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,2
Departments of
*
Nutritional Science and Dietetics, and
Biochemistry, University of Nebraska at Lincoln, Lincoln, NE
2To whom correspondence should be addressed. E-mail: jzempleni2{at}unl.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • endoplasmic reticulum • interleukin-2 • Jurkat cells • riboflavin • stress response
Mammalian cells convert riboflavin into flavin mononucleotide (FMN)3 and FAD, which serve as coenzymes for numerous oxidases and dehydrogenases (1 ). Some proteins designated for secretion into the extracellular space contain intracellular disulfide bonds; these bonds are formed by oxidative folding in the endoplasmic reticulum (ER) before secretion. Evidence has been provided that oxidative folding of proteins in yeast depends on flavins; oxidative folding of secretory proteins is mediated by Ero1p, a flavin-dependent enzyme located in the ER (2 ). Theoretically, riboflavin deficiency might impair proper folding and secretion of proteins, causing accumulation of unfolded proteins in the ER. The human homolog of Ero1p has been named Ero1-L (GenBank accession number AF081886); it is uncertain whether Ero1-L depends on FAD.
Accumulation of unfolded proteins in the ER triggers cellular stress response systems. For example, cells respond to intracellular accumulation of proteins by altering rates of protein synthesis and by increasing the transcription of genes involved in the degradation of unfolded proteins (3 ). Continued generation of unfolded proteins may cause increased expression of genes involved in cell growth arrest (e.g., GADD153) and apoptosis (4 ), leading to decreased net proliferation of cells.
Cells of the immune system (and other tissues) use cytokines, e.g., interleukin (IL)-2, to communicate with each other (5 ). Cells secrete cytokines in response to stimulation of the immune system, e.g., by antigens. After secretion, cytokines bind to receptors located on the surface of target cells, triggering processes such as proliferation and differentiation. Ultimately, cytokine-receptor complexes are endocytosed and degraded to avoid continued and excessive stimulation of immune cells. IL-2 is a cytokine that is secreted primarily by activated TH1 lymphocytes. In IL-2, cysteine-58 and cysteine-105 form a disulfide bond before secretion; this disulfide bond is essential for the secretion and biological activity of IL-2 (6 ). In the present study, a human lymphoid cell line (Jurkat cell) was used to determine whether riboflavin supply affects IL-2 metabolism and cellular stress response systems.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Jurkat cells (clone E6–1), a human leukemia T-cell line (American Type Culture Collection, Manassas, VA), were cultured in riboflavin-defined media containing the following components: 1) 90% (by volume) riboflavin-free RPMI-1640 (Atlanta Biologicals, Norcross, GA); 2) 10% riboflavin-depleted fetal bovine serum (FBS) (prepared by dialyzing FBS against four changes of 20 volumes of distilled water at 4°C). Riboflavin-depleted FBS contained 6 nmol/L riboflavin as determined by HPLC (see below); FAD and FMN were below limits of detection (i.e., <3 and 1 nmol/L, respectively). Given that culture media contained 10% FBS, serum contributed 0.6 nmol/L of riboflavin to total flavins in media; 3) 100,000 U/L penicillin plus 100 mg/L streptomycin (final concentrations); and 4) riboflavin to produce the following final concentrations (including the riboflavin in FBS): 0.85 nmol/L (denoted "severely deficient"), 3.1 nmol/L (denoted "moderately deficient"), 12.6 nmol/L (denoted "physiologic") and 300.6 nmol/L (denoted "pharmacologic"). Cells cultured in physiologic medium were considered the control group.
Riboflavin concentrations were chosen on the basis of the following lines of reasoning: 300.6 nmol/L represents the riboflavin concentration in plasma from riboflavin-supplemented adults (7 ); 12.6 nmol/L represents the riboflavin concentration in normal human plasma (7 ); 3.1 nmol/L represents the riboflavin concentration observed in plasma from moderately deficient pregnant women (8 ); and 0.85 nmol/L represents plasma concentrations of riboflavin as they may occur in severely deficient patients with cystic fibrosis (9 ) and in preterm infants treated with phototherapy (10 ).
