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, Interleukin-1ß, and 3-Methylcrotonyl-CoA Carboxylase, and Decreases Expression of the Gene Encoding Interleukin-4 in Human Peripheral Blood Mononuclear Cells1
,**
,2
Departments of
*
Nutritional Science and Dietetics, and
Biochemistry, University of Nebraska at Lincoln, Lincoln, NE, and
Department of Pediatrics, and
**
Genetic Microarray Core Facility, University of Nebraska Medical Center, Omaha, NE
2To whom correspondence should be addressed. E-mail: jzempleni2{at}unl.edu.
 |
ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, interleukin-1ß, and 3-methylcrotonyl-CoA carboxylase was 4.3, 5.6 and 8.9 times greater, respectively, after supplementation with biotin compared with before supplementation. In contrast, the abundance of mRNA encoding interleukin-4 was 6.8 times greater before supplementation than after supplementation. These data suggest that biotin supplementation affects gene expression in human immune cells. Effects of biotin on gene expression are likely to modulate the response of immune cells to antigens.
KEY WORDS: • biotin • cytokines • humans • 3-methylcrotonyl-CoA carboxylase • peripheral blood mononuclear cells
In mammals, biotin serves as a covalently bound coenzyme for four carboxylases: acetyl-CoA carboxylase (isoforms 
and ß; EC 6.4.1.2); pyruvate carboxylase (EC 6.4.1.1); propionyl-CoA carboxylase (EC 6.4.1.3); and 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) (1
). These carboxylases catalyze essential steps in the metabolism of glucose, amino acids and fatty acids. Furthermore, eukaryotic cells can covalently attach biotin to histones (DNA-binding proteins) in an enzyme-mediated reaction (2
,3
). Biotinylation of histones might play a role in processes such as cell proliferation and DNA repair (3
,4
).
Evidence is accumulating that biotin plays a role in the regulation of gene expression in addition to its role as a covalently bound group in carboxylases and histones. For example, the expression of genes encoding glucokinase, phosphoenolpyruvate carboxykinase and ornithine transcarbamylase decreases in response to biotin deficiency (5
). Recently, evidence has been provided that the expression of genes encoding the cytokine interleukin-2 (IL-2)3 and IL-2 receptor correlate with biotin status in human lymphoid cells (6
).
Cytokines are messenger proteins of the immune system that are secreted by immune cells in response to stimulation by antigens (7 ). After secretion, cytokines bind to receptors on the surface of target cells such as T cells and natural killer cells. Some cells that are not immune cells may also secrete or bind cytokines (7 ). Binding of cytokines to receptors triggers intracellular signaling cascades (7 ,8 ), which play important roles in processes such as cellular growth, proliferation, differentiation and apoptosis (7 –9 ). Effects of biotin on cytokine metabolism may account for the essential role of biotin in immune function that has been observed in previous studies (10 –12 ).
Regulation of the immune response by cytokines is a complex network, involving >60 cytokines and cytokine receptors (7
). Cytokines exhibit a substantial amount of cross-talk, e.g., IL-2 receptor serves as receptor for IL-2, IL-4, IL-7, IL-9, and IL-15 (7
,8
). DNA microarrays permit simultaneous screening for the expression of a large number of genes; thus, microarrays are a powerful tool with which to investigate the effects of nutrients on complex networks of gene expression such as in cytokine metabolism. In the present study, DNA microarrays were used to determine whether supplementation of healthy adults with biotin affects 1) the expression of genes that play a role in immune function (cytokine metabolism, signal transduction, cell proliferation, stress response and apoptosis), and 2) the expression of genes that play a role in biotin utilization.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Healthy Caucasian adults (1 man, 5 women) aged 25–55 y participated in this study. All subjects were nonsmokers; none had knowingly consumed any vitamin supplements for at least 3 wk before initiation of the study. Pregnant women and individuals treated with anticonvulsants were not eligible for study participation (13 ). This study was approved by the Institutional Review Board at the University of Nebraska-Lincoln.
Study design.
