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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA).

Methods

Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques.

Results

The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells.

Conclusion

The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown cause (1, 2). Studies have shown that the apoptosis-inducing death receptor Fas plays an important role in peripheral deletion of potentially autoreactive T cells and B cells (3–5) and that mice harboring mutations in Fas or Fas ligand develop characteristic systemic autoimmune diseases, including polyarthritis (6–8). However, with the exception of human autoimmune lymphoproliferative syndrome (9), a disease characterized by massive lymphadenopathy and autoimmune phenomena, mutations in Fas or Fas ligand (10) are not commonly recognized as a cause of human systemic autoimmunity. Nonetheless, defects in apoptosis may be critical to the pathogenesis of systemic autoimmunity, as exemplified by a recent finding that anti–death receptor 5 (anti–DR-5), but not Fas or anti–DR-4 antibody, significantly ameliorated synovial overgrowth and experimental arthritis in mice (11).

We have studied the contribution of DR-3 to the pathogenesis of RA, with reference to transactivation of the DR3 gene. DR-3 is a member of the apoptosis-inducing tumor necrosis factor (TNF) receptor superfamily that includes Fas (12–16), and its promoter region near the translation start site contains GATA, Oct-1, and two Sp1 binding sites, but without containing either the TATA or the CAAT box. Sp1 is a ubiquitously expressed zinc-finger transcription factor that supports messenger RNA (mRNA) expression in a variety of eukaryotic genes that lack a functional TATA box (17, 18), and this may be important for synovial cell activation.

Since plenty of CpG motifs exist in the promoter region of DR3 and since apoptotic signaling pathways are often the targets for epigenetic gene silencing (19), the present study was designed to clarify the contribution of the methylation status of the DR3 promoter to the transcriptional regulation of gene expression in patients with RA as compared with healthy individuals and patients with osteoarthritis (OA). We studied the methylation status of the CpG island in relation to promoter activity and DR-3 protein expression. Our findings indicate that DR3 promoter CpG motifs are hypermethylated in rheumatoid synovial cells; this is discussed in relation to transactivation of the DR3 gene and the pathogenesis of RA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients and controls.

Synovial tissue from 7 RA patients and 7 OA patients who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria (20, 21) were studied. Synovial tissue was obtained during joint replacement surgery in accordance with the guidelines of the Kobe University Hospital Institutional Review Board. Of the RA patients, 1 was a man, and 6 were women. Their mean ± SD age was 63.3 ± 13.7 years (range 34–77 years), and their mean ± SD disease duration was 14.1 ± 8.3 years (range 6–30 years). Of the OA patients, 3 were men, and 4 were women. Their mean ± SD age was 77.4 ± 6.0 years (range 71–87 years), and their mean ± SD disease duration was 2.5 ± 1.3 years (range 1–5 years). Disease-modifying antirheumatic drugs prescribed for the 7 RA patients were gold sodium thiomalate (25 mg/month; n = 1), auranofin (6 mg/day; n = 1), methotrexate (16 mg/week; n = 5), and prednisolone (5 mg/day; n = 4).

Blood samples were obtained from 9 RA patients and 9 healthy control subjects. Four of the control subjects were men, and 5 were women. Their mean ± SD age was 25.8 ± 6.4 years (range 22–37 years).

Preparation of synovial cells and peripheral blood mononuclear cells (PBMCs) and extraction of DNA.

Synovial tissues were minced and stirred with 1 mg/ml of collagenase (Nissui, Tokyo, Japan) in serum-free Dulbecco's modified Eagle's medium (DMEM; Nissui) for 1 hour, filtered through nylon mesh, and washed extensively. Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), as described previously (22). First- or second-passage RA synovial cells and first-passage OA synovial cells were used in the studies.

Peripheral blood mononuclear cells (PBMCs) were derived from blood samples obtained from RA patients and healthy control subjects. PBMCs were isolated by Ficoll-Hypaque centrifugation.

DNA was extracted from the PBMCs and the synovial cells using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) according to the manufacturer's instructions.

Methylation-specific polymerase chain reaction (PCR).

After denaturation in 0.3N NaOH at 37°C for 30 minutes, DNA (1 μg) was treated with 1.6N sodium bisulfite and 0.5 mM hydroquinone, pH 5, at 55°C for 18 hours (23, 24). For desulfonation, purified DNA was treated with 0.2N NaOH at 37°C for 10 minutes, followed by a conventional ethanol-precipitation procedure.

