Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α

ObjectiveTo examine the impact of 5-Aza-2ʹ-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.ResultsAfter 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).ConclusionMethylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.     

TRPV4 Channel Inhibits TGF-β1-Induced Proliferation of Hepatic Stellate Cells

TRPV4, one of the TRP channels, is implicated in diverse physiological and pathological processes including cell proliferation. However, the role of TRPV4 in liver fibrosis is largely unknown. Here, we characterized the role of TRPV4 in regulating HSC-T6 cell proliferation. TRPV4 mRNA and protein were measured by RT-PCR and Western blot in patients and rat model of liver fibrosis in vivo and TGF-β1-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPV4 were dramatically increased in liver fibrotic tissues of both patients and CCl₄-treated rats. Stimulation of HSC-T6 cells with TGF-β1 resulted in increase of TRPV4 mRNA and protein. However, TGF-β1-induced HSC-T6 cell proliferation was inhibited by Ruthenium Red (Ru) or synthetic siRNA targeting TRPV4, and this was accompanied by downregulation of myofibroblast markers including α-SMA and Col1α1. Moreover, our study revealed that miR-203 was downregulated in liver fibrotic tissues and TGF-β1-treated HSC-T6 cell. Bioinformatics analyses predict that TRPV4 is the potential target of miR-203. In addition, overexpression of miR-203 in TGF-β1-induced HSC significantly reduced TRPV4 expression, indicating TRPV4, which was regulated by miR-203, may function as a novel regulator to modulate TGF-β1-induced HSC-T6 proliferation.     

Anti-Inflammatory Effects of Ginsenoside-Rh2 Inhibits LPS-Induced Activation of Microglia and Overproduction of Inflammatory Mediators Via Modulation of TGF-β1/Smad Pathway

Microglia activation plays an important role in neuroinflammation and contributes to several neurological disorders. Hence, inhibition of both microglia activation and pro-inflammatory cytokines may lead to the effective treatment of neurodegenerative diseases. In this study, we found that GRh2 inhibited the inflammatory response to lipopolysaccharide (LPS) and prevented the LPS-induced neurotoxicity in microglia cells. GRh2 significantly decreased the generation of nitric oxide production, and tumor necrosis factor-α, interleukin (IL)-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase in LPS-induced activated microglia cells. Furthermore, GRh2 (20 and 50 μM) significantly increased TGF-β1 expression and reduced the expression of Smad. These results suggest that GRh2 effectively inhibits microglia activation and production of pro-inflammatory cytokines via modulating the TGF-β1/Smad pathway.     

Genipin Inhibits TNF-α-Induced Vascular Smooth Muscle Cell Proliferation and Migration via Induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.     

IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.     

Inflammatory Mediators and Modulation of Blood–Brain Barrier Permeability

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood–brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed.2. Methods for measuring blood–brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved.3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions.4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling).5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood–brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized.6. Bradykinin (Bk)² increases blood–brain barrier permeability by acting on B₂ receptors. The downstream events reported include elevation of [Ca²⁺]_i, activation of phospholipase A₂, release of arachidonic acid, and production of free radicals, with evidence that IL-1 potentiates the actions of Bk in ischemia.7. Serotonin (5HT) has been reported to increase blood–brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT₂ receptors and a calcium-dependent permeability increase.8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood–brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H₂ receptors and elevation of [Ca²⁺]_i and an H₁ receptor-mediated reduction in permeability coupled to an elevation of cAMP.9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood–brain barrier permeability. The effects are mediated predominantly via a P_2U (P2Y₂) G-protein-coupled receptor causing an elevation of [Ca²⁺]_i; a P2Y₁ receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations.10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood–brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways.11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood–brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood–brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.     

Inhibition of the K+ Channel KCa3.1 Reduces TGF-β1-Induced Premature Senescence,Myofibroblast Phenotype Transition and Proliferation of Mesangial Cells

Objective

K_Ca3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K_Ca3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

Methods & Materials

Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K_Ca3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K_Ca3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05.

Results

K_ca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G₀-G₁ phase and the expression of K_ca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01).

Conclusion

Targeted disruption of K_Ca3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.     

MACC1 Down-Regulation Inhibits Proliferation and Tumourigenicity of Nasopharyngeal Carcinoma Cells through Akt/β-Catenin Signaling Pathway

The present study was aimed at investigating the expression of metastasis-associated in colon cancer 1 (MACC1) in nasopharyngeal carcinoma (NPC), its relationship with β-catenin, Met expression and the clinicopathological features of NPC, and its roles in carcinogenesis of NPC. Our results showed that MACC1 expression was higher in NPC cells and tissues than that in normal nasopharyngeal cells and chronic inflammation of the nasopharynx tissues, respectively. MACC1 expression was closely related to the clinical stage (p = 0.005) and the N classification (p<0.05) of NPC. Significant correlations between MACC1 expression and Met expression (p = 0.003), MACC1 expression and β-catenin abnormal expression (p = 0.033) were found in NPC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, and colony formation, but induced apoptosis in NPC cells compared with the control group. Furthermore, MACC1 down-regulation inhibited phosphorylated-Akt (Ser473) and β-catenin expression in NPC cells, but phosphorylated-Erk1/2 expression was not altered. Further study showed that phosphotidylinsitol-3-kinase inhibitor downregulated β-catenin and Met expression in NPC cells. There was a significant relationship between MACC1 expression and phosphorylated-Akt expression (p = 0.03), β-catenin abnormal expression and phosphorylated-Akt expression (p = 0.012) in NPC tissue, respectively. In addition, Epstein Barr virus-encoded oncogene latent membrane protein 1 upregulated MACC1 expression in NPC cells. Our results firstly suggest that MACC1 plays an important role in carcinogenesis of NPC through Akt/β-catenin signaling pathway. Targeting MACC1 may be a novel therapeutic strategy for NPC.     

Col1A1 Production and Apoptotic Resistance in TGF-β1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells

Recent studies have suggested that proliferating cholangiocytes have an important role in the induction of fibrosis, either directly via epithelial-to-mesenchymal transition (EMT), or indirectly via activation of other liver cell types. Transforming growth factor beta 1 (TGF-β1), a critical fibrotic cytokine for hepatic fibrosis, is a potent EMT inducer. This study aimed to clarify the potential contributions of TGF-β1-induced EMT-like cholangiocyte phenotype to collagen production and cell survival of cholangiocytes in vitro. Mouse cholangiocytes (603B cells) were treated with TGF-β1 and EMT-like phenotype alterations were monitored by morphological changes and expression of EMT-associated genes. Alterations in Col1A1 gene, Col1A1-associated miR-29s, and pro-apoptotic genes were measured in TGF-β1-treated 603B cells. Snail1 knockdown was achieved using shRNA to evaluate the contribution of EMT-associated changes to Col1A1 production and cell survival. We found TGF-β1 treatment induced partial EMT-like phenotype transition in 603B cells in a Snail1-dependent manner. TGF-β1 also stimulated collagen α1(I) expression in 603B cells. However, this induction was not parallel to the EMT-like alterations and independent of Snail1 or miR-29 expression. Cells undergoing EMT-like changes showed a modest down-regulation of multiple pro-apoptotic genes and displayed resistance to TNF-α-induced apoptosis. TGF-β1-induced apoptosis resistance was attenuated in Snail1 knockdown 603B cells. TGF-β1-induced Col1A1 production seems to be independent of EMT-like transition and miR-29 expression. Nevertheless, TGF-β1-induced EMT may contribute to the increased survival capacity of cholangiocytes via modulating the expression of pro-apoptotic genes.     

L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-κB Translocation in Cortical Neurons/Glia Cocultures

In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Influence of Gangliosides on the IL-2– and IL-4–Dependent Cell Proliferation

Ganglioside-induced apoptosis in the cells of IL-2–dependent cytotoxic murine cell line CTLL-2 was shown to be caspase dependent: GM1-, GM2-, and GD3-induced suppression of cell proliferation was cancelled by a general caspase inhibitor Z-VAD-FMK. Ganglioside-induced apoptosis pathways are different for different individual glycolipids; the differences exist both at the initiation and effector stages of the caspase cascade. Only for GM1-induced process, molecular mechanisms of signal transduction coincide with the ones for CD95 and TNF: the participation of both the main initiation caspases 8, 1, and 4, and caspases 3 and 9 as well, has been shown. Caspase 3 participates in the pathway induced by GM3, GD1a, GD1b, and GT1b, but not by GM2. As morphological features show, tumor-associated ganglioside GM2 is also a stimulus of programmed cell death (PCD) for CTLL-2 cell line: addition of GM2 into cell culture has resulted in appearance of annexin V-positive cells and in accumulation of DNA breaks (shown by the TUNEL direct dyeing of the open ends). But a caspase 3 inhibitor Z-DEVD-FMK did not restore the cell proliferation suppressed by GM2, and addition of a fluorescent substrate of caspase 3 Ac-DEVD-AFC did not result in the fluorescence development. So caspase 3 does not participate in downstream pathways of GM2-induced cell apoptosis, and a PCD-effector system other than the apoptosome-mediated one is involved here.     

