Chia-Ron Yang

Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A, a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action

TL1A is a member of the tumor necrosis factor superfamily and plays an important role in regulating endothelial cell apoptosis. A previous study shows TL1A is able to interact with death receptor 3 and decoy receptor 3 (DcR3). Here, we demonstrate that DcR3 is able to induce angiogenesis in human umbilical vein endothelial cells (HUVECs). DcR3 promotes HUVEC proliferation and migration and up-regulates matrix metalloproteinase-2 mRNA expression and enzyme activity. Furthermore, DcR3 enhances EC differentiation into cord vascular-like structures in vitro, as well as neovascularization in vivo. The effects of DcR3 on HUVECs are also mimicked by anti-TL1A and antideath receptor 3 antibodies. In contrast, human aortic endothelial cells, which do not express TL1A, are not responsive to DcR3 treatment, including cell proliferation, migration, and angiogenic differentiation. These data demonstrate DcR3 might not only help tumor cells to escape immune surveillance but also induce angiogenesis by blocking TL1A action in endothelial cells. The pathological role of DcR3 in promoting cancer progress raises the possibility to target DcR3 for antiangiogenic therapy in the future.

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells

AbstractRecent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-α release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-α synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

Decoy Receptor 3 Increases Monocyte Adhesion to Endothelial Cells via NF-κB-Dependent Up-Regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 Expression

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT and TL1A, is highly expressed in cancer cells. We show that pretreatment of HUVECs with DcR3 enhances the adhesion of THP-1 and U937 cells and primary monocytes. A similar stimulatory effect of DcR3 on THP-1 adhesion was also observed in human microvascular endothelial cells (HMVECs). Flow cytometry and ELISA showed that DcR3-treated HUVECs exhibited significant increases in ICAM-1 and VCAM-1 expression. We also demonstrate the ability of DcR3 to stimulate the secretion of IL-8 by HUVECs. RT-PCR and reporter assays revealed that the expression of adhesion molecules and IL-8 are regulated at the level of gene transcription. Experiments with pyrrolidine dithiocarbamate indicated the involvement of an NF-κB signaling pathway. DcR3 was found to induce IκB kinase activation, IκB degradation, p65 nuclear translocation, and NF-κB DNA-binding activity. The enhancement by DcR3 of cell adhesion to HUVECs was not mimicked by the TL1A-Ab, which has been shown in our previous work to be a neutralizing Ab against TL1A, thereby inducing HUVECs angiogenesis. Moreover, DcR3-induced cell adhesion could be detected in human aortic endothelial cells (ECs) in which TL1A expression is lacking. Together, our data demonstrate that DcR3 increases monocyte adhesion to ECs via NF-κB activation, leading to the transcriptional up-regulation of adhesion molecules and IL-8 in ECs. This novel action appears not to be due to TL1A neutralization, but occurs through an as yet undefined target(s). This study implicates DcR3 in the relationship between inflammation and cancer development.

Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways

ObjectivesNatural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages.MethodsWe used nitrate and prostaglandin E2 (PGE2) assays to examine inhibitory effect of aciculatin on nitric oxide (NO) and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis.ResultsAciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM). Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).ConclusionOur results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1β signaling in osteoarthritis fibroblast-like synoviocytes

OBJECTIVE MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). METHOD MiRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. RESULTS IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. CONCLUSIONS Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

Intracerebral Transplantation of Neural Stem Cells Combined With Trehalose Ingestion Alleviates Pathology in a Mouse Model of Huntington's Disease

The present investigation examined the neuroprotective benefits for combined trehalose administration with C17.2 neural stem cell transplantation in a transgenic mouse model of Huntingtons disease (HD), R6/2. C17.2 neural stem cells have the potential of differentiating into a neuronal phenotype in vitro and have been shown to be effective in the treatment of a variety of lysosomal lipid storage disorders in the nervous system. In this study, we transplanted these cells into the lateral ventricle of R6/2 transgenic mice in order to examine the efficacy of using these cells for correcting the accumulated polyglutamine storage materials in HD. To improve efficacy, animals were fed with a diet rich in trehalose, which has been shown to be beneficial to retard aggregate formation. The combined treatment strategy not only decreased ubiquitin‐positive aggregation in striatum, alleviated polyglutamine aggregation formation, and reduced striatal volume, but also extended life span in the R6/2 animal model. Behavioral evaluation showed that the combination treatment improved motor function. Statistical analysis revealed that the combination treatment was more effective than treatment with trehalose alone on the basis of the above biochemical and behavioral criteria. This study provides a strong a basis for further developing an effective therapeutic strategy for HD.

Denbinobin upregulates miR-146a expression and attenuates IL-1β-induced upregulation of ICAM-1 and VCAM-1 expressions in osteoarthritis fibroblast-like synoviocytes

Interleukin-1β (IL-1β) upregulates intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions in osteoarthritis fibroblast-like synoviocytes (OA-FLS) via nuclear factor (NF)-κB-mediated mechanism; enhancement of leukocyte infiltration and upregulation of proinflammatory mediators play a crucial role in OA pathophysiology. MicroRNA (miR)-146a suppresses inflammatory responses by inhibiting NF-κB activity and target gene expression, and epigenetic mechanisms are reportedly involved in miR expression regulation. Here, we aimed to verify the inhibition of ICAM-1/VCAM-1 expression in OA-FLS on denbinobin treatment and to determine whether this inhibition was due to the miR-146a-dependent pathway. We also assessed the epigenetic regulation caused by histone acetyltransferases involved in denbinobin action. Denbinobin attenuated the upregulation of IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS. The mechanism underlying the inhibitory effects of denbinobin involved miR-146a induction, which in turn inhibited NF-κB signaling. This is because miR-146a inhibitor abrogated the inhibitory effects of denbinobin. Furthermore, histone acetyltransferase inhibitor attenuated the denbinobin-induced upregulation of miR-146a expression and inhibited the acetylation of NF-κB-binding sites located within the miR-146a promoter region. These data suggest that an epigenetic mechanism plays a crucial role in the upregulation of miR-146a expression in response to denbinobin treatment. Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.Key message• Denbinobin inhibited IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS.• It was due to denbinobin increased miR-146a level, which in turn inhibited NF-κB signaling.• Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.

Decoy Receptor 3 Expression in AsPC-1 Human Pancreatic Adenocarcinoma Cells via the Phosphatidylinositol 3-Kinase-, Akt-, and NF-κB-Dependent Pathway

Many cancers develop different means of escaping destruction by the immune system, such as resistance to Fas ligand (FasL)-Fas interaction-mediated apoptotic signals. Decoy receptor 3 (DcR3), a soluble receptor for FasL, is highly expressed in cancer cells and plays a significant role in immune suppression and tumor progression. However, how DcR3 expression is modulated is unclear. In this study, immunoprecipitation and ELISA using human pancreatic cancer cells showed the presence of high levels of DcR3 protein in AsPC-1 cells, but not in PANC-1 cells. Treatment with herbimycin A (a tyrosine kinase inhibitor), LY294002 or wortmannin (PI3K inhibitors), pyrrolidine dithiocarbamate (an NF-κB inhibitor), or AG1024 (an insulin-like growth factor-1 inhibitor) significantly reduced endogenous DcR3 levels in AsPC-1 cells. Furthermore, transfection of AsPC-1 cells with Akt or IκBα dominant-negative plasmids also markedly reduced DcR3 levels. In contrast, 48-h transfection of PANC-1 cells with a constitutively active Akt induced DcR3 expression. Flow cytometry assays indicated that apoptosis was not seen in AsPC-1 cells incubated with soluble FasL or membrane-bound FasL, but was seen when DcR3 small interfering RNA-transfected AsPC-1 cells underwent the same treatment. In addition, PANC-1 cell incubation with conditioned medium from AsPC-1 cells transfected with dominant-negative Akt or IκBα plasmids or DcR3 small interfering RNA showed increased soluble FasL-mediated apoptosis compared with the control group. Our results show that insulin-like growth factor-1-induced activation of the PI3K/Akt/NF-κB signaling pathway is involved in the modulation of endogenous DcR3 expression in AsPC-1 cells, and that reducing endogenous DcR3 levels increases FasL-induced apoptosis of human pancreatic cancer cells.

Potent anti-inflammatory effects of denbinobin mediated by dual inhibition of expression of inducible no synthase and cyclooxygenase 2.

Inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) have been suggested to play important roles in various inflammatory diseases. We explored the anti-inflammatory potential of a natural compound, denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone), by examining its effects on the expression and activity of iNOS and COX-2 in LPS-activated macrophages. Denbinobin markedly decreased the LPS (1 &mgr;g/mL)-induced increase in iNOS and COX-2 gene and protein expression, as well as levels of the downstream products NO and prostaglandin E2, in a concentration-dependent manner (0.3-3 &mgr;M). In clarifying the mechanisms involved, denbinobin was found not only to inhibit LPS-induced nuclear factor &kgr;B (NF-&kgr;B) activation, an effect highly correlated with its inhibitory effect on LPS-induced inhibitory &kgr;B kinase activation, inhibitory &kgr;B degradation, NF-&kgr;B phosphorylation, and binding of NF-&kgr;B to the &kgr;B motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of mitogen-activated protein kinases. Reporter gene assays and Western blotting revealed that denbinobin significantly suppressed NF-&kgr;B activation. Furthermore, denbinobin also downregulated the LPS-mediated CD14/toll-like receptor 4 complex level and TNF-&agr;, IL-1&bgr;, and IL-10 mRNA expression. Our results demonstrate that denbinobin exerts potent anti-inflammatory activity, suggesting that it might provide a new therapeutic approach to inflammatory diseases.

Thiazolidinediones inhibit TNF-alpha-mediated osteoclast differentiation of RAW264.7 macrophages and mouse bone marrow cells through downregulation of NFATc1.

TNF-&agr; plays critical roles in bone-resorbing diseases, such as rheumatoid arthritis. Recent evidence suggests that thiazolidinediones (TZDs), which are peroxisome proliferator-activated receptor &ggr; agonists, have anti-inflammatory effects. The aim of this study was to evaluate the effect of TZDs on the TNF-&agr;-mediated osteoclastogenesis of osteoclast precursor cells. TNF-&agr; treatment of RAW264.7 murine macrophages or mouse bone marrow cells induced significant multinuclear osteoclast formation, and these differentiated osteoclast cells possessed bone-resorbing activity. The TZD drugs, rosiglitazone and pioglitazone, significantly inhibited TNF-&agr;-induced osteoclast differentiation from both cell types and subsequent bone resorption. Reverse transcriptase-polymerase chain reaction, reporter gene assays, and Western blot results revealed that TZD treatment significantly suppressed NFATc1 expression. Moreover, GW9662 (a peroxisome proliferator-activated receptor &ggr; antagonist) prevented the inhibitory effect of TZDs on NFATc1 expression and osteoclast differentiation. In summary, our results demonstrate that TZDs inhibit TNF-&agr;-mediated osteoclast differentiation by downregulation of NFATc1 expression. This observation increases the therapeutic applications of TZDs in inflammatory bone-resorbing diseases.