trichostatin-a and Arthritis--Rheumatoid

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn

Topics: Animals; Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Benzamides; Binding Sites; Carbolines; Cell Line, Tumor; Collagen Type II; HEK293 Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mice, Inbred DBA; Molecular Docking Simulation; Zebrafish

This study was undertaken to explore the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, on the viability, apoptosis, and invasiveness of hypoxic rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs). RA FLSs were exposed to hypoxia for 24 h in the presence or absence of 2 μM TSA and tested for cell viability, apoptosis, invasion, and gene expression. The involvement of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway was checked. TSA significantly inhibited the viability and induced apoptosis of hypoxic RA FLSs, compared to vehicle control. TSA blocked hypoxia-induced invasion of RA FLSs during Matrigel invasion assays and reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9) and PI3K and phosphorylation of Akt. Overexpression of constitutively active Akt reversed TSA-mediated suppression of invasiveness and downregulation of MMP-2 and MMP-9. Our results indicate the antisurvival and antiinvasive activities of TSA in hypoxic RA FLSs, which is associated with inactivation of PI3K/Akt signaling.

Topics: Arthritis, Rheumatoid; Cell Hypoxia; Cells, Cultured; Fibroblasts; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Synoviocytes

In rheumatoid arthritis (RA), synovial fibroblasts (RA-SFs) accumulate in affected joints, where they play roles in inflammation and joint destruction. RA-SFs exhibit tumor-like proliferation and are resistant to apoptosis. Although RA-SF activation is well described, negative regulators of RA-SF activation are unknown. We previously reported that histone deacetylase (HDAC) inhibitors facilitate apoptosis in RA-SFs. Here we found that RA-SFs treated with the HDAC inhibitor Trichostatin A (TSA) exhibited an upregulation of the immediate early response gene X-1 (IEX-1). IEX-1 has roles in apoptosis sensitivity, cell-cycle progression, and proliferation, and is reported to be involved in immune responses, inflammation, and tumorigenesis, and to have anti-arthritic properties. To investigate IEX-1's role in RA-SFs, we used in vitro-cultured synovial fibroblasts from RA and osteoarthritis (OA) patients. We confirmed that TSA upregulated the IEX-1 protein and mRNA expressions in RA-SFs by western blotting and quantitative RT-PCR. Inhibiting HDAC1, 2, and 3 (but not 6 or 8) also upregulated IEX-1. The IEX-1 mRNA levels were higher in RA-SFs than in OA-SFs, and were further upregulated in RA-SFs by the pro-inflammatory cytokines TNFα and IL-1β. The staining of surgical specimens showed that IEX-1 was present in the pannus from affected RA joints. Si-RNA-mediated IEX-1 knockdown upregulated the lipopolysaccharide (LPS)-induced expression of TNFα and various chemokine mRNAs, indicating that IEX-1 downregulates TNFα and chemokines. Furthermore, apoptosis analysis showed that IEX-1 knockdown protected RA-SFs from apoptosis induced by TSA or by an anti-Fas mAb, indicating that IEX-1 is pro-apoptotic in RA-SFs. Collectively, our results showed that IEX-1 is induced by TNFα and IL-1β in RA-SFs, in which it suppresses TNFα and chemokine production and induces apoptosis; thus, IEX-1 negatively regulates RA-SF activation. Further investigation of IEX1's functions in RA-SFs may lead to new therapeutic approaches for RA.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Arthritis, Rheumatoid; Cells, Cultured; Chemokines; Fibroblasts; Humans; Hydroxamic Acids; Membrane Proteins; Osteoarthritis; Synovial Membrane; Tumor Necrosis Factor-alpha; Up-Regulation

To characterize the role of histone deacetylase (HDAC) activity in rheumatoid arthritis (RA) and to evaluate the effects of MI192, a novel HDAC-3-selective inhibitor, compared with the established nonselective HDAC inhibitor trichostatin A (TSA), on proinflammatory cytokine production.. Activity of HDAC and histone acetyltransferase was measured in peripheral blood mononuclear cells (PBMCs) from RA patients by spectrophotometric assay, prior to and after 12 weeks of etanercept therapy. The effects of HDAC inhibitor treatment on cytokine production in both RA and healthy PBMCs were assessed by enzyme-linked immunosorbent assay.. RA PBMCs exhibited significantly increased HDAC activity (P = 0.007) compared to PBMCs from healthy individuals, and the increase was unaltered after 12 weeks of etanercept therapy. TSA was a potent inhibitor of tumor necrosis factor (TNF) and interleukin-6 (IL-6) production in both RA and healthy PBMCs and of interferon-γ (IFNγ) production in healthy PBMCs; IFNγ was not produced by RA PBMCs. MI192 inhibited TNF production at high concentrations and dose-dependently inhibited IL-6 in RA PBMCs but not healthy PBMCs, across a dose range of 10 μM-5 nM.. HDAC activity is dysregulated in RA PBMCs and is a potential target for therapeutic intervention, as it is not affected by conventional anti-TNF treatment with etanercept. Both the selective and the nonselective HDAC inhibitors (MI192 and TSA, respectively) were found to regulate cytokine production from PBMCs, but their effects were cell type and compound specific. HDAC inhibitors have potential in the treatment of RA, and HDAC-selective inhibition may improve the therapeutic margin of safety; however, further clinical characterization and evaluation for adverse effects is needed.