A heparinized blood sample (
150 mL) was collected from each subject before biotin supplementation (denoted "presupplementation"). A second blood sample (denoted "postsupplementation") was collected 4 h after subjects had completed a 21-d supplementation with biotin; one pill of "Solaray" (Park City, UT) was taken per day. Biotin content of the supplement was determined by avidin-binding assay as described previously (14
) with modifications (15
); the biotin content was 8.8 µmol/pill (2150 µg). The normal dietary intake of biotin is 0.4 µmol/d (16
). Previous studies have suggested that 3 wk of biotin supplementation (3.1 µmol/d) are sufficient to achieve new steady-state levels of biotin in immune cells (17
) and to affect secretion of cytokines by immune cells (15
,18
).
Cell culture.
Peripheral blood mononuclear cells (PBMC) were isolated aseptically from blood as previously described (19
); plasma was saved for determination of biotin concentration and for use as a culture supplement as described below. PBMC (3.5 x 109 cells/L) were suspended in custom-manufactured RPMI-1640 (Atlanta Biologicals, Norcross, GA) that was compounded from pure ingredients without biotin; the culture medium was supplemented with 0.1 L of autologous plasma/L of final medium, 1 x 105 IU/L penicillin and 100 mg/L streptomycin (final concentrations). Antibiotics and culture medium from the same stock solutions and powder, respectively, were used in all experiments.
For cell culturing, we attempted to simulate the likely plasma levels of biotin before and during supplementation of subjects. The biotin concentration in culture media was adjusted to 0.25 nmol/L for presupplementation PBMC and to 10 nmol/L for postsupplementation PBMC, on the basis of plasma concentrations of biotin plus biotin metabolites (specifically catabolites) observed in our previous studies (18 ). Concanavalin A at a final concentration of 20 mg/L was added to culture media immediately after suspending the cells to stimulate expression of genes encoding cytokines; concanavalin A from the same stock solution was used in all experiments. Twenty-one hours after stimulation, cells were collected by centrifugation (250 x g for 10 min) for isolation of RNA.
Plasma biotin.
Biotin and biotin metabolites in plasma were quantified by avidin-binding assay as described. The concentration of biotin plus biotin metabolites was 25 ± 6.9 times greater (range 19–37) in postsupplementation plasma (54.9 ± 33.8 nmol/L) than in presupplementation plasma (2.0 ± 0.8 nmol/L; P < 0.05), suggesting that subjects complied with the supplementation protocol. Note that the avidin-binding assay used here is not absolutely specific for biotin but may also bind biotin metabolites and other compounds (20 ). Thus, the avidin-binding assay is likely to overestimate the true concentration of biotin in plasma.
Isolation of RNA.
Total RNA was extracted from PBMC by using the RNeasy mini kit and the RNase-free DNase set (Qiagen, Valencia, CA). RNA samples were analyzed by polymerase chain reaction (PCR) to detect potential contaminations with genomic DNA; primers specific for intronic sequences of the IL-2 gene were used for PCR: 5'-GCA TTG CAC TAA GTC TTG CAC TTG TCA-3' and 5'-AGT AGA CAT GAA ACT GAA TCA CAA ACA-3' (GenBank accession number X00695). PCR was conducted using the following temperatures and times per cycle: 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 30 cycles plus one final extension step (72°C for 10 min) (21 ). Samples were electrophoresed using agarose gels and stained with ethidium bromide as described previously (21 ); no bands were visible under UV light, consistent with the absence of genomic DNA.
Absorbances at 260 and 280 nm were measured for each RNA sample; the absorbance ratio (260-to-280 nm) was 1.9 ± 0.2, consistent with the absence of significant contamination with protein. To confirm integrity of RNA, 22 µg of RNA were electrophoresed using a formaldehyde-agarose gel (22 ); RNA was stained with ethidium bromide and visualized using a Kodak EDAS 290 Documentation and Analysis System (Rochester, NY). Two major ribosomal bands (28S and 18S rRNA) but no degraded RNA were detected (data not shown).
DNA microarrays.
