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Arthritis Research & Therapy
Published in: Arthritis Research & Therapy 1/2018

Open Access 01-12-2018 | Research article

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Authors: Kohjin Suzuki, Kohsuke Yoshida, Takeshi Ueha, Kenta Kaneshiro, Ayako Nakai, Naonori Hashimoto, Koto Uchida, Teppei Hashimoto, Yoshiko Kawasaki, Nao Shibanuma, Natsuko Nakagawa, Yoshitada Sakai, Akira Hashiramoto

Published in: Arthritis Research & Therapy | Issue 1/2018

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Abstract

Background

Effects 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.

Methods

Under 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.

Results

MTX (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.

Conclusions

We 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.
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Metadata
Title
Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts
Authors
Kohjin Suzuki
Kohsuke Yoshida
Takeshi Ueha
Kenta Kaneshiro
Ayako Nakai
Naonori Hashimoto
Koto Uchida
Teppei Hashimoto
Yoshiko Kawasaki
Nao Shibanuma
Natsuko Nakagawa
Yoshitada Sakai
Akira Hashiramoto
Publication date
01-12-2018
Publisher
BioMed Central
Published in
Arthritis Research & Therapy / Issue 1/2018
Electronic ISSN: 1478-6362
DOI
https://doi.org/10.1186/s13075-018-1552-9

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