The DR3 promoter region was amplified by PCR from sodium bisulfite–treated DNA with the use of methylation-specific primer sets (23). Amplified products were subjected to electrophoresis in nondenaturing 8% polyacrylamide gels, stained with ethidium bromide, and visualized under ultraviolet illumination. Methylated or unmethylated DNA showed 1 positive band. Partially methylated DNA showed 2 positive bands (1 for the methylation-specific primer set and 1 for the unmethylation-specific primer set).

The primer pairs for amplifying methylated DNA for the region −139 to +53 were 5′-GTTTTATTTGGTTTGTTCGTTGTC-3′ (forward) and 5′-CGTACTCTCTACCCGTCGTAA-3′ (reverse). The primer pairs for amplifying unmethylated DNA for the region −137 to +53 were 5′-TTTATTTGGTTTGTTTGTTGTTGTT-3′ (forward) and 5′-ACTCCATACTCTCTACCCATCATAA-3′ (reverse). These primers, which encompass the CpG island of the DR3 promoter, were searched with MethPrimer software (available at http://www.urogene.org/methprimer/index1.html) (25).

Bisulfite genomic sequencing.

Sodium bisulfite–treated DNA was extracted as described above and used as a template for amplifying the DR3 gene promoter. PCR amplification was conducted in a 20-μl reaction volume containing 0.2 μg of DNA, 1× PCR buffer, 200 μM of each dNTP, 200 nM of each primer, 1.5 mM MgCl2, and 1 unit of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) (24). The mixture was reacted at 95°C for 10 minutes and then amplified for 40 cycles of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. This procedure results in the conversion of unmethylated cytosine to thymine, whereas methylated cytosine is unaffected. Accordingly, the ratios of methylated CpG and unmethylated CpG was defined as the ratio of the count of C to C and the ratio of the count of C to T, respectively, at each CpG site within region −196 to +88 (26).

The primer pairs for the region −467 to −72 were 5′-TGGAATTGTAGGTGTGGTATTATTA-3′ (forward; CL1) and 5′-ACCAAATACCCCCTCTACTC-3′ (reverse; CR1). The primer pairs for the region −191 to +178 were 5′-ACAACTCTATCCTATACCCCTAA-3′ (forward; CL2) and 5′-TTTTTGGGATAGGGTTTAAAGT-3′ (reverse; CR2).

PCR products were cloned into pT7Blue vector (Novagen, Madison, WI). Products were sequenced using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI 377 sequencer (Applied Biosystems).

Luciferase reporter gene construct.

Genomic DNA obtained from PBMCs derived from healthy subjects was amplified by PCR to generate a series of luciferase reporters. The primer pairs used for amplifying the 5′-flanking region of the DR3 promoter were as follows: for −441/+139pDR3 (−441 to +139), 5′-CCGGGTACCAAGTAGAGACGGCATTTCACC-3′ (forward; pDR3-F1) and 5′-CCCAAGCTTAGCCTTTAACGAGATCGGAAAGG-3′ (reverse; pDR3-R1); for −239/+139pDR3 (−239 to +139), 5′-CCGGGTACCTTGAAGTGGTTCTCAGGGTT-3′ (forward; pDR3-F2) and 5′-CCCAAGCTTCTCTTGGGACAGGGCTCAAAGCT-3′ (reverse; pDR3-R2); for −112/+139pDR3 (−112 to +139), 5′-CCGGGTACCTAATGAGCTCAGGTCTAGGCCG-3′ (forward; pDR3-F3) and 5′-CCCAAGCTTCTCTTGGGACAGGGCTCAAAGCT-3′ (reverse; pDR3-R2); for +1/+139pDR3 (+1 to +139), 5′-CCGGGTACCCGGGCCCTGCGGGCGCGGGGCTGAA-3′ (forward; pDR3-F4) and 5′-CCCAAGCTTCTCTTGGGACAGGGCTCAAAGCT-3′ (reverse; pDR3-R2); and for −441/−294pDR3 (−441 to −294), 5′-CCGGGTACCAAGTAGAGACGGCATTTCACC-3′ (forward; pDR3-F1) and 5′-CCCAAGCTTGAGGTTGGGCCAAAAGTACC-3′ (reverse; pDR3-R3).

The Kpn I and Hind III recognition sequences were added to the 5′ ends of the forward and reverse primers, respectively. The PCR products were cloned into the Kpn I–Hind III site of the pGL3-Enhancer vector (Promega). We used TFSearch software (available at http://mbs.cbrc.jp/research/db/TFSEARCH.html) to search for transcription factor binding sites.

In vitro DNA methylation.

The DR3 reporter gene constructs (−441/+139pDR3, −239/+139pDR3, and −112/+139pDR3) were methylated in vitro using bacterial methylase Sss I (New England Biolabs, Beverly, MA). Briefly, the DR3 reporter gene construct (40 μg) was incubated at 37°C for 2 hours with 40 units of Sss I in 50 mM NaCl, 10 mM Tris HCl, 10 mM MgCl2, and 1 mM dithiothreitol (pH 7.9) supplemented with 160 μM S-adenosylmethionine. Methylated plasmids were purified with the use of the Wizard DNA Clean-Up system (Promega).

Luciferase reporter assay.

COS-7 and 293T cells (1 × 105) were transfected with 200 ng of pGL3-Enhancer vector containing the DR3 reporter gene constructs (−441/+139pDR3, −239/+139pDR3, −112/+139pDR3, +1/+139pDR3, or −441/−294pDR3) by using Lipofectamine Plus reagent (Invitrogen, San Diego, CA) (27). As an internal control for transfection efficiency, 5 ng of pRL-TK (Promega) containing the herpes simplex virus thymidine kinase promoter in the upstream of Renilla luciferase was cotransfected. Cell lysate (20 μl) was analyzed for luciferase activity using the Dual-Luciferase Reporter Assay (Promega) in a Luminoskan (Labsystems, Tokyo, Japan). Activities of both firefly and Renilla luciferases were measured, and the activity of firefly luciferase was normalized to that of Renilla luciferase. Data were expressed as the mean ± SD of 2 independent experiments.

Western blotting.

Cells (1 × 106) were suspended in 100 μl of hypotonic lysis buffer (25 mM Tris, pH 8.0, 1% Nonidet P40, 150 mM NaCl, 1.5 mM EGTA, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium orthovanadate, and 3 mg/ml of aprotinin), maintained on ice for 10 minutes, and the protein fraction was obtained by centrifugation at 15,000 revolutions per minute for 10 minutes (28). Protein (20 μg) was subjected to electrophoresis in 10–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels (Biocraft, Tokyo, Japan) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was then incubated with mouse IgG anti-human DR-3 antibody (eBioscience, San Diego, CA) and then reacted with horseradish peroxidase–conjugated anti-mouse IgG antibody and ECL enhanced chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ). The density of the blots was scanned and quantified using NIH Image/Image J software (National Institutes of Health, Bethesda, MD; available at http://rsbweb.nih.gov/ij/).

Statistical analysis.

The chi-square test and Student's 2-tailed independent t-test were used to determine the significance of differences between groups, as appropriate.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Methylation status of the DR3 promoter.

PCR amplification of the CpG island encompassing the region −196 to +139 by use of methylation-specific and unmethylation-specific primers revealed that this region was consistently unmethylated in PBMCs from healthy controls and patients with RA (Figure 1). In contrast, synovial cells from patients with OA were either unmethylated or partially methylated. Furthermore, synovial cells from patients with RA were mostly methylated. Table 1 summarizes the individual methylation status of PBMCs from RA patients and healthy controls and of synovial cells from RA and OA patients, as examined by methylation-specific and unmethylation-specific PCR.

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Figure 1. Methylation status of the DR3 gene promoter, as determined by methylation-specific polymerase chain reaction (PCR). Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) obtained from healthy controls and patients with rheumatoid arthritis (RA) as well as from synovial cells obtained from patients with RA and osteoarthritis (OA), treated with bisulfite, and then amplified by PCR using unmethylation-specific (U) and methylation-specific (M) primers encompassing the region −137/−139 to +53. PCR products were visualized in 8% polyacrylamide gels under nondenaturing conditions. Shown are the results from 3 representative subjects per group.

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Table 1. Methylation status of the DR3 promoter, as determined by the methylation-specific polymerase chain reaction method*
 No. of subjectsMethylation statusP
UnmethylatedPartially methylatedMethylated
  • *

    Values are the number (%) of subjects. PBMCs = peripheral blood mononuclear cells; RA = rheumatoid arthritis; OA = osteoarthritis.

  • Determined by chi-square test.