Equal amounts of RNA from all six subjects were pooled to produce two samples: presupplementation and postsupplementation RNA. RNA (30 µg) was reverse transcribed into cDNA in the presence of amino allyl dUTP to allow for indirect labeling of the samples with fluorophores Cy3 (presupplementation cDNA) and Cy5 (postsupplementation cDNA) using the Fairplay labeling system (Stratagene, LaJolla, CA). Equal amounts of presupplementation and postsupplementation cDNA were pooled, dried under vacuum and resuspended in 18 µL of a hybridization solution containing 0.5 L formamide, 0.5 L 5X SSC buffer (0.75 mol/L sodium chloride, 0.075 mol/L sodium citrate, pH 7) and 3.5 mmol/L lauryl sulfate; 20 µg of human Cot1 DNA and 20 µg of polyA DNA were added to the hybridization solution to prevent nonspecific binding of labeled cDNA to microarrays. The cDNA was denatured at 95°C for 3 min, cooled on ice and hybridized to DNA microarrays (see below) at 42°C for 16 h. Subsequently, microarrays were washed in the following buffers in this sequential order: 1) 1X SSC buffer (0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate, pH 7), containing 6.9 mmol/L lauryl sulfate at 42°C for 5 min; 2) 0.1X SSC (15 mmol/L sodium chloride, 1.5 mmol/L sodium citrate, pH 7), containing 6.9 mmol/L lauryl sulfate at room temperature for 5 min; and 3) 0.1X SSC at room temperature for 5 min. Slides were scanned using an Axon GenePix 4000A scanner (Axon Instruments, Union City, CA). Images were analyzed using GenPix 4.0 software (Axon). Microarray analysis was carried out twice, beginning with reverse transcription of RNA; both arrays produced essentially identical results.
The DNA microarrays used in these studies was printed in the Genetic Microarray Core Facility (University of Nebraska Medical Center, Omaha, NE). The printer used was an Affymetrix (GMS) 417 printer (Affymetrix, Santa Clara, CA); the spots correspond to PCR products amplified from the Resgen Sequence Validated Human cDNA Library (Research Genetics, Huntsville, AL). This chip contains 1896 spots in all and is enriched with PCR products corresponding to 475 genes encoding proteins that play roles in processes such as cytokine metabolism, signal transduction, cell proliferation, cellular stress response and apoptosis.
Reverse transcriptase PCR.
Reverse transcriptase PCR was used to ultimately determine whether the expression of a given gene was affected by biotin supplementation. Genes were analyzed by reverse transcriptase PCR if at least one of the following selection criteria applied: 1) signal intensity in postsupplementation samples was at least two times the intensity in presupplementation samples in DNA microarrays; 2) signal intensity in postsupplementation samples was <0.5 times the intensity in presupplementation samples in DNA microrarrays; 3) the gene encodes for IL-4, whose cellular secretion is affected by biotin (6
,15
,18
); and 4) the gene encodes for propionyl-CoA carboxylase ( chain) or 3-methylcrotonyl-CoA carboxylase ( chain), whose expression might be affected by biotin status (23
).
Equal amounts of RNA from six subjects were pooled to produce presupplementation and postsupplementation samples; PCR analyses were conducted at least twice for each gene. RNA was reverse transcribed into cDNA as described previously (21 ). Gene expression was quantified by quantitative PCR as described previously (21 ), using the customized primers (Integrated DNA Technologies, Coralville, IA) listed in Table 1 . Commercial primers were used for the control gene ß-actin, whose expression was not affected by biotin (Clontech, Palo Alto, CA). PCR-amplified samples were collected at timed intervals for up to 60 PCR cycles. Equal volumes (10 µL) of each sample were chromatographed using agarose gels (15 g agarose/L). DNA was stained with ethidium bromide and analyzed using the Kodak EDAS 290 Documentation and Analysis System; only values from within the exponential phase of PCR amplification (typically <42 PCR cycles) were considered for data analysis. The abundances of mRNA were normalized by the abundance of mRNA encoding ß-actin. Genes were considered affected by biotin supplementation if the abundance of mRNA increased by at least 100% in postsupplementation samples compared with presupplementation samples, or if the abundance of RNA decreased by at least 50% in postsupplementation samples compared with presupplementation samples.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DNA microarray data were consistent with the hypothesis that biotin supplementation affects gene expression in human immune cells. Hybridization signals of genes encoding for IL-1ß, endoglin and interferon- were 2.0, 3.6 and 5.7 times greater, respectively, in postsupplementation samples than in presupplementation samples. Expression profiles of these three genes were further analyzed by reverse transcriptase PCR as described below. Also, the following genes were included in analysis by reverse transcriptase PCR, based on the reasoning provided in Subjects and Methods: IL-4, propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase.