MicroRNA-153 Inhibits Osteosarcoma Cells Proliferation and Invasion by Targeting TGF-β2

Increasing evidence indicates that microRNAs (miRNAs), a class of small noncoding RNAs, participate in almost every step of cellular processes. MiRNAs are aberrantly expressed in human cancers and contribute to cancer development and progression. Study of miRNAs may provide a new clue for understanding the mechanism of carcinogenesis and a new tool for cancer treatment. In the present study, miR-153 was downregulated in human osteosarcoma tissues and cell lines. Introduction of miR-153 mimics into the MG-63 cells inhibited cell proliferation and invasion. Our results further revealed that transforming growth factor beta 2 (TGF-β2) was negatively regulated by miR-153. Furthermore, overexpression of miR-153 decreased p-SMAD2, p-SMAD3, epidermal growth factor receptor (EGFR) and insulin-like growth factor binding protein-3 (IGFBP-3) expressions, which were the downstream signaling molecules of TGF-β. Furthermore, miRNA-153 suppressed TGF-β-mediated MG-63 proliferation and migration. Therefore, our results suggest that miR-153 may act as a tumor suppressor in osteosarcoma through targeting TGF-β2.     

Caspase-1 Cleavage of the TLR Adaptor TRIF Inhibits Autophagy and β-Interferon Production during Pseudomonas aeruginosa Infection

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β Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs β Cell Proliferation and Insulin Secretion

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Proliferation of rabbit chondrocyte and inhibition of IL-1β-induced apoptosis through MEK/ERK signaling by statins

Chondrocyte plays a critical role in endochondral ossification and cartilage repair by maintaining the cartilaginous matrix. Statins have been widely used to lower the cholesterol level in patients with cardiovascular disorders. Previous research has demonstrated potential role of statins in chondrocyte proliferation. This study addresses the proliferation-regulatory effect of lovastatin in rabbit chondrocytes as well as the underlying signaling mechanisms, thereby exploring its potential application in chondrocyte-related disorders, such as cartilage damage and osteoarthritis. Rabbit chondrocytes were treated with lovastatin at multiple concentrations, and the proliferation rate was measured by CCK-8 test. The results showed significant increase in chondrocyte proliferation under lovastatin treatment. Using real-time quantitative PCR, it was observed that the expression levels of COL2A1, SOX-9, Caspase-3, and MMP-3 genes were significantly changed by lovastatin treatment. Western blotting analysis showed that the abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Caspase-3, and MMP-3 proteins was also significantly influenced by lovastatin treatment. Interleukine-1 beta (IL-1β) is involved in the progression of osteoarthritis (OA) by inducing articular cartilage and chondrocyte aging and senescence. In this study, we observed that lovastatin treatment inhibited IL-1β-induced chondrocyte apoptosis, while the combined treatment of lovastatin and U0126 evidently offset the apoptosis-inhibiting effect of lovastatin in chondrocyte proliferation. The expressional level and protein abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, caspase-3, and MMP-3 genes showed significant alterations under the combined treatment. Together, our results suggested that lovastatin significantly promoted proliferation and inhibited the IL-1β-induced apoptosis in rabbit chondrocytes, which was mediated by the MEK/ERK signaling.     

FAS1 Domain Protein Inhibits VEGF165-Induced Angiogenesis by Targeting the Interaction between VEGFR-2 and αvβ3 Integrin

It is known that VEGF receptors (VEGFR) and integrins interact with each other to regulate angiogenesis. We reported previously that the fasciclin 1 (FAS1) domain-containing protein, TGFBIp/βig-h3 (TGF-β-induced protein) is an angiogenesis regulator that inhibits both endothelial cell migration and growth via αvβ3 integrin. In an attempt to target the interaction between VEGFR-2 and αvβ3 integrin, we determined whether the FAS1 domain region of TGFBIp/βig-h3 (FAS1 domain protein) can block the interaction between the two receptors, leading to the suppression of angiogenesis. In this study, we showed that FAS1 domain protein inhibits VEGF(165)-induced endothelial cell proliferation and migration via αvβ3 integrin, resulting in the inhibition of VEGF(165)-induced angiogenesis. We also defined a molecular mechanism by which FAS1 domain protein blocks the association between αvβ3 integrin and VEGFR-2, showing that it binds to αvβ3 integrin but not to VEGFR-2. Blocking the association of these major angiogenic receptors with FAS1 domain protein inhibits signaling pathways downstream of VEGFR-2. Collectively, our results indicate that FAS1 domain protein, in addition to its inhibitory effect on αvβ3 integrin-mediated angiogenesis, also inhibits VEGF(165)-induced angiogenesis. Thus, FAS1 domain protein can be further developed into a potent anticancer drug that targets two principal angiogenic pathways. Mol Cancer Res; 10(8); 1010-20. ?2012 AACR.