Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts
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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.
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.
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.
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.
KeywordsRheumatoid arthritis Synovial fibroblasts Methotrexate Proline and acidic amino acid-rich basic leucine zipper Period2 Bcl-2 interacting killer
Bcl-2 interacting killer
Brain and muscle Arnt-like protein-1
Circadian locomotor output cycles kaput
D site of the albumin promoter binding protein
Dulbecco’s modified Eagle’s medium
E4-binding protein 4
Fetal bovine serum
Hepatic leukemia factor
- PAR bZIP
Proline and acidic amino acid-rich basic leucine zipper
Polymerase chain reaction
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Standard error of the mean
Small interfering RNA
TATA box binding protein
Thyrotroph embryonic factor
Tumor necrosis factor
Methotrexate (MTX) is a folic acid antagonist widely used as an anchor drug in treating various cancers [1, 2], as well as rheumatoid arthritis (RA) . For cancer cells, MTX competitively inhibits dihydrofolate reductase (DHFR) to block purine and pyrimidine biosynthesis, and thus it inhibits DNA replication and cell proliferation. For RA, low-dose MTX shows anti-inflammatory effects by inducing extracellular adenosine, which binds to adenosine receptors . Moreover, it has been reported that MTX induced apoptosis in synovial fibroblasts in both in-vivo and in-vitro experiments . Although MTX induced apoptosis in synovial fibroblast within 24–48 h [6, 7], the precise mechanism of how MTX expresses antiproliferative effects on synovial fibroblasts remains incompletely understood .
RA is a chronic arthritis characterized by ‘tumor-like’ synovial cell growth . 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  and that tumor necrosis factor (TNF)-α significantly disturbed the oscillation of biological clocks of synovial fibroblasts . Fibroblasts usually demonstrate daily rhythms of circadian clock, while Haas et al.  pointed out that the expression rhythm of clock genes disappears in RA synovial fibroblasts presumably due to prolonged inflammation.
The circadian rhythm in human cells is mainly regulated by the core clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Rant-like protein-1 (Bmal1), period (Per), and cryptochrome (Cry) [19, 20, 21, 22]. The circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP) includes the D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). PAR bZIP regulates gene expression by binding to a consensus sequence of D-box (5′-TTAXGTAA-3′; X = T or C) on the promoter region [23, 24, 25]. It has been reported that PAR bZIP can regulate the transcription of Per2 gene by binding two D-box sequences existing on its promoter (D-box 1, 5′-TTATGTAA-3′, −372 to −365; and D-box 2, 5′-TTACGTAA-3′, −47 to −40) . In contrast, E4-binding protein 4 (E4bp4) also binds to the D-box to suppress the transcription of Per2 [26, 27, 28] (see Additional file 1).
It is noted that Bcl-2 interacting killer (Bik) possesses D-box (5′-TTAAGTCA-3′, −285 to −277) on its promoter region , resembling the arrangement of Per2 genes. Bik, a member of the BH3-only subfamily, acts as an important signaling molecule upstream of the Bcl-2 and Bax subfamily . The BCL-2 family has been known to be central players in regulating physiological activities of mitochondria, with Bcl-2, Bcl-xL, and Mcl-1 suppressing mitochondria-related apoptosis, whereas Bax and Bak induce it. Among these, Bik directly binds to these family proteins to induce apoptosis [29, 30].
In this study, we explored novel pharmacological effects of MTX on circadian clock genes and apoptosis induction in RA synovial fibroblasts.
Synovial fibroblast culture
Characterization of patients
59.3 ± 3.4
Disease duration (years)
22.7 ± 3.2
C-reactive protein (mg/dl)
1.2 ± 0.4
2.6 ± 0.4
7.4 ± 1.4
3.7 ± 0.9
2/10 (2, 1.5 mg)
Synchronization of synovial fibroblasts by serum shock
Cell viability assay
Synovial fibroblasts (3.0 × 103 cells) were cultured in serum-free DMEM with or without MTX (1, 10, and 100 nM, 1 μM; Sigma Aldrich, St. Louis, MO, USA). After incubating for 24–72 h, cell viabilities were measured as the 450-nm absorbance of reduced WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitriphenyl)–5-(2, 4-disulphonyl)-2H–tetrazolium, monosodium salt) using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The values were represented relative to MTX-untreated cells (control).
