Abstract
BackgroundEffects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts.MethodsUnder treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression.ResultsMTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high.ConclusionsWe present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.
Highlights
Effects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood
Since MTX concentrations below 100 nM were conceivable in sera of RA patients, we focused on the effect of MTX on circadian clock genes and circadian transcriptional factor PAR bZIP genes and their relations to mitochondria-related apoptosis of synovial fibroblasts
Since circadian clock genes were reported to be closely related to the pathogenesis of arthritis [16, 37] and excessive expression of Per2 could induce apoptosis [38], we examined the effect of MTX on expression of Per2, brain and muscle Rant-like protein-1 (Bmal1), circadian locomotor output cycles kaput (Clock), and Cry1 that were regarded as “core” clock genes [20,21,22]
Summary
Effects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. RA is a chronic arthritis characterized by ‘tumor-like’ synovial cell growth [9] Another remarkable feature of RA is the circadian variation of disease-related symptoms, such as morning stiffness, increased production of proinflammatory cytokines at night time, and peaked secretion of immunoglobulin (Ig) A/IgM types of rheumatoid factor in the morning [10,11,12,13,14,15]. Since these rheumatic symptoms possess a daily rhythm, we have previously shown that the action of the biological clock was significantly disturbed in the mouse model of collagen antibody-induced arthritis [16] and that tumor necrosis factor (TNF)-α significantly disturbed the oscillation of biological clocks of synovial fibroblasts [17]. Fibroblasts usually demonstrate daily rhythms of circadian clock, while Haas et al [18] pointed out that the expression rhythm of clock genes disappears in RA synovial fibroblasts presumably due to prolonged inflammation
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