Reverse transcriptase PCR.
Analysis by PCR confirmed the microarray findings that expression of genes encoding IL-1ß and interferon- increased in response to biotin supplementation: the abundances of mRNA encoding IL-1ß and interferon- were 5.6 and 4.3 times greater, respectively, in postsupplementation samples than in presupplementation samples (Fig. 1
). In contrast, the abundance of mRNA encoding endoglin was not affected by biotin supplementation, as judged by reverse transcriptase PCR; this observation suggests that analysis by DNA microarrays might have produced a false positive result for endoglin.
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chain) increased in response to biotin supplementation: the abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase was 8.9 times greater in postsupplementation samples than in presupplementation samples. Expression of propionyl-CoA carboxylase was not affected by biotin supplementation. Taken together, biotin supplementation affected the expression of 0.8% of the genes (4 of 478) that were investigated in human PBMC. |
DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and IL-4 in human PBMC. These cytokines play the following important roles in cells of both the immune system and other tissues. 1) IL-1ß is secreted by macrophages and various epithelial cells; after secretion, IL-1ß binds to receptors located on the surface of target cells (7
). Binding of IL-1ß to receptors causes activation of TH lymphocytes, maturation and clonal expansion of B lymphocytes, activation of natural killer cells and attraction of macrophages and neutrophils to sites of inflammatory response. In addition IL-1ß induces synthesis of acute-phase proteins by hepatocytes, osteoblast proliferation and prostaglandin production in osteoblasts (24
). 2) Interferon- is secreted by TH1 cells and natural killer cells. Binding of interferon- to receptors induces expression of genes that play important roles in antiviral defense, e.g., 2',5'-oligoadenylate synthase (7
). 3) IL-4 is secreted by TH2 and TH0 lymphocytes, mast cells and bone marrow stromal cells; target cells include antigen-primed B cells and activated B cells (7
). Binding of IL-4 to its receptors mediates B-cell activation and class switch in B lymphocytes, i.e., the change from the expression of one immunoglobulin class to another.
Biotin supplementation also caused increased expression of the gene encoding 3-methylcrotonyl-CoA carboxylase ( chain). This is consistent with a previous study that provided evidence that expression of genes encoding biotin-dependent carboxylases correlates with biotin supply in HepG2 cells (23
). In that study, the following mechanism was proposed to explain effects of biotin on gene expression. An intermediate of biotin metabolism, biotinyl-AMP, activates soluble guanylate cyclase, leading to increased production of cGMP. cGMP stimulates protein kinase G, leading to phosphorylation of proteins that stimulate transcription of carboxylase genes.
Effects of biotin on the expression of the gene encoding 3-methylcrotonyl-CoA carboxylase may enhance function of the immune system. Mitogen-induced proliferation of PBMC is paralleled by an up to 180% increase in 3-methylcrotonyl-CoA carboxylase activity (25 ) and an up to 620% increase in cellular uptake of the coenzyme biotin (26 ). Proliferation of human lymphoid cells correlates with biotin supply (15 ).
Increased expression of genes encoding cytokines such as IL-1ß and interferon- is not necessarily paralleled by increased levels of these cytokines in extracellular fluids. Previous studies have shown that biotin supplementation of healthy adults (18
) and lymphoid cells (Jurkat cells) in culture (15
) may cause decreased net secretion of cytokines by PBMC and Jurkat cells. The following model has been proposed to explain decreased net secretion of cytokines despite increased abundance of mRNA encoding these cytokines (6
). Binding of cytokines to receptors triggers both intracellular signaling cascades, and internalization and degradation of cytokines. Biotin supplementation causes increased synthesis of cytokine receptors, leading to increased endocytosis and, thus, decreased extracellular accumulation of cytokines.