In a pre-phase to this study, Jurkat cells were cultured in medium containing 12.6 nmol/L riboflavin for a period of 5 wk to adjust the cells to physiologic concentrations of riboflavin. Medium was replaced with fresh medium every 48 h. Cells were transferred into riboflavin-defined media (0.85, 3.1, 12.6 and 300.6 nmol/L riboflavin) and culturing was continued for 5 wk before analyses. Cell viability was monitored at timed intervals by using trypan blue as described previously (11 ); viability was 98–100%.
Flavin analysis.
Concentrations of riboflavin, FMN and FAD in FBS were quantified by HPLC. Flavins were extracted from dialyzed FBS using acetonitrile and chloroform as described previously (12 ); extracts were chromatographed using a 0.46 x 25 cm C18 column and the following binary gradient system (buffer A = methanol, buffer B = 10 mmol/L acetic acid, pH 4.5): 2 min 28% A and 72% B; linear increase of A to 40% over 2.5 min; 4.5 min 40% A and 60% B; instantaneous change to 28% A; 8 min 28% A and 72% B. Flow rate was 1.00 mL/min. Wavelength settings at the fluorescence detector were 450 nm (excitation) and 530 nm (emission).
Riboflavin transport.
Rates of riboflavin transport into Jurkat cells were determined using a physiologic concentration of [3H]riboflavin (10 nmol/L) as described previously (11 ).
Glutathione metabolism.
After 5 wk of culturing in riboflavin-defined media, Jurkat cells were harvested and lysed using BugBuster Protein extraction reagent (Novagen, Madison, WI). Activities of glutathione reductase in lysates from 1.86 x 106 cells were measured as described previously (13 ). Concentrations of glutathione in lysates from 3.75 x 106 cells were determined by reduction of 5,5'-dithiobis-2-nitrobenzoic acid as described previously (14 ).
Northern blot analysis.
Expression of two genes that play a role in stress response was investigated. These are UBE1, an enzyme that catalyzes the activation of ubiquitin to ubiquitin adenylate, which is the first step in the ubiquitin-dependent degradative pathway of unfolded proteins (15 ), and X-box binding protein (XBP1), a transcription factor that binds to the unfolded protein response elements in genes that play a role in the cellular response to ER stress (see Discussion). Expression of XBP1 increases in response to ER stress (16 ).
Total RNA was isolated from cells with Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). RNA (25 µg/lane) was electrophoresed using formaldehyde-agarose gels and blotted onto nylon membranes as described previously (17 ). Cellular abundance of 18S RNA remains unchanged over a wide range of conditions (18 ) and was used as a loading control.
Gene-specific probes for mRNA were generated as follows: human cDNA from liver [for cloning of the gene encoding ubiquitin-activating enzyme E1 (UBE1)], lymphocytes (for the gene encoding IL-2), and Jurkat cells (for the gene encoding XBP1) was amplified by PCR using the following primers: 1) 5'-TGC ACT AAG TCT TGC ACT TGT CAC AAA-3' and 5'-CCA TCT GTT CAG AAA TTC TAC AAT GGT-3' for IL-2 (GenBank accession number XM_035511); 2) 5'-TGG TAG ATT TAG AAG AAG AGA ACC AAA-3' and 5'-AAT CAG CTG GGG AAA GAG TTC ATT GGC-3' for XBP1 (GenBank accession number AB_76384); and 3) 5'-CTC GCC GCT GTC CAA GAA ACG TCG CGT-3' and 5'-TCC TCC TCC CGC AGG TAG AAC TGG GAG-3' for UBE1 (GenBank accession number XM_033895). All polymerase chain reactions (PCR) (
35 cycles) were carried out using the following temperatures and times per cycle: 94°C for 1 min (denaturating), 55°C for 1 min (annealing), and 72°C for 2 min (extending); PCR was completed by a final extension step (72°C for 10 min).
