The clock gene Bmal1 controls inflammatory mediators in rheumatoid arthritis fibroblast-like synoviocytes.

OBJECT: To clarify the involvement of clock genes in the production of inflammatory mediators from RA-FLS, we examined the role of Bmal1, one of the master clock genes. METHODS: RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), IL-6 (0, 20 ng/mL), IL-17 (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expression of Bmal1, MMP-3, CCL2, IL-6, IL-7 and IL-15 by qPCR and immunofluorescence staining. After silencing Bmal1, RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expressions of inflammatory mediators; MMP-3, CCL2, IL-6 and IL-15 by qPCR, ELISA and immunofluorescence staining. RESULTS: Bmal1 expressions were increased by IL-1ß, TNF-α and IFN-γ stimulations. Under stimulations with TNF-α, IL-1ß, and IFN-γ, mRNA and protein expressions of MMP-3, CCL2 and IL-6 were suppressed by siBmal1. CONCLUSION: Results indicate that Bmal1 contributes the production of MMP-3, CCL2, and IL-6 from RA-FLS, implying Bmal1 is involved in the pathogenesis of RA by regulating the inflammation.

Tocilizumab suppresses NF-kappa B activation via toll-like receptor 9 signaling by reducing cell-free DNA in rheumatoid arthritis.

Endogenous DNA is released into the bloodstream as cell-free DNA (cfDNA) following cell death and is associated with various pathological conditions. However, their association with therapeutic drugs against rheumatoid arthritis (RA) remains unknown. Therefore, we investigated the significance of cfDNA in RA treated with tocilizumab and tumour necrosis factor inhibitor (TNF-I). Biological DMARDs (bDMARDs), including tocilizumab and TNF-I, were administered to 77 and 59 RA patients, respectively. Plasma cfDNA levels were measured at weeks 0, 4, and 12 by quantitative polymerase chain reaction. Disease activity was evaluated at the same time point using DAS28ESR. cfDNA levels from RA synovial cells treated with tocilizumab or etanercept for 24 h were measured. Human toll-like receptor 9 (hTLR9)-expressing HEK293 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon NF-κB activation, were stimulated by cfDNA from RA patients, and subsequently, SEAP levels were determined. NF-κB translocation was evaluated by immunofluorescence staining with or without tocilizumab. The DAS28ESR significantly improved in both bDMARD groups at week 12. However, plasma cfDNA levels significantly decreased in the tocilizumab group at week 12 compared to that in week 0. cfDNA levels correlated with DAS28ESR in biological treatment-naïve patients administered tocilizumab. cfDNA levels in synovial cells were significantly suppressed by tocilizumab treatment and unaltered with etanercept. HEK293 cells released SEAP upon cfDNA stimulation, and the observed NF-κB nuclear translocation was suppressed by tocilizumab. Tocilizumab suppressed inflammation via the TLR9 pathway by decreasing cfDNA levels. Regulation of cfDNA may be a therapeutic target for RA.

Chronotherapy targeting cytokine secretion attenuates collagen-induced arthritis in mice.

OBJECTIVE: Diurnal variation of symptoms are observed in rheumatoid arthritis, especially in productions of cytokines that show peak concentrations during mid night. In contrast, cytokines of collagen-induced arthritis (CIA) mice increase in daytimes under Mid-light condition. By using chronotherapy, differences in drug efficacies according to administration time of Baricitinib, a wide ranged cytokine blocker, were examined in CIA mice. METHODS: CIA mice were administered a dose of 3 mg/kg of Baricitinib once a day at zeitgeber time (ZT) 0 or ZT12 for 21 days. Arthritis scores, histopathology and factors related to joint destruction in sera were examined. Phosphorylation of STAT3 in liver, expressions of cytokines in spleen, and Interleukin (IL)-6 and tumor necrosis factor (TNF)-α in sera were measured. RESULTS: In CIA mice, diurnal variations were observed both in expressions of cytokines and phosphorylation of STAT3. Arthritis scores of ZT0/12 group decreased from day3 as compared to untreated mice, and those of ZT0 group significantly decreased as compared to ZT12 group from day12. Pathological findings, immunohistochemistry of cytokines and Receptor activator of nuclear factor kappa-Β ligand (RANKL)/osteoprotegerin ratio in sera well reflected results of arthritis scores. Diurnal variation of STAT3 phosphorylation was suppressed in ZT0 group. At ZT2, expressions of IL-6/Interferon-γ/TNF/granulocyte-macrophage colony-stimulating factor in ZT0 group were significantly decreased as compared to untreated mice, though not in ZT12 group. In ZT0 group, IL-6 and TNF-α in sera were decreased for longer time than that in ZT12 group. CONCLUSION: Chronotherapy using Baricitinib targeting cytokine secretions is effective in CIA mice. Clinical applications of chronotherapy can be expected to enhance the drug efficacy.

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

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.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

TNF-α induces expression of the circadian clock gene Bmal1 via dual calcium-dependent pathways in rheumatoid synovial cells.

Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca2+ chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca2+ influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca2+ influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca2+ influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA.

Role of the MICA polymorphism in systemic lupus erythematosus.