PBMCs     
 Healthy controls99 (100)00 
 RA patients99 (100)00 
Synovial cells     
 OA patients72 (29)5 (71)00.21
 RA patients706 (86)1 (14) 

Bisulfite genomic sequencing confirmed that the CpG island (−196 to +139) was consistently unmethylated in PBMCs from all healthy individuals and the patients with RA (Figure 2A). The CpG island, however, was highly methylated in synovial cells from patients with RA and to a lesser extent in synovial cells from patients with OA. When the percentage of methylation in the region −196 to +88 (ATG translation start site) was plotted, we found that synovial cells from RA patients were highly methylated as compared with those from OA patients (Figure 2B). Table 2 summarizes the individual methylation status of PBMCs from RA patients and healthy controls and of synovial cells from RA and OA patients, as examined by the bisulfite sequencing method. The results showed that synovial cells from patients with RA were predominantly hypermethylated as compared with those of OA (P = 0.008), which indicates that the CpG island in the DR3 gene promoter is hypermethylated in RA synovial cells in a disease-specific manner. Furthermore, the lymphocytes infiltrating to the rheumatoid synovium were also significantly methylated, with samples from 4 of 12 patients (33%) being unmethylated, 7 of 12 (58%) being partially methylated, and 1 of 12 (8%) being methylated, as determined by the methylation-specific PCR method.

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Figure 2. Methylation status of the DR3 gene promoter, as determined by bisulfite sequencing. A, Promoter construct, showing the CpG island, additional CpG motifs, the ATG translation start site, and position +1, the latter 2 of which were designed. Bisulfite-treated genomic DNA derived from peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects and patients with rheumatoid arthritis (RA) and derived from synovial cells obtained from patients with RA and osteoarthritis (OA) was cloned into pT7Blue vector and then sequenced. A total of 7–10 genomic clones of the DR3 promoter were obtained from each subject (numbered vertically). Each circle represents a CpG dinucleotide; intervening bases were omitted. B, Ratios of methylated and unmethylated CpG motifs. In the CpG island encompassing −196 to +88 (ATG translation start site), the ratios of C to C and of C to T were determined in each 5–10 clones derived from PBMCs from healthy subjects (n = 2) and RA patients (n = 2) and in those derived from synovial cells from RA patients (n = 7) and OA patients (n = 7). The presence of >65% C to C was arbitrarily designated as methylated, the presence of >65% C to T was designated as unmethylated, and the presence of C to C and of C to T at ≥35% but ≤65% was designated as partially methylated.

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Table 2. Methylation status of the DR3 promoter, as determined by the bisulfite sequencing method*
 No. of subjectsMethylation statusP
UnmethylatedPartially methylatedMethylated
  • *

    Values are the number (%) of subjects. PBMCs = peripheral blood mononuclear cells; RA = rheumatoid arthritis; OA = osteoarthritis.

  • Determined by chi-square test, comparing only the unmethylated versus the methylated group.

PBMCs     
 Healthy controls22 (100)00 
 RA patients22 (100)00 
Synovial cells     
 OA patients73 (43)4 (57)00.008
 RA patients703 (43)4 (57) 

Promoter activity.

COS-7 and 293T cells were transfected with luciferase reporter gene constructs driven by the truncated DR-3 promoters −441/+139pDR3, −239/+139pDR3, −112/+139pDR3, +1/+139pDR3, and −441/−294pDR3 and assayed for luciferase activity (Figure 3A). Comparable levels of promoter activity were detectable for promoter regions −441/+139pDR3, −239/+139pDR3, and −112/+139pDR3, whereas promoter regions +1/+139pDR3 and −441/−294pDR3 were essentially inert (Figure 3B, top). When the −441/+139pDR3, −239/+139pDR3, and −112/+139pDR3 constructs of the DR3 promoter were experimentally methylated in vitro by the use of bacterial methylase Sss I, the promoter activities were essentially abolished (Figure 3B, bottom). These findings indicate that promoter activity in the CpG island upstream of exon 1 is essential for the transactivation of the DR3 gene.

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Figure 3. Luciferase promoter assays. A, DR3 gene map, showing the positions of the CpG island, the transcription factor binding sites, the additional CpG motifs, the ATG translation start site, and the DR3 reporter gene constructs encompassing the region −441 to +139 (−441/+139pDR3, −239/+139pDR3, −112/+139pDR3, +1/+139pDR3, and −441/−294pDR3), which were used in the dual-luciferase (LUC) reporter assay. B, The DR3 reporter constructs were examined for luciferase activity in COS-7 and 293T cells using a dual-luciferase reporter assay (top). The DR3 reporter constructs were then either left untreated or were treated with Sss I for 2 hours to increase DNA methylation and were assayed for luciferase activity in COS-7 and 293T cells (bottom). Values are the mean ± SD. P values were determined by Student's 2-tailed independent t-test. ND = not done.

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Methylation status and protein expression.

We next studied the relationship between the methylation status of the CpG island and protein expression. Western blotting studies using PBMCs from healthy individuals and synovial cells from RA patients showed that the expression of DR-3 protein was decreased in RA synovial cells (n = 5) as compared with healthy PBMCs (n = 5) (Figure 4A). We also confirmed that the level of expression of DR-3 protein in PBMCs was comparable in the RA patients and healthy controls (data not shown). The expression of DR-3 protein was significantly decreased in association with methylation of the CpG island (P = 0.008), as demonstrated by comparison of the PBMCs from healthy controls with the synovial cells from RA patients (Figure 4B). Furthermore, the expression of DR-3 protein was also decreased in the RA synovial cells (n = 3) as compared with OA synovial cells (n = 3) (P = 0.03) (Figures 4C and D).

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Figure 4. Methylation status and DR-3 protein expression. A, Western blotting for DR-3 protein (top) and for the methylation status of the DR3 CpG island (bottom) in peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects (n = 5) and in synovial cells obtained from patients with rheumatoid arthritis (RA) (n = 5). Findings in the PBMCs and synovial samples were consistent with each other. B, Densitometry data derived from the Western blots shown in A, as determined with the use of the National Institutes of Health (NIH) Image/Image J software. C, Western blotting for DR-3 protein (top) and for the methylation status of the DR3 CpG island (bottom) in synovial cells from patients with RA (n = 3) and patients with osteoarthritis (OA) (n = 3). D, Densitometry data derived from the Western blots shown in C, as determined with the use of the NIH Image/Image J software. Horizontal lines in B and D indicate the mean; P values were determined by Student's 2-tailed independent t-test.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We found that the CpG island of the DR3 gene promoter was highly methylated in RA synovial cells as compared with OA synovial cells. Previous studies have shown that plenty of CpG motifs exist in the promoter region of apoptosis-inducing TNF receptor superfamily genes (29) and that apoptotic signaling pathways are often subjected to epigenetic gene silencing (19, 30). However, with the exception of the study by Petak et al (31), who showed an inverse correlation between the methylation status of the Fas gene promoter and Fas protein expression by comparing different tumor cell lines, the promoter activity of apoptosis-inducing genes was not directly studied in the same cell. Thus, the molecular relationship among the methylation status of the CpG island, the expression of the apoptosis-promoting gene, and the activity of the disease remained unclear.

In the present study, we found that transactivation of the DR3 gene was down-modulated by methylation of the promoter CpG island and the expression of DR-3 protein was significantly decreased in association with the decreased methylation status of the promoter CpG island. Several transcription factors, such as GATA, Oct-1, or Sp1, can bind the CpG island. In particular, Sp1 is a ubiquitously expressed zinc-finger transcription factor that supports mRNA expression in a variety of eukaryotic genes that lack the functional TATA box (17, 18). Among Sp family proteins, Sp1 and Sp3 were shown to be ubiquitously expressed in human tissues, and transcriptional activity enhanced by Sp1 was shown to be suppressed by Sp3 (32). Therefore, in such genes, CpG motifs may play an essential role in gene expression, especially in the expression of the DR3 gene (33, 34). This idea needs further investigation.

Expression of DR-3 has been shown to be increased in lymphocytes (14), which may be dependent on the fact that the CpG island of the DR3 promoter region was almost completely unmethylated in PBMCs from patients with RA and healthy individuals, as shown in Figure 2A and Tables 1 and 2. In contrast, our results indicated that the CpG island was highly methylated in RA synovial cells and in a synovial tissue lymphocyte fraction as well, which indicates that inflammatory conditions unique to rheumatoid joints could be responsible for the heightened CpG methylation in RA. Furthermore, our finding that expression of DR-3 was down-regulated in RA synovial cells as compared with the noninflammatory OA synovial cells (see Figures 4C and D) may be responsible for the defect in apoptosis induction against TNF-like molecule 1A (TL1A), a physiologic ligand of DR-3, both in vitro (35) and in vivo (36). Our finding may also be related to findings of other studies shown that apoptosis is defective in vivo in rheumatoid synovium (37, 38).

In summary, we found one CpG island in the DR3 promoter region that was highly methylated in RA synovial cells. The CpG island is essential for the transactivation of the DR3 gene, and the expression of DR-3 protein was decreased in association with the increased methylation of the promoter CpG island of the DR3 gene.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. M. Lamphier for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES