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


CONCLUSIONS
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.
Takami
Nozomi
N
Kobe University FHS School of Medicine, Sumaku, Japan.
Osawa
Kayo
K
Miura
Yasushi
Y
Komai
Koichiro
K
Taniguchi
Mariko
M
Shiraishi
Masahiko
M
Sato
Keizo
K
Iguchi
Tetsuhiro
T
Shiozawa
Kazuko
K
Hashiramoto
Akira
A
Shiozawa
Shunichi
S
D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008745:Methylation; D008875:Middle Aged; D010003:Osteoarthritis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D053219:Receptors, Tumor Necrosis Factor, Member 25; D013583:Synovial Membrane / Q000166:cytology*
D018124:Receptors, Tumor Necrosis Factor; D053219:Receptors, Tumor Necrosis Factor, Member 25; C506033:TNFRSF25 protein, human