OBJECTIVE: To study the genetic contribution of major histocompatibility complex class I polypeptide-related sequence A (MICA), important in natural killer (NK) cell function, in patients with systemic lupus erythematosus (SLE). METHODS: Japanese patients with SLE (n=716), those with rheumatoid arthritis (RA) (n=327), and healthy control subjects (n=351) were genotyped for the Val129 Met polymorphism (rs1051792) and transmembrane (TM) alanine-encoding GCT repeats, termed A4, A5, A5.1, A6, and A9, in the MICA gene. Recombinant human MICA-GST fusion proteins were tested on the NK cell line NK92MI for the expression of NK group 2, member D (NKG2-D), NK cell-mediated cytotoxicity, and interferon-γ (IFNγ) production. RESULTS: The MICA 129Met allele, TMA9 allele, and 129Met/Met genotype were positively associated with SLE (corrected P [Pcorr]=0.01 and odds ratio [OR] 1.3, Pcorr=0.003 and OR 1.6, and Pcorr=0.02 and OR 1.8, respectively), while the MICA 129Val allele was negatively associated with SLE (Pcorr=0.01, OR 0.8). The MICA 129Met;A9 haplotype was also associated with SLE (Pcorr=0.0006, OR 1.8), and there was an additive genetic effect between the MICA 129Met;A9 haplotype and HLA-DRB1*15:01. When NK92MI cells were incubated in vitro with recombinant human disease-associated 129Met;A9 (the combination of polymorphisms at 129Met and TMA9), expression of NKG2-D on NK92MI cells and cytotoxicity of the NK cells were inhibited, but production of IFNγ from NK92MI cells was enhanced. CONCLUSION: The MICA polymorphism is genetically associated with SLE, and MICA appears to contribute to the pathogenesis of SLE by modulating NK cell function.

Heat shock protein 90 maintains the tumour-like character of rheumatoid synovial cells by stabilizing integrin-linked kinase, extracellular signal-regulated kinase and protein kinase B.

OBJECTIVE: To clarify the contribution of heat shock protein 90 (HSP90) to the pathogenesis of RA, we studied the effects of geldanamycin (GA), an inhibitor of HSP90, on excessive cellular extension and resistance to apoptosis induction of rheumatoid synovial cells. METHODS: Expression of integrin-α5ß1 and integrin-linked kinase (ILK) in synovial cells was determined by western blot. The peripheral localization of ILK, reorganization of F-actin, complex formation of ILK with particularly interesting new cysteine-histidine protein (PINCH) and α-parvin, and activation of Rac/cdc42 in synovial cells were examined by using immunohistochemistry and immunoprecipitation. Apoptosis induction by GA treatment was analysed by nuclear staining, cell proliferation assay and western blot of caspase. Effects of GA on mitogen-activated protein kinase (MAPK), PI-3K/protein kinase B (Akt) pathway, mitochondrial Bcl-2 pathway and activation of nuclear factor-κB (NF-κB) were examined by western blot and ELISA. RESULTS: HSP90 was overexpressed in synovial cells while GA decreased the expression of integrin-α5ß1 and ILK. The peripheral localization of ILK, reorganization of F-actin, complex formation of ILK with PINCH and α-parvin, and activation of Rac/cdc42 in synovial cells were all inhibited by GA treatment. We found that HSP90 stabilized and regulated the MAPK and PI-3K/Akt pathway, thereby inhibiting HSP90-potentiated synovial apoptosis by stimulating caspases and the mitochondrial Bcl-2 pathway on the one hand and inhibiting the activation of NF-κB on the other. CONCLUSION: The contribution of HSP90 is important in the pathogenesis of RA that potentiates a tumour-like synovial overgrowth by stabilizing ILK, extracellular signal-regulated kinase and Akt.

Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3(+) CD69(+) T cells and a higher production of TNF-alpha from spleen cells. When arthritis is induced, Cry1(-/-)Cry2(-/-) mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-alpha, IL-1beta and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1(-/-)Cry2(-/-) mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti-TNF-alpha Ab significantly reduced the severity and halted the progression of the arthritis of Cry1(-/-)Cry2(-/-) mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1(-/-)Cry2(-/-) mice significantly reduced the trans activation of TNF-alpha gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative, but independent, signaling via ERK/MAPK and phosphatidylinositol 3-kinase/Akt.

OBJECTIVE: To determine whether angiopoietin 1 (Ang-1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell-signaling mechanisms of Ang-1 in the rheumatoid joint. METHODS: Expression of Ang-1, TIE-2 (a receptor for Ang-1), and matrix metalloproteinase 3 (MMP-3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3-kinase/Akt pathways and of NF-kappaB was determined by Western blotting and an NF-kappaB p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin-Rho and p21-activated kinase 1. Matrix degradation was examined by induction of proMMP-3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay. RESULTS: Ang-1 stimulated the ERK/MAPK and PI 3-kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang-1 activated NF-kappaB via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang-1 directly potentiated the extension of synovial cells in an ERK- and Akt-dependent manner by up-regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang-1 induced proMMP-3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix. CONCLUSION: Ang-1 stimulates the ERK/MAPK and PI 3-kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang-1/TIE-2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint.