Mingcheng Huang

Bromodomain and extra-terminal domain bromodomain inhibition prevents synovial inflammation via blocking IκB kinase–dependent NF-κB activation in rheumatoid fibroblast-like synoviocytes

OBJECTIVE To explore the roles of the bromodomain (Brd) and extra-terminal domain (BET) of chromatin adaptors in regulating synovial inflammation in RA. METHODS Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissue from RA patients. A specific BET inhibitor, JQ1, and short hairpin RNA (shRNA) for Brd2 or Brd4 were used to evaluate the role of the BET Brd in inflammatory responses. Protein expression was measured by western blot or immunofluorescence staining. Nuclear factor kappa B (NF-κB) gene activity was detected by luciferase assay. The secretion and gene expression of cytokines and MMPs were evaluated by ELISA and real-time PCR, respectively. FLS proliferation was detected by BrdU incorporation. RESULTS Four Brd proteins, including Brd2, Brd3, Brd4 and Brdt, were expressed in FLSs from patients with RA and OA; however, the expression of Brd2 and Brd4 was increased in RA compared with that in OA. Treatment with JQ1, Brd2 shRNA or Brd4 shRNA decreased the production of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and IL-8), MMPs expression (MMP-1, MMP-3 and MMP-13) and proliferation by RA FLSs. BET inhibition downregulated TNFα-induced NF-κB-dependent transcription and expression of the NF-κB target genes. JQ1 suppressed the phosphorylation of IκB kinaseβ and IκBα, and nuclear translocation of p65. Intraperitoneal injection of JQ1 in mice with collagen-induced arthritis reduced synovial inflammation, joint destruction and serum levels of the anti-CII antibodies TNFα and IL-6. CONCLUSION This study implicates BET Brds as important regulators of IκB kinase/NF-κB-mediated synovial inflammation of RA and identifies BET proteins as novel therapeutic targets in inflammatory arthritis.

Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

BET Bromodomain Suppression Inhibits VEGF-induced Angiogenesis and Vascular Permeability by Blocking VEGFR2-mediated Activation of PAK1 and eNOS

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a critical modulator of angiogenesis. Increasing evidence indicate the important role of bromodomain and extra-terminal domain (BET) of chromatin adaptors in regulating tumor growth and inflammatory response. However, whether BET proteins have a role in angiogenesis and endothelial permeability is unclear. In this study, we observed that treatment with JQ1, a specific BET inhibitor, suppressed in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis in a Matrigel plug and oxygen-induced retinopathy neovascularization. JQ1 attenuated the VEGF-induced decrease in TEER in HUVECs and prevented Evans blue dye leakage in the VEGF-induced Miles assay in athymic Balb/c nude mice. BET inhibition with JQ1 or shRNA for Brd2 or Brd4 suppressed VEGF-induced migration, proliferation, and stress fiber formation of HUVECs. Furthermore, BET inhibition suppressed phosphorylation of VEGFR2 and PAK1, as well as eNOS activation in VEGF-stimulated HUVECs. Inhibition with VEGFR2 and PAK1 also reduced migration and proliferation, and attenuated the VEGF-induced decrease in TEER. Thus, our observations suggest the important role of BET bromodomain in regulating VEGF-induced angiogenesis. Strategies that target the BET bromodomain may provide a new therapeutic approach for angiogenesis-related diseases.

Inhibitory effects of niclosamide on inflammation and migration of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

ObjectivesThis study evaluated the anti-inflammatory effect of niclosamide in tumor necrosis factor (TNF)-α-stimulated human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and inhibitory effects on migration and invasion in RA FLS and investigated the signal mechanism, and further explored the treatment activity of niclosamide on collagen-induced arthritis (CIA).MethodsThe levels of interleukin (IL)-1β, IL-6, IL-8, IL-10,IL-17A and interferon (IFN)-γ in cultural supernatants were measured by multiplex cytokine assay kits. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction proteins expression was measured by western blot. The in vivo suppressive effects of niclosamide were elucidated on CIA in a mouse model.ResultsNiclosamide reduced the secretion of IL-1β, IL-6, IL-8, IL-17A and IFN-γ from TNF-α-induced RA FLS in a dose-dependent manner. Niclosamide inhibits FBS-induced migration and invasion and exhibits F-actin alterations in RA FLS. Niclosamide decreased the phosphorylation of c-Jun N-terminal kinase and ERK in TNF-α-stimulated RA FLS and blocked TNF-α-induced IKK, IκBα phosphorylation and translocation of p65. Niclosamide treatments reduced the severity of CIA model.ConclusionsOur data suggest for the first time that niclosamide posses the anti-inflammatory effect in RA both in vitro and in vivo.