Expression of cytokine genes in immune cells is regulated in a timed fashion. For example, peak levels of mRNA encoding IL-2 are achieved within 6 h of stimulation with antigen or mitogen, whereas peak levels of mRNA encoding various adhesion molecules are achieved >48 h after stimulation (27
). Thus, the data reported here must be interpreted within the context of the period of time (21 h) that was chosen for stimulation with concanavalin A. The timing of sample collection can help to explain the following observations. 1) Previous studies have provided evidence that expression of the gene encoding IL-2 increases in response to biotin supplementation (6
). In contrast, the abundance of mRNA encoding IL-2 was low in the present study, and was not affected by biotin supplementation. Likely, samples were collected after an abundance of IL-2 mRNA had reached peak levels, preventing detection of the effects of biotin. 2) Theoretically, some of the effects of biotin on gene expression might be caused by a shift in the time courses of mRNA abundance. For example, biotin supplementation might have shifted peak IL-4 expression to an earlier time, rather than actually decreasing expression of the IL-4 gene. 3) Biotin may not directly affect expression of genes encoding IL-1ß, interferon-, IL-4 and 3-methylcrotonyl-CoA carboxylase; effects of biotin might be mediated by upstream events such as changes in the expression of early-response cytokines, which may affect late-response cytokines.
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FOOTNOTES |
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3 Abbreviations used: IL, interleukin; PCR, polymerase chain reaction; PBMC, peripheral blood mononuclear cells.
Manuscript received 14 October 2002. Initial review completed 26 November 2002. Revision accepted 4 December 2002.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Zempleni, J. & Mock, D. M. (1999) Biotin biochemistry and human requirements. J. Nutr. Biochem. 10:128-138.
2. Hymes, J., Fleischhauer, K. & Wolf, B. (1995) Biotinylation of histones by human serum biotinidase: assessment of biotinyl-transferase activity in sera from normal individuals and children with biotinidase deficiency. Biochem. Mol. Med. 56:76-83.[Medline]
3. Stanley, J. S., Griffin, J. B. & Zempleni, J. (2001) Biotinylation of histones in human cells: effects of cell proliferation. Eur. J. Biochem. 268:5424-5429.[Medline]
4. Peters, D. M., Griffin, J. B., Stanley, J. S., Beck, M. M. & Zempleni, J. (2002) Exposure to UV light causes increased biotinylation of histones in Jurkat cells. Am. J. Physiol. 283:C878-C884.
5. Dakshinamurti, K. (2003) Regulation of gene expression by biotin, vitamin B6 and vitamin C. Daniel, H. Zempleni, J. eds. Molecular Nutrition 2003 CABI Publishing Oxfordshire, UK. .
6. Rodriguez-Melendez, R., Camporeale, G., Griffin, J. B. & Zempleni, J. (2003) Interleukin-2 receptor -dependent endocytosis depends on biotin in Jurkat cells. Am. J. Physiol. (in press).
7. Klein, J. & Horejsi, V. (1997) Immunology 1997 Blackwell Science Oxford, UK.
8. Sugamura, K., Asao, H., Kondo, M., Tanaka, N., Ishii, N., Ohbo, K., Nakamura, M. & Takeshita, T. (1996) The interleukin-2 receptor chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179-205.[Medline]
9. Russell, S. M., Johnston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedman, M., Berg, M., McVicar, D. W., Witthuhn, B. A., Silvennoinen, O., Goldman, A. S., Schmalstieg, F. C., Ihle, J. N., O’Shea, J. J. & Leonard, W. J. (1994) Interactions of IL-2Rß and c chains with Jak1 and Jak3: implications for XSCID and XCID. Science (Washington, DC) 266:1042-1045.