Real-time polymerase chain reaction
Synovial fibroblasts (8.0 × 104 cells) were cultured in serum-free DMEM with or without MTX (10, 100 nM) for 24–32 h. After incubation, total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Then, reverse transcription was performed with the ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan) and analyzed on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The TaqMan probes used were: Hs00154147_m1 for Bmal1, Hs00231857_m1 for Clock, Hs00256143_m1 for Per2, Hs00172734_m1 for Cry1, Hs00609747_m1 for Dbp, Hs00171406_m1 for Hlf, Hs01115720_m1 for Tef, Hs00993282_m1 for E4bp4, Hs00154189_m1 for Bik, and Hs00427621_m1 for TATA box binding protein (Tbp). Expression levels were normalized to Tbp.
Synovial fibroblasts (5.0 × 105 cells) were cultured in serum-free DMEM with or without MTX (10, 100 nM) for 24–48 h, and lysed with RIPA buffer (Wako) to obtain cytoplasmic proteins. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA), probed with antibodies, and developed by ImmunoStar® LD (Wako). Antibodies (Abs) used were: anti-PER2 Ab (sc-101,105; Santa Cruz, Dallas, TX, USA), anti-CYTOCHROME C Ab (ab13575; Abcam, Cambridge, UK), anti-BIK Ab (NB100–56109; Novus, Littleton, CO, USA), anti-Actin Ab (sc-1616; Santa Cruz), anti-mouse IgG Ab (NA9310; GE Healthcare, Chicago, IL, USA), and anti-rabbit IgG Ab (NA9340; GE Healthcare).
ELISA for PER2 and CYTOCHROME C
Synovial fibroblasts (3.0 × 103 cells) were cultured in serum-free DMEM with or without MTX (10, 100 nM) for 32–48 h, and protein expression of PER2 and CYTOCHROME C was measured by the In Cell ELISA test kit (3440–02; Thermo). Antibodies were as already described.
Luciferase reporter gene construction
Genomic DNA was obtained from RA synovial fibroblasts and amplified by PCR to generate the luciferase reporters. The primer pairs used for amplifying human Bik promoter containing D-box (−780 to +176) were 5′-TGGCCTAACTGGCCGTAAACAAGCTTTGCCGTGC-3′ (forward) and 5′-CGCCGAGGCCAGATCATGCTGGCAGCGTCTGTA-3′ (reverse). The primer pairs used for amplifying human Bik promoter without D-box (−260 to + 323) were 5′-TGGCCTAACTGGCCGCCTCTTGGAGCCTCGGTT-3′ (forward) and 5′-CGCCGAGGCCAGATCTTGCTGGAGCGGTAAAACC-3′ (reverse). The KpnI recognition sequence was added to the 5′ ends of forward primers and the BglII recognition sequence was added to the 5′ ends of reverse primers. The PCR products were cloned into the KpnI and BglII site of the pGL4.10 (luc2) vector (Promega, Madison, WI, USA) using In-Fusion® HD Cloning Plus (Takara, Shiga, Japan). The luciferase reporters containing Per2 promoters were generated with reference to Yoshida et al. . D-box motifs of Per2 promoter were mutated from 5′-TTATGTAA-3′ to 5’-CGCCAGGC-3′ (−372 to −365) and from 5′-TTACGTAA-3′ to 5′-CAGCGTAA-3′ (−47 to −40) (see Additional file 3).
Transient transfection and luciferase reporter assay
Synovial fibroblasts (4 × 104 cells) were transfected with 500 ng of the pGL4.10 (luc2) vector containing various Per2 and Bik promoters using Lipofectamine 3000 Transfection Reagent (Thermo). As an internal control, 35 ng of pRL-TK (Promega) containing the herpes simplex virus thymidine kinase promoter driving Renilla luciferase was cotransfected. After 24 h of incubation for transfection, cells were treated with 10 and 100 nM MTX for 24 h, and analyzed for luciferase activity using the Dual-Luciferase Reporter Assay (Promega). Activities of both firefly and Renilla luciferases were measured, and the activity of firefly luciferase was normalized by Renilla luciferase. The values were shown as relative variations to MTX-untreated cells.
