increased slightly as the culture became more confluent (Fig. 4) . The reason for the apparent difference in regulation of FR in MA-104 cells is unclear. Our findings with this cell line are also different than those of Lark et al. (18 ), who found that FR transport decreased as growth slowed in these cells. The reason for the discrepancy between the previous findings and the results presented here, again, is not completely clear. However, Lark et al. used [³H]5-MTHF rather than [³H]folic acid to assess FR function. Other investigators have shown that 5-MTHF, even when supplied in nmol/L concentrations, is taken up by both FR and the RFC (28 ). Thus, experiments conducted with this ligand reflect the combined action of both folate transporters. The other difference between the present study and the previous one using MA-104 cells was the time span over which the growth rates were varied. Lark et al. measured FR function at 3, 6 and 9 d of growth, whereas we measured it at 4, 6 and 8 d (18 ). It is likely that the differences in growth rates were more pronounced in the previous study and, therefore, may have had a more dramatic effect on FR function.

Quantitation of cellular folate levels revealed that the amount of this vitamin per cell decreased as each of the cell lines grew to confluence and beyond. Although FR function was also downregulated as the culture matured, this decrease does not appear to be attributable to loss of FR-mediated folate uptake. Rather, the depletion of cellular folates most likely occurred because there was very little extracellular folic acid available in the medium to replenish folate pools reduced through cell division. In most cells, the maximum amount of folate transported by FR before being downregulated is considerably less than the cellular folate pool. Therefore, changes in FR function would be expected to alter cellular pools only by small percentages over a short time period (i.e., days). The fact that the overall level of folate in the culture remained constant suggests that there was no significant catabolism of this vitamin by the cells.

All three cell lines that normally express FR showed increased expression of this receptor as growth slowed (r between -0.998 and -0.999, P < 0.05). Almost all of the receptor was involved in endocytosis at the subconfluent stage of growth. But, as cellular growth rate slowed, there was a significant increase in the amount of FR in an intracellular compartment. The utility of this large pool of apparently inactive receptor and its subcellular location is not known and is the subject of ongoing research in our laboratory. However, the inverse relationship between the response of FR function and receptor levels to slower growth indicates that the amount of this protein, either in the cell or involved in endocytosis, does not regulate its activity.

FR levels were regulated differently in cells that had been transfected with this protein. Both FRGPI-16 and FRTM-8 cells expressed very high levels of FR at subconfluent growth, with these levels decreasing by 40% as growth slowed. In contrast to what was found with the cells that endogenously express FR, a significant amount of the FR in the transfected cells was not actively involved in endocytosis at the faster, subconfluent growth rates. These differences between the endogenous and exogenous receptor suggest that the expression of FR is normally regulated through mechanisms that utilize the promoter and other elements of the gene not present in the transfected cells. Alternatively, if only a fixed amount of FR can be accommodated in the endocytic pathway, then the large amount of inactive receptor in the transfected cells may result from the active pool being saturated due to the high level of expression of this protein. The lowest level of cycling receptor in either of the transfected cell lines (5 pmol/mg protein in postconfluent FRGPI-16 cells) is in the same range as the highest level found in any of the cell lines that normally express FR under any growth conditions (8 pmol/mg in PC MA-104 cells). Thus, the cellular machinery needed to process FR to the active form may be saturated in the transfected cells.

Comparison of GPI-anchored FR and TM-FR receptor function indicates these two forms of the receptor are similarly regulated, suggesting the GPI anchor does not play a major role in this regulation. A greater percentage of the TM-FR and a higher absolute amount (up to 47 pmol/mg protein for FRTM-8 cells) is actively involved in endocytosis as compared with the GPI-anchored FR, suggesting that the GPI anchor may play a role in limiting the amount of FR actively involved in the endocytic process. However, our ability to define the role of the GPI anchor in the regulation of FR function and levels using these transfected cells is limited by the fact that they appear to regulate the receptor differently than cells that normally express FR.

FOOTNOTES

¹ Supported by March of Dimes Grant no. 1-FY00-320 (to V.L.S.) and National Institutes of Health Training Grant no. 5-22472 (M.M.D.).

² Presented in part as an abstract at Experimental Biology 2000, San Diego, CA, April 2000 [Doucette M.M., Kristyanne E.S. & Stevens V.L. Cellular requirements for folates regulate folate receptor function and levels. FASEB J. 14: A154.2].

⁴ Abbreviations used: BCA, bicinchoninic acid; 5-MTHF, 5-methyltetrahydrofolate; FR, folate receptor; GPI, glycosylphosphatidylinositol; L. casei, Lactobacillus casei; PBS, phosphate-buffered saline; PI-PLC, phosphatidylinositol-specific phospholipase C; RFC, reduced folate carrier; TM, transmembrane.

Manuscript received 24 April 2001. Initial review completed 26 June 2001. Revision accepted 9 August 2001.

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