Topics: Aged, 80 and over; Arthritis, Rheumatoid; Dose-Response Relationship, Drug; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Interferon-gamma; Interleukin-6; Leukocytes, Mononuclear; Male; Middle Aged

Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue.. To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model.. RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1β, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays.. HDACi (0.25-1.0 μM) suppressed RA FLS IL-6 production induced by IL-1β, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1β stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1β stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages.. Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblasts; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation Mediators; Interleukin-6; Macrophages; MAP Kinase Signaling System; NF-kappa B; RNA, Messenger; Signal Transduction; Synovial Membrane; Transcription Factor AP-1

Histone deacetylase inhibitors have shown suppressive effects on tumor growth and in some autoimmune diseases. However, the molecular mechanisms of their effects are not very clear. The purpose of this study was to investigate the effects of trichostatin A (TSA) on collagen-induced rheumatoid arthritis (CIA) in a mouse model and its underlying mechanisms.. CIA was induced in DBA/1 mice with type II collagen. Paws were scored to assess disease severity. Inflammation of joints was evaluated by histological examination. Real-time PCR was used to determine cytokine mRNA levels. Cytokine production in serum and in supernatants from dendritic to T cell co-cultures was measured by ELISA. T cell proliferation was determined using [(3)H] incorporation. Intracellular cytokine staining was used to measure interferon gamma (IFN-γ)- and interleukin (IL)-4-producing T cells in splenocytes. Chromatin immunoprecipitation was used to examine histone H3 and H4 acetylation.. TSA potently suppressed the severity of arthritis and type II collagen-specific T cell responses in CIA. IFN-γ expression was high in CIA mice, but was inhibited by TSA treatment either at the same time as immunization or at the onset of arthritis manifestation. T cells from TSA-treated mice produced higher levels of IL-4 than cells from the control group. TSA predominantly suppressed Th1 cell proliferation in vitro by induction of apoptosis. In addition, TSA enhanced IL-4 gene expression of in vitro differentiated Th2 cells, and the mechanism is associated with an increased level of histone acetylation in the IL-4 gene promoter.. While TSA selectively suppresses a Th1 response by inducing apoptosis, it upregulates IL-4 expression probably by increasing histone H3 and H4 acetylation of the IL-4 gene promoter. We conclude that TSA can induce a Th1/Th2 balance in vivo and exert protective effects on CIA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Proliferation; Coculture Techniques; Dendritic Cells; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Immunohistochemistry; Interferon-gamma; Interleukin-4; Joints; Mice; Mice, Inbred DBA; Polymerase Chain Reaction; RNA, Messenger; Th1 Cells; Th1-Th2 Balance; Th2 Cells

Macrophages contribute significantly to the pathology of many chronic inflammatory diseases, including rheumatoid arthritis (RA), asthma, and chronic obstructive pulmonary disease. Macrophage activation and survival are tightly regulated by reversible acetylation and deacetylation of histones, transcription factors, and structural proteins. Although histone deacetylase (HDAC) inhibitors (HDACis) demonstrate therapeutic effects in animal models of chronic inflammatory disease, depressed macrophage HDAC activity in patients with asthma, chronic obstructive pulmonary disease, or RA may contribute to inflammation in these diseases, potentially contraindicating the therapeutic administration of HDACis. In this study, we directly examined whether HDACis could influence the activation of macrophages derived from the inflamed joints of patients with RA. We found that inhibition of class I/II HDACs or class III sirtuin HDACs potently blocked the production of IL-6 and TNF-alpha by macrophages from healthy donors and patients with RA. Two HDACis, trichostatin A and nicotinamide, selectively induced macrophage apoptosis associated with specific downregulation of the antiapoptotic protein Bfl-1/A1, and inflammatory stimuli enhanced the sensitivity of macrophages to HDACi-induced apoptosis. Importantly, inflammatory and angiogenic cytokine production in intact RA synovial biopsy explants was also suppressed by HDACis. Our study identifies redundant, but essential, roles for class I/II and sirtuin HDACs in promoting inflammation, angiogenesis, and cell survival in RA.

Topics: Adult; Aged; Apoptosis; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation; Interleukin-6; Macrophages; Male; Middle Aged; Minor Histocompatibility Antigens; Niacinamide; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Tumor Necrosis Factor-alpha

The therapeutic effects of histone deacetylase (HDAC) inhibitor combined with ultrasound (US) (1 MHz, 10% duty factor, 0.1 or 0.2 W/cm(2)) in RA synovial fibroblasts (RASFs) were examined.. RASFs were isolated from rheumatoid synovial tissues obtained from patients with RA during total knee arthroplasty. RASFs were treated with an HDAC inhibitor, trichostatin A (TSA), with or without US. Cell viability was estimated using the Trypan blue dye exclusion test and cell cycle was examined by flow cytometry using propidium iodide (PI) staining. Gene expression of cell cycle-related genes cyclin D, cyclin A, cyclin B and p21(WAF1/Cip1) was analysed by semi-quantitative RT-PCR. Detection of apoptosis was examined by flow cytometry using annexin V-FITC and PI staining. Microarray analysis was carried out to profile gene expression of inflammation-related genes.. Dose-dependent decreases in cell viability, cell cycle arrest and apoptosis in RASFs due to TSA were observed. US treatment in the presence of microbubbles increased cellular uptake, but did not induce cell cycle arrest or apoptosis. The combination of TSA and US modulated cell cycle-related gene expression and significantly decreased S phase cells and increased G(2)-M phase cells. US also further enhanced TSA-induced RASF apoptosis and regulated expression of inflammation-related genes.. HDAC inhibitor in combination with US effectively reduces cell viability and induces apoptosis in RASFs. The combination therapy could be useful to control synovial proliferation and inflammation, since US can be easily applied to targeted joints as local physiotherapy.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Cycle; Cell Survival; Cells, Cultured; Combined Modality Therapy; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Genes, cdc; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Microbubbles; Oligonucleotide Array Sequence Analysis; Polysaccharides; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Ultrasonic Therapy

Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure.. To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL-induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF).. Apoptotic cells were detected after co-treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 micromol/l) by flow cytometry using propidium iodide/annexin-V-FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor-2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA.. Co-treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids.. Because TSA sensitises RASF for TRAIL-induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.

Topics: Aged; Apoptosis; Apoptosis Regulatory Proteins; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Synergism; Female; Fibroblasts; Flow Cytometry; Humans; Hydroxamic Acids; Male; Membrane Glycoproteins; Middle Aged; Osteoarthritis; Synovial Membrane; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

To clarify the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the growth and survival of rheumatoid arthritis synovial fibroblasts (RA-SF).. Cell viability was assessed using a WST-8 assay and direct cell counting. Apoptosis was detected by annexin V staining on a flow cytometer. Protein and mRNA expression was determined by Western blotting, flow cytometry, and RT-PCR.. TSA suppressed cell growth of RA-SF in a dose-dependent manner, as determined by WST-8 assay and direct cell counting. Other histone deacetylase inhibitors also showed inhibitory effects on RA-SF proliferation. TSA upregulated p21(WAF1/CIP1) cell cycle inhibitor, suggesting that cell cycle arrest is involved in the reduction of cell numbers. In addition, TSA cooperated with Fas-induced pathway to induce cell death, determined by WST-8 assay and annexin V staining. TSA reduced FLICE inhibitory protein (FLIP) expression but not Bcl-2, Bcl-XL, and Fas expression, indicating that the synergistic effect may be through downregulation of FLIP.. TSA has antirheumatic effects on RA-SF and might be a potential therapeutic tool for the treatment of RA.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Survival; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Enzyme Inhibitors; fas Receptor; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Hydroxamic Acids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Synovial Membrane

Rheumatoid arthritis (RA) is characterized by progressive destruction of the affected joints. The pathophysiology results from genetic susceptibility and autoimmune phenomena, leading to tissue inflammation and synovial hyperplasia termed pannus, which irreversibly destroys cartilage and bone. The current treatment options, which suppress immune responses or ameliorate inflammation, do not halt the destructive process. We found that the histone deacetylase (HDAC) inhibitors (phenylbutyrate and trichostatin A) causing histone hyperacetylation to modulate multiple gene expression not only induced the expression of p21(Cip1) and p16(INK4) in synovial cells but also inhibited the expression of tumor necrosis factor-alpha in affected tissues in adjuvant arthritis, an animal model of RA. Based on the observations that joint swelling is reduced, subintimal mononuclear cell infiltration is decreased, synovial hyperplasia is inhibited, pannus formation is suppressed, and no cartilage or bone destruction is seen, the HDAC inhibitors may represent a new class of compounds for the treatment of RA by simultaneously, coordinately, synergistically, or epigenetically modulating multiple molecular targets in the pathogenesis of RA.

Topics: Acetylation; Animals; Arthritis, Rheumatoid; Blotting, Western; Cell Division; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Immunohistochemistry; Inflammation; Leukocytes, Mononuclear; Male; Phenylbutyrates; Rats; Rats, Long-Evans; Time Factors; Tumor Necrosis Factor-alpha