10. Rabin, B. S. (1983) Inhibition of experimentally induced autoimmunity in rats by biotin deficiency. J. Nutr. 113:2316-2322.
11. Báez-Saldaña, A., Díaz, G., Espinoza, B. & Ortega, E. (1998) Biotin deficiency induces changes in subpopulations of spleen lymphocytes in mice. Am. J. Clin. Nutr. 67:431-437.[Abstract]
12. Petrelli, F., Moretti, P. & Campanati, G. (1981) Studies on the relationships between biotin and the behaviour of B and T lymphocytes in the guinea pig. Experientia 37:1204-1206.[Medline]
13. Zempleni, J. (2001) Biotin. Bowman, B. A. Russell, R. M. eds. Present Knowledge in Nutrition 2001 ILSI Press Washington, DC. .
14. Mock, D. M. (1997) Determinations of biotin in biological fluids. McCormick, D. B. Suttie, J. W. Wagner, C eds. Vitamins and Coenzymes, Part I, Methods in Enzymology 1997:265-275 Academic Press New York, NY. .
15. Manthey, K. C., Griffin, J. B. & Zempleni, J. (2002) Biotin supply affects expression of biotin transporters, biotinylation of carboxylases, and metabolism of interleukin-2 in Jurkat cells. J. Nutr. 132:887-892.
16. National Research Council (1998) Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic acid, biotin, and choline (Institute of Medicine, Food and Nutrition Board, ed.) 1998 National Academy Press Washington, DC .
17. Zempleni, J. & Mock, D. M. (1999) The efflux of biotin from human peripheral blood mononuclear cells. J. Nutr. Biochem. 10:105-109.
18. Zempleni, J., Helm, R. M. & Mock, D. M. (2001) In vivo biotin supplementation at a pharmacologic dose decreases proliferation rates of human peripheral blood mononuclear cells and cytokine release. J. Nutr. 131:1479-1484.
19. Zempleni, J. & Mock, D. M. (1998) Uptake and metabolism of biotin by human peripheral blood mononuclear cells. Am. J. Physiol. 275:C382-C388.
20. Green, N. M. (1975) Avidin. Adv. Protein Chem. 29:85-133.[Medline]
21. Zempleni, J., Stanley, J. S. & Mock, D. M. (2001) Proliferation of peripheral blood mononuclear cells causes increased expression of the sodium-dependent multivitamin transporter gene and increased uptake of pantothenic acid. J. Nutr. Biochem. 12:465-473.[Medline]
22. Rodriguez-Melendez, R., Cano, S., Mendez, S. T. & Velazquez, A. (2001) Biotin regulates the genetic expression of holocarboxylase synthetase and mitochondrial carboxylases in rats. J. Nutr. 131:1909-1913.
23. Solorzano-Vargas, R. S., Pacheco-Alvarez, D. & Leon-Del-Rio, A. (2002) Holocarboxylase synthetase is an obligate participant in biotin-mediated regulation of its own expression and of biotin-dependent carboxylases mRNA levels in human cells. Proc. Natl. Acad. Sci. U.S.A. 99:5325-5330.
24. Kim, C. H., Kang, B. S., Lee, T. K., Park, W. H., Kim, J. K., Park, Y. G., Kim, H. M. & Lee, Y. C. (2002) IL-1ß regulates cellular proliferation, prostaglandin E2 synthesis, plasminogen activator activity, osteocalcin production, and bone resorptive activity of the mouse calvarial bone cells. Immunopharmacol. Immunotoxicol. 24:395-407.[Medline]
25. Stanley, J. S., Griffin, J. B., Mock, D. M. & Zempleni, J. (2002) Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities. J. Nutr. 132:1854-1859.
26. Zempleni, J. & Mock, D. M. (1999) Mitogen-induced proliferation increases biotin uptake into human peripheral blood mononuclear cells. Am. J. Physiol. 276:C1079-C1084.
27. Goldsby, R. A., Kindt, T. J. & Osborne, B. A. (2000) Immunology 2000 W. H. Freeman and Company New York, NY.
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