Synovial fibroblasts (6.0 × 103 cells) were cultured in serum-free DMEM with or without MTX (10 nM) for 24 h. Then, cells were fixed with 4% formaldehyde, stained with anti-PER2 Ab, anti-BIK Ab, anti-CYTOCHROME C Ab, anti-mouse IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 594 Conjugate) (#8890; Cell Signaling Technology, Danvers, MA, USA), anti-rabbit IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 488 Conjugate) (#4412; Cell Signaling Technology), and DAPI (0.5 μg/ml; Sigma, St. Louis, MO, USA). Protein expression and morphological changes of the nucleus were examined under fluorescence microscopy.
Per2 small interfering (si) RNA (s16931; Life Technologies) and Bik siRNA (s1990; Life Technologies) were transfected into synovial fibroblasts (3.0 × 103 cells) using Lipofectamine™ RNAiMAX (Life Technologies) for 48 h. Noncoding siRNA (4,390,843; Life Technologies) was also used as controls. After that, cells were cultured in serum-free DMEM with 10 nM of MTX for 24 h to measure cell viabilities using the Cell Counting Kit-8. The viabilities were represented relative to those of noncoding siRNA without MTX treatment.
In dynamite-plunger plots, values were expressed as the mean ± standard error of the mean (SEM). In boxplots, values were expressed with 10th, 25th, 50th (median), 75th, and 90th percentiles, and the single values were superimposed on the boxplots using black symbols.
For statistical analyses, a one-sample Kolmogorov–Smirnov test was used to test the normality, A one-sample t test was used to compare one single control value and others, a paired t test was used to compare differences between two experimental groups, the Tukey test was used to compare differences between more than three experimental groups, and Dunnett’s test was used to compare differences between the control and others. All statistical tests were two-sided and p < 0.05 was considered statistically significant.
The statistical analyses were performed by EZR, based on R and R commander .
MTX inhibited viabilities of synovial fibroblasts
MTX accelerated the expression of circadian clock gene Per2
MTX increased the expression of PAR bZIP and Bik
Next, we examined the mRNA expression of the proapoptotic factor Bik, since the PAR bZIP-binding site also exists on the promoter region of Bik . As shown in Fig. 3b, the expression of Bik mRNA was significantly increased by 10 nM of MTX treatment for 32 h.
Finally, we observed cytosolic release of CYTOCHROME C since Bik interacted with BCL-2 family proteins to induce mitochondria-related apoptosis. As a result, expression of CYTOCHROME C was increased by 10 nM of MTX treatment for 48 h, but not by 100 nM (Fig. 3c, d).
Transcription of Per2 and Bik was enhanced via D-box
PER2, BIK, and CYTOCHROME C were highly expressed in apoptotic cells
Per2 and Bik are critical genes for MTX-induced apoptosis
When we examined the long-term expression of Per2 and Bik mRNA (Fig. 6b), they gradually increased from 8 h until 24 or 32 h after synchronized circadian oscillations by serum shock, consistent with our previous report that the expression of Per2 on synovial fibroblasts was increased after synchronization and peaked at 24 h . Since these genes were responsible for MTX-induced cell death, synovial fibroblasts were separately treated by 10 nM of MTX at 0–8 h (the lower Per2/Bik expression period) and at 24–32 h (the higher Per2/Bik expression period), and viabilities of synovial fibroblasts were again measured. As shown in Fig. 6c, the reduction rate of cell viabilities was significantly increased by 24–32 h of MTX treatment compared with 0–8 h of treatment, depending on the expression levels of Per2 and Bik.
We present here a novel pharmacological action of MTX in the viewpoint of circadian clock genes, circadian transcriptional factor PAR bZIP, and proapoptotic molecule Bik. In addition, a crucial mechanism of MTX-induced apoptosis on rheumatoid synovial fibroblasts was further traced.