PCR products were cloned using the AdvanTAge PCR cloning kit (Clontech, Palo Alto, CA). Nucleotide sequences of the clones were 99–100% identical to the published sequences, as determined by sequence analyses at the DNA core facility at the University of Nebraska-Lincoln. For generation of probes for Northern blots, plasmids were diluted, amplified by PCR as described above and labeled with [32P]dCTP (specific radioactivity = 111 TBq/mmol) using the Megaprime DNA labeling system (Amersham Biosciences, Piscataway, NJ). RNA was probed with [32P]-labeled cDNA as previously described (19 ). Autoradiography film was exposed to the membranes and developed using a Konica SRX101 developer (Wayne, NJ).
Reporter-gene constructs.
The following constructs were used to determine whether riboflavin supply affects transcriptional activity of the gene encoding IL-2: 1) a construct of the regulatory region of the IL-2 gene (321 bases upstream of the transcription start site) linked to the luciferase gene [denoted "p(-321)IL2-Luc"] was provided by L. P. Freedman (Memorial Sloan-Kettering Cancer Center) (20 ); 2) a construct of the Rous sarcoma virus (RSV) promoter linked to the ß-galactosidase reporter gene (denoted "RSV ßgal") was provided by B. R. White (University of Nebraska-Lincoln) and was used as control for transfection efficiency.
After 5 wk in riboflavin-defined media, 15 x 106 cells were cotransfected with p(-321)IL2-Luc and RSVßgal using SuperFect (Qiagen, Palo Alto, CA). Twenty-four hours after transfection, 50 µg/L of phorbol 12-myristate 13-acetate (PMA) and 2 mg/L of phytohemagglutinin (PHA) (final concentrations) were added to culture media to stimulate the 5'-flanking region of the IL-2 gene; cells were harvested 6 h after stimulation. Luciferase activity was quantified using the LucLite Plus assay kit (Packard Instrument, Meriden, CT) in aliquots containing 2.3 x 106 cells; ß-galactosidase activity was quantified using a commercial assay kit (Promega, Madison, WI) and Emax Microwell Plate Reader (Molecular Devices, Sunnyvale, CA) in aliquots containing 1.15 x 106 cells. Luciferase activities were normalized by ß-galactosidase activities.
Proliferation rates.
Proliferation rates of Jurkat cells were quantified by measuring the cellular uptake of [3H]thymidine (ICN, Irvine, CA) as described previously (21 ).
Quantification of interleukin-2.
IL-2 secretion into media and intracellular concentrations of IL-2 were determined using ELISA ("hIL-2 EASIA," Biosource, Camarillo, CA) as described previously (22 ). For determination of extracellular IL-2, 106 cells were stimulated with 50 µg/L of PMA and 2 mg/L of PHA in a final volume of 260 µL for 17 h. For determination of intracellular IL-2, 20 x 106 cells were stimulated with PMA and PHA for 6 h (final volume of 5.2 mL). Cells were harvested by centrifugation (250 x g for 10 min) and were washed twice with PBS to remove secreted IL-2; cells were lysed using 0.5% nonionic detergent (Igepal, Sigma, St. Louis, MO) and suspended in 300 µL of PBS for analysis. IL-2 was quantified in aliquots (100 µL) containing 6.7 x 106 lysed cells.
Cellular uptake of [125I]-labeled IL-2.
Radiolabeled IL-2 was used to determine whether riboflavin supply affects receptor-mediated endocytosis of IL-2. Recombinant human IL-2 (2 µg) (Biosource) was radiolabeled with 125I using a commercially available kit according to the manufacturer’s instructions (Iodo-Beads; Pierce, Rockford, IL). Bovine serum albumin was added after iodination (5 g/L final concentration) to prevent adsorption of IL-2 to plastic surfaces; unbound 125I was removed by gel exclusion chromatography (D-Salt desalting column; Pierce). Specific radioactivity of the final product was 11.5 GBq/mmol. Jurkat cells (107) were stimulated with 50 µg/L of PMA and 2 mg/L of PHA in a volume of 2.6 mL for 17 h to induce expression of IL-2 receptor genes (23
). Endocytosis of [125I]IL-2 was measured as described previously (24
).
