TNFα Modulates the Expression of Circadian Clock Gene, Per2, Via D-Box Motif in the Promoter Region in Rheumatoid Synovial Cells

Kohsuke Yoshida¹, Akira Hashiramoto², Takaichi Okano³, Nao Shibanuma⁴ and Shunichi Shiozawa⁵, ¹Hyogo Prefectural Rehabilitation Center at Nishi-harima, Tatsuno, Japan, ²Department of Internal Medicine, Kobe University Graduate School of Medicine / The Center for Rheumatic Diseases, Kobe University Hospital, Kobe, Japan, ³The Center for Rheumatic Diseases,, Kobe University Hospital, Kobe, Japan, ⁴The Center for Rheumatic Diseases, Kobe University Hospital / Departmant of Orthopaedic Surgery, Kobe Kaisei Hospital, Kobe, Japan, ⁵Department of Medicine, Kyushu University Beppu Hospital, Beppu, Japan

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Rheumatoid arthritis (RA), synovial cells, synovial fluid and tumor necrosis factor (TNF)

Session Information

Title: Rheumatoid Arthritis - Human Etiology and Pathogenisis

Session Type: Abstract Submissions (ACR)

Background/Purpose:

The mammalian clock genes including Clock (circadian locomotor output cycles kaput), Bmal1 (brain and muscle Arnt-like protein 1), Per (Period) and Cry (Cryptochrome) regulate the circadian rhythm. We previously showed that arthritis was significantly accelerated in Cry1^-/-Cry2^-/- mice due to the activation of TNFα (tumor necrosis factor α) transcription, and TNFα inhibited the expression of Per2 mRNA in primary cultured human rheumatoid synovial cells. Here, we tried to elucidate the effects of TNFα on the transcription of Per2 gene in rheumatoid synovial cells.

Methods: Primary cultured rheumatoid synovial cells were synchronized upon incubation with 50% horse serum for 2 hours, and then stimulated with 10 ng/ml TNFα. Total RNA was extracted from synovial cells every 8 hrs until 32 hrs’ culture period, and mRNA expression of D-box binding protein genes, including Dbp (D site of albumin promoter binding protein), Hlf (hepatic leukemia factor), Tef (thyrotroph embryonic factor) and E4BP4 (E4-binding protein 4), were analyzed by real-time PCR. Synovial cells were transfected with the luciferase reporter vector containing the human Per2 promoter to measure the transcriptional activity of Per2 gene.

Results: The expression of Dbp, Hlf, and Tef mRNA were significantly inhibited (P <0.01), while those of E4bp4 mRNA was significantly increased (P <0.01) upon incubation with TNFα in rheumatoid synovial cells. Since Dbp, Hlf, Tef and E4bp4 genes could transactivate and suppress the expression of Per2 gene, respectively, by binding to D-box motif in the promoter region, we next introduced site-directed mutations into the D-box 1 (TTATGTAA, -372 to -365) and/or D-box 2 (TTACGTAA, -47 to -40) motif in the promoter, and then transfected rheumatoid synovial cells with luciferase reporter gene constructs driven by the Per2 promoter. As results, TNFα inhibited the transcriptional activity of the wild type of Per2 gene. However, when the promoter containing a mutated both D-box 1 and D-box 2 motif was transfected, TNFα-mediated transcriptional inhibition did not observed as compared with the wild type and the D-box 1 mutated promoter (P <0.05 and P<0.05, respectively).

Conclusion: TNFα significantly modulates the expression of Per2 gene via D-box binding protein, DBP, HLF, TEF and E4BP4, in rheumatoid synovial cells, and thereby may contribute to the pathogenesis of RA.

Disclosure:

K. Yoshida,
None;

A. Hashiramoto,
None;

T. Okano,
None;

N. Shibanuma,
None;

S. Shiozawa,
None.

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