In this study, the effect of MTX on cell viabilities did not show a concentration-dependent manner, with a wide range of MTX concentrations from 1 nM to 1 μM. In addition to our result, we found that MTX concentrations lower than 1 nM did not decrease cell viability (see Additional file 4). MTX is transferred almost equally to synovial fluid and sera, and the concentration of MTX in human sera can reach 200 nM at the peak and immediately decreases to less than 10 nM , although pharmacological effects on cytotoxicity or cellular viability may not necessarily increase in a concentration-dependent manner. As reported previously, viabilities of human lymphoblastic leukemia cells and epithelial cells of rat decreased in a concentration-dependent manner at low MTX concentrations, but did not show a significant decrease at high MTX concentrations [34, 35]. Moreover, MTX did not affect viability in a concentration-dependent manner on T cells and RA synovial fibroblasts, in contrast to those of dose dependency observed in osteoarthritis synovial fibroblasts [6, 36]. Thus, further studies are required for the action of MTX correlated with drug dosage or cell types.
Since MTX concentrations below 100 nM were conceivable in sera of RA patients, we next 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 , we examined the effect of MTX on expression of Per2, Bmal1, Clock, and Cry1 that were regarded as “core” clock genes [20, 21, 22]. We examined mRNA expression of circadian clock genes over time, and found that the controls and 10/100 nM of MTX showed almost the same expression rhythms, and MTX influenced their expression levels (see Additional file 5).
As described, both Per2 and Bik genes have D-box in their promoter regions, and PAR bZIP proteins regulate the expression of these genes by binding to D-box [24, 39]. Indeed, promoter activities of Per2 and Bik were upregulated by 10 nM of MTX when the D-box sequence exists and were cancelled without D-box. Moreover, both PER2 and BIK were highly expressed in MTX-induced apoptotic cells, while inhibitions of Per2 and Bik synergistically attenuated the effect of MTX on cellular viabilities. As it has been reported that PAR bZIP and Bik mediate oxidative stress-induced apoptosis in fibroblasts , we consider Per2 and Bik as essential factors for MTX-dependent synovial cell death and propose here that two independent pathways can mediate these death signals: the PAR bZIP–Per2 transcriptional pathway and the PAR bZIP–Bik transcriptional pathway (see Additional file 6). However, as shown in Fig. 2a, downregulation of Clock and Cry1 appeared to be less effective on cell viabilities after treatment with 100 nM of MTX. Further in-vivo study should be required for clinical application by investigating the cross-talk of clock genes.
Last, because of the similarity of their promoter region containing D-box, we supposed Bik might show oscillation as has been reported for Per2 [17, 31]. For the further understanding of circadian manifestation in rheumatoid arthritis, we showed that the expression of Bik, as well as Per2, was gradually increased from 8 h until 24 or 32 h, and MTX was significantly effective in situations when Per2 and Bik were highly expressed. It has been reported that administration of MTX at bedtime, as an optimal dosing time associated with the oscillation of TNF-α production, could reduce the disease activities of patients with RA [40, 41]. Thus, we considered that expression levels of Per2 and Bik could also be a critical biomarker for chronotherapy of MTX not only for RA, but also for other diseases such as acute lymphocytic leukemia, non-Hodgkin’s lymphoma, osteosarcoma, and breast cancer, in accordance with a previous report that circadian clock genes could be a biomarker for chronotherapy .
We propose here that MTX induces apoptosis in synovial fibroblasts through the binding of PAR bZIP to D-box of two different genes, Per2 and Bik, and these dual pathways work independently but synergistically. In consequence, MTX acts as a modulator of circadian environments of synovial fibroblasts in relation to biological clock genes and circadian transcriptional factors.
The authors declare that they have no funding.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
KS performed and analyzed most of the experiments, and wrote the manuscript. KY, TU, and KK provided technical support for the experiments. AN, NH, and KU maintained synovial fibroblasts and exchanged useful arguments on this manuscript. TH, YK, NS, and NN collected synovial samples. YS supervised the project. AH conceived the idea for the project, supervised the experiments, and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study has been approved by the ethics committee of Kobe University Graduate School of Health Sciences (#579–1) and Kobe Kaisei Hospital (#0072), in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient before study enrolment.
Consent for publication
The authors declare that they have no competing interests.
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