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Induced Activation (induced + activation)
Selected AbstractsLoss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration,HEPATOLOGY, Issue 2 2007Yongzhi Cui Growth hormone controls many facets of a cell's biology through the transcription factors Stat5a and Stat5b (Stat5). However, whole body deletion of these genes from the mouse does not provide portentous information on cell-specific cytokine signaling. To explore liver-specific functions of Stat5, the entire Stat5 locus was deleted in hepatocytes using Cre-mediated recombination. Notably, Stat5-mutant mice developed fatty livers and displayed impaired proliferation of hepatocytes upon partial hepatectomy (PHx). Loss of Stat5 led to molecular consequences beyond the reduced expression of Stat5 target genes, such as those encoding suppressor of cytokine signaling 2 (SOCS2), Cish, and insulin-like growth factor 1 (IGF-1). In particular, circulating growth hormone levels were increased and correlated with insulin resistance and increased insulin levels. Aberrant growth hormone (GH)-induced activation of the transcription factors Stat1 and Stat3 was observed in mutant livers. To test whether some of the defects observed in liver-specific Stat5 deficient mice were due to aberrant Stat1 expression and activation, we generated Stat1,/, mice with a hepatocyte-specific deletion of Stat5. Concomitant loss of both Stat5 and Stat1 restored cell proliferation upon PHx but did not reverse fatty liver development. Thus the molecular underpinnings of some defects observed in the absence of Stat5 are the consequence of a deregulated activation of other signal transducers and activators of transcription (STAT) family members. Conclusion: Aberrant cytokine-Stat5 signaling in hepatocytes alters their physiology through increased activity of Stat1 and Stat3. Such cross-talk between different pathways could add to the complexity of syndromes observed in disease. (HEPATOLOGY 2007.) [source] Differential regulation of TGF-, signal in hepatic stellate cells between acute and chronic rat liver injuryHEPATOLOGY, Issue 1 2002Yoshiya Tahashi During chronic liver injury, transforming growth factor , (TGF-,) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-,,induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-, signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-, increased plasminogen activator inhibitor type 1 (PAI-1) and ,2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-, were observed in HSCs. Thereafter, Smad 7 induced by TGF-, negatively regulated the Smad 2 action. These results indicated that endogenous TGF,,mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-, signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-, signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-, signal requiring Smad 2 phosphorylation by activated TGF-, receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-, under a low level of Smad 7 could be involved in the progression of liver fibrosis. [source] Osteoblast-Derived TGF-,1 Stimulates IL-8 Release Through AP-1 and NF-,B in Human Cancer Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008Yi-Chin Fong Abstract Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-,1 is associated with osteolytic bone diseases. Materials and Methods: IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-,1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c- fos, c- jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-,B, and activator protein (AP)-1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-,B promoter in human cancer cells. Osteoblasts were transfected with TGF-,1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-,1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-,1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-,B DNA-protein binding and MAPKs after TGF-,1 treatment was shown, and TGF-,1,induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-,1, BMP-2, and IGF-1. TGF-,1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-,B on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis. [source] HSP70 interacts with TRAF2 and differentially regulates TNF, signalling in human colon cancer cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2010Shengming Dai Abstract Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNF,-induced signalling pathway have not been fully elucidated. We report here that HSP70 over-expression in human colon cancer cells can inhibit TNF,-induced NF,B activation but promote TNF,-induced activation of c-Jun N-terminal kinase (JNK) through interaction with TNF receptor (TNFR)-associated factor 2 (TRAF2). We provide evidence that HSP70 over-expression can