TNF-alpha modulates expression of the circadian clock gene Per2 in rheumatoid synovial cells

Objectives: To study the effect of tumour necrosis factor (TNF)-alpha, responsible for the inflammation and circadian rhythm of rheumatoid arthritis (RA), on the expression of circadian clock genes in primary cultured human rheumatoid synovial cells. Method: The expression of circadian clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Arnt-like protein-1 (Bmal1), period (Per) 1/2, and cryptochrome (Cry) 1/2, and the proline and acidic amino acid-rich basic leucine zipper (PAR bZip) genes, a transcriptional activator of Per2, including D site of albumin promoter binding protein (Dbp), hepatic leukaemia factor (Hlf), and thyrotroph embryonic factor (Tef), and a transcriptional repressor of Per2, E4-binding protein 4 (E4bp4), in TNF-alpha-stimulated synovial cells was determined by real-time polymerase chain reaction (PCR). The D-box motifs in the Per2 promoter were mutated by site-directed mutagenesis, and the promoter activity of the Per2 gene was examined using the luciferase assay. Results: TNF-alpha enhanced the mRNA expression of Bmal1 and Cry1 but did not affect that of Clock, Per1, or Cry2. However, TNF-alpha inhibited the mRNA expression of the Per2 gene, as well as Dbp, Hlf, and Tef, but enhanced the mRNA expression of E4bp4. Furthermore, TNF-alpha inhibited the transcriptional activity of the wild-type Per2 gene in a manner dependent on the D-box 1 and D-box 2 motifs in the Per2 promoter. Conclusions: TNF-alpha modulates the expression of the Per2 gene through the D-box binding proteins DBP, HLF, TEF, and E4BP4, in rheumatoid synovial cells, and thereby may contribute to the pathogenesis of RA.