Increased phosphorylation of ezrin is associated with the migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE Increasing evidence indicates that the cytoskeletal protein ezrin may play a critical role in cell motility. This study aims to investigate the role of ezrin in regulating the migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with RA. METHODS Synovial tissues were obtained from 12 patients with RA and 6 with OA, and then FLSs were separated from synovial tissues. The expression of ezrin and phosphorylated ezrin (p-ezrin) was examined by Western blotting or IF staining. A specific inhibitor of ezrin phosphorylation and small interference RNA-mediated ezrin knockdown were used to inhibit the phosphorylation of ezrin. Migration and invasion of FLSs in vitro were measured by the Boyden chamber assay. RESULTS Increased expression of p-ezrin protein was found in synovial tissue and FLSs in patients with RA compared with patients with OA. Stimulation with TNF-α and IL-1β increased ezrin phosphorylation in RA FLSs. Inhibition of p-ezrin protein by a specific inhibitor of phosphorylation of ezrin and small interfering RNA-mediated knockdown reduced in vitro migration and invasion, as well as actin stress fibre formation in RA FLS. Furthermore, rho kinase and p38 mitogen-activated protein kinase (MAPK) signal pathways were involved in the phosphorylation of ezrin and invasion of RA FLSs. CONCLUSION Increased expression of p-ezrin may contribute to aberrant aggressive behaviours of RA FLSs, which are mediated by rho kinase and the p38 MAPK pathway. This suggests a novel strategy targeting phosphorylation of ezrin to prevent synovial invasiveness and joint destruction in RA.

Inhibition of 6‐phosphofructo‐2‐kinase suppresses fibroblast‐like synoviocytes‐mediated synovial inflammation and joint destruction in rheumatoid arthritis

Abnormal glycolytic metabolism contributes to joint inflammation in rheumatoid arthritis (RA). The aims of this study were to investigate the role of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase 3 (PFKFB3), a bifunctional enzyme that controls the glycolytic rate, in regulating fibroblast‐like synoviocyte (FLS)‐mediated synovial inflammation and invasiveness in RA.

Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis

Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow–derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4+ T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

Niclosamide induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes.

To explore the effects of niclosamide on the viability and apoptosis of rheumatoid arthritis of fibroblast-like synoviocytes (rheumatoid arthritis (RA)fibroblast-like synoviocytes (FLS)), FLS obtained from RA patients were treated with niclosamide. Niclosamide significantly inhibited the viability of RA FLS in a concentration-dependent manner. Niclosamide treated FLS showed a significant increase in the percentage of apoptosis and higher intracellular ROS levels. N-acetyl-l-cysteine (NAC) pretreatment significantly attenuated niclosamide-induced apoptosis. The apoptotic response was due to the up-regulation of pro-apoptotic protein, Bax, and down-regulation of antiapoptotic protein, B cell lymphoma 2 (Bcl-2). The activation of mitochondrial pathway in niclosamide-treated RA FLS induced the cytochrome C, cleavage of caspase-9 and caspase-3. Additionally, niclosamide inhibited the phosphorylation of Akt. Collectively, our results reveal that niclosamide inhibits cell proliferation and induces mitochondrial apoptosis of RAFLSs, which is associated with the modulation of Akt signaling pathways.

Role of protein arginine methyltransferase 5 in inflammation and migration of fibroblast-like synoviocytes in rheumatoid arthritis.

To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast‐like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme‐linked immunosorbent assay (ELISA) and real‐time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up‐regulated by stimulation with IL‐1β and TNF‐α. Inhibition of PRMT5 by EPZ015666 and siRNA‐mediated knockdown reduced IL‐6 and IL‐8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseβ and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF‐κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.

The suppression of bromodomain and extra‐terminal domain inhibits vascular inflammation by blocking NF‐κB and MAPK activation

There is increasing evidence indicating that bromodomain and extra‐terminal domain (BET) proteins play a critical role in the regulation of immune and inflammatory responses; however, their contribution to vascular inflammation has not yet been elucidated. In this study, we investigated the effect of inhibiting BET bromodomain on vascular inflammation and the underlying mechanisms.