Statistics.
Homogeneity of variances among groups was tested using Bartlett’s test (25 ). When variances were heterogeneous, data were log-transformed before further statistical analysis. Significance of differences among groups was tested by one-way ANOVA. Fisher’s Protected Least Significant Difference procedure was used for post-hoc testing (25 ). StatView 5.0.1 (SAS Institute, Cary, NC) was used to perform all calculations. Differences were considered significant if P < 0.05. Data are expressed as means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transport rates of riboflavin correlated negatively with riboflavin supply in media (Fig. 1 ). For example, when cells were cultured in media that were severely and moderately riboflavin deficient, transport rates were 374 ± 92% and 224 ± 30%, respectively, of controls (=100%) cultured in medium containing a physiologic concentration of riboflavin. These findings are consistent with the hypothesis that cells respond to riboflavin deficiency by increasing rates of riboflavin transport.
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Riboflavin deficiency caused increased intracellular accumulation of IL-2 but did not affect secretion of IL-2 into culture media. Concentrations of intracellular IL-2 were quantified in Jurkat cells after 5 wk of culturing in riboflavin-defined media. When cells were cultured in severely riboflavin-deficient medium, concentrations of intracellular IL-2 were 209 ± 20% of control values measured in cells cultured in physiologic medium (Fig. 3B ). In contrast, secretion of IL-2 into the extracellular media was not significantly different among treatment groups (data not shown), suggesting that cells maintain normal secretion of IL-2 despite increased intracellular accumulation.
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Intracellular accumulation of IL-2 in severely riboflavin-deficient cells was not caused by increased receptor-mediated endocytosis of IL-2. Rates of endocytosis of radiolabeled IL-2 were quantified after culturing cells in riboflavin-defined media for 5 wk; internalization of IL-2, as judged by cellular uptake of [125I]IL-2, was not significantly different among treatment groups [units = Bq/(107 cells · h)]: 1.4 ± 0.1 (severely deficient medium); 1.4 ± 0.2 (moderately deficient medium); 1.2 ± 0.3 (physiologic medium); and 1.5 ± 0.5 (pharmacologic medium; P > 0.05 among groups).
Cellular stress response.
Accumulation of IL-2 in riboflavin-deficient Jurkat cells triggered stress response systems. Abundance of mRNA encoding UBE1 was substantially greater in severely riboflavin-deficient cells compared with other treatment groups, and was slightly greater in moderately riboflavin-deficient cells compared with cells cultured in physiologic and pharmacologic medium (Fig. 4 ). Similarly, the abundance of mRNA encoding XBP1 was substantially greater in severely riboflavin-deficient cells compared with other treatment groups. The abundance of 18S rRNA (loading control) was not affected by riboflavin supply (data not shown). These findings are consistent with the hypotheses that Jurkat cells respond to riboflavin deficiency with increased ubiquitin-dependent elimination of unfolded proteins, and that accumulation of unfolded proteins in riboflavin-deficient cells causes increased synthesis of the transcription factor XBP1, leading to increased expression of stress response genes.
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Riboflavin supply correlated with rates of cell proliferation, as judged by cellular uptake of [3H]thymidine [units = fmol of thymidine/(106 cells · h)]: 52 ± 6 (severely deficient medium) vs. 58 ± 1 (moderately deficient medium), vs. 76 ± 2 (physiologic medium), vs. 62 ± 1 (pharmacologic medium). Uptake of thymidine into severely and moderately deficient cells was significantly lower than in other treatment groups (P < 0.03).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mechanism leading to increased expression of the IL-2 gene in riboflavin-deficient cells compared with physiologic controls is uncertain. Previous studies in HeLa cells have suggested that abnormal protein folding triggers dissociation of the transcription factor nuclear factor (NF)-
B from its inhibitory binding partner IB in the cytoplasm, followed by translocation of NF-B to the nucleus (3
,27
). Increased nuclear concentrations of NF-B might cause increased transcription of the IL-2 gene. Consistent with this hypothesis, the 5'-flanking region of the IL-2 gene contains a NF-B binding site (28
).
Increased expression of genes encoding UBE1 and XBP1 suggests that severely riboflavin-deficient cells utilize at least two mechanisms to reduce the abundance of unfolded proteins. First, unfolded proteins are conjugated to ubiquitin and degraded in proteasomes (29 ). Second, synthesis of the transcription factor XBP1 is up-regulated in response to riboflavin deficiency. XBP1 binds to ER stress response elements in 5'-flanking regions of genes that play important roles in the cellular response to accumulation of unfolded proteins (16 ), leading to increased transcriptional activity of these genes. For example, expression of the gene encoding glucose-regulated protein 78 (HSPA5, BiP) increases in response to elevated concentrations of XBP1 (16 ); glucose-regulated protein 78 binds misfolded proteins in the ER, mediating refolding or degradation of the misfolded proteins (30 ).
In the present study, folding of IL-2 and cellular stress response were affected only if Jurkat cells were cultured in severely riboflavin-deficient medium; effects of moderate riboflavin deficiency were quantitatively minor. We hypothesize that Jurkat cells are relatively resistant to developing riboflavin deficiency based on the following lines of reasoning. Jurkat cells secrete only two proteins in response to stimulation with PMA and PHA: interferon-
(which does not contain disulfide bonds) and IL-2 (which contains only one disulfide bond). Thus, the flavin requirement for oxidative folding might be small in Jurkat cells. In contrast, cells other than Jurkat cells may secrete substantial amounts of proteins that contain more than one disulfide bond, e.g., secretion of apolipoprotein B by liver cells (31
) and secretion of immunoglobulins by B cells (6
). Theoretically, moderate riboflavin deficiency might impair folding of secretory proteins in these cells, leading to accumulation of unfolded proteins and cellular stress.
Do the findings presented here have any physiologic significance? Previous studies have provided evidence that severe riboflavin deficiency may occur in preterm newborns treated with phototherapy (10 ) and in patients with cystic fibrosis (9 ). Cells from these individuals are likely to be exposed to increased levels of stress triggered by unfolded proteins, based on the data presented here. It is uncertain whether riboflavin supplementation beyond physiologic levels has beneficial effects on folding and secretion of proteins. In the present study, supplementation of cells with pharmacologic concentrations of riboflavin did not produce evidence for increased secretion of proteins or decreased cellular stress compared with physiologic controls.
The present study suggests a new role for riboflavin in human intermediary metabolism that goes beyond the classical roles for FMN and FAD in the respiratory chain, glutathione metabolism and the metabolism of nutrients such as lipids and some vitamins (26 ). Future studies will 1) identify the mechanism leading to increased IL-2 transcription in riboflavin-deficient cells; 2) determine whether the human homolog (Ero1-L) of Ero1p is a flavin-dependent protein; and 3) determine whether riboflavin supply affects folding of proteins and cellular stress in vivo.
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FOOTNOTES |
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3 Abbreviations used: ER, endoplasmic reticulum; FBS, fetal bovine serum; FMN, flavin mononucleotide; IB, inhibitory B; IL-2, interleukin-2; NF-B, nuclear factor-B; PCR, polymerase chain reaction; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; UBE1, ubiquitin-activating enzyme E1; XBP1, X box-binding protein 1.
Manuscript received 31 October 2002. Initial review completed 20 November 2002. Revision accepted 26 November 2002.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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