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Hypermethylated promoter region of DR3 the death receptor 3 gene in rheumatoid arthritis synovial cells.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 3, March 2006, pp 779–787
DOI 10.1002/art.21637
© 2006, American College of Rheumatology
Hypermethylated Promoter Region of DR3,
the Death Receptor 3 Gene,
in Rheumatoid Arthritis Synovial Cells
Nozomi Takami,1 Kayo Osawa,1 Yasushi Miura,2 Koichiro Komai,1 Mariko Taniguchi,1
Masahiko Shiraishi,3 Keizo Sato,4 Tetsuhiro Iguchi,4 Kazuko Shiozawa,5
Akira Hashiramoto,2 and Shunichi Shiozawa6
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 downmodulated 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.
Dr. Osawa’s work was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (14657117). Dr. Shunichi Shiozawa’s work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (13204059) and by a grant from the 21st
Century Center of Excellence Program (Center of Excellence for
Signal Transduction Disease: Diabetes Mellitus as Model).
1
Nozomi Takami, MSc, Kayo Osawa, PhD, Koichiro Komai,
PhD, Mariko Taniguchi, BSc: Kobe University FHS School of Medicine, and Kobe University Graduate School of Medicine, Kobe, Japan;
2
Yasushi Miura, MD, PhD, Akira Hashiramoto, MD, PhD: Kobe
University FHS School of Medicine, Kobe University Graduate School
of Medicine, and Kobe University Hospital, Kobe, Japan; 3Masahiko
Shiraishi, PhD: National Cancer Center Research Institute, Tokyo,
Japan; 4Keizo Sato, MD, Tetsuhiro Iguchi, MD, PhD: Kobe Rosai
Hospital, Kobe, Japan; 5Kazuko Shiozawa, MD, PhD: KonanKakogawa Hospital, Kakogawa, Japan; 6Shunichi Shiozawa, MD, PhD:
Kobe University FHS School of Medicine, Kobe University Graduate
School of Medicine, Kobe University Hospital, and the 21st Center of
Excellence, (CDE) Japan, Kobe, Japan.
Address correspondence and reprint requests to Shunichi
Shiozawa, MD, PhD, Department of Rheumatology, Kobe University
FHS School of Medicine, 7-10-2 Tomogaoka, Sumaku, Kobe 654-0142,
Japan. E-mail: shioz@kobe-u.ac.jp.
Submitted for publication May 11, 2005; accepted in revised
form November 10, 2005.
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
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TAKAMI ET AL
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
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). Diseasemodifying 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 FicollHypaque 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 ␮g) 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 ethanolprecipitation 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 methylationspecific 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-␮l reaction volume
containing 0.2 ␮g of DNA, 1⫻ PCR buffer, 200 ␮M 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
HYPERMETHYLATED DR3 PROMOTER IN RA SYNOVIAL CELLS
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 methylationspecific (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.
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
781
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; pDR3R1); for ⫺239/⫹139pDR3 (⫺239 to ⫹139), 5⬘-CCGGGTACCTTGAAGTGGTTCTCAGGGTT-3⬘ (forward; pDR3F2) 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 ␮g) 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 ␮M S-adenosylmethionine. Methylated plasmids were
purified with the use of the Wizard DNA Clean-Up system
(Promega).
Table 1. Methylation status of the DR3 promoter, as determined by the methylation-specific polymerase
chain reaction method*
Methylation status
PBMCs
Healthy controls
RA patients
Synovial cells
OA patients
RA patients
No. of
subjects
Unmethylated
Partially
methylated
Methylated
9
9
9 (100)
9 (100)
0
0
0
0
7
7
2 (29)
0
5 (71)
6 (86)
0
1 (14)
P†
0.21
* Values are the number (%) of subjects. PBMCs ⫽ peripheral blood mononuclear cells; RA ⫽
rheumatoid arthritis; OA ⫽ osteoarthritis.
† Determined by chi-square test.
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TAKAMI ET AL
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 ⱖ35%
but ⱕ65% was designated as partially methylated.
HYPERMETHYLATED DR3 PROMOTER IN RA SYNOVIAL CELLS
Table 2.
783
Methylation status of the DR3 promoter, as determined by the bisulfite sequencing method*
Methylation status
PBMCs
Healthy controls
RA patients
Synovial cells
OA patients
RA patients
No. of
subjects
Unmethylated
Partially
methylated
Methylated
2
2
2 (100)
2 (100)
0
0
0
0
7
7
3 (43)
0
4 (57)
3 (43)
0
4 (57)
P†
0.008
* 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.
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 ␮l) 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 ␮l 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 ␮g) 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
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 unmethylationspecific PCR.
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
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TAKAMI ET AL
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.
indicates that the CpG island in the DR3 gene promoter
is hypermethylated in RA synovial cells in a diseasespecific 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.
Promoter activity. COS-7 and 293T cells were
transfected with luciferase reporter gene constructs driven
HYPERMETHYLATED DR3 PROMOTER IN RA SYNOVIAL CELLS
785
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.
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.
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
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TAKAMI ET AL
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).
DISCUSSION
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 apoptosispromoting 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 downregulated 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.
ACKNOWLEDGMENT
We thank Dr. M. Lamphier for critical reading of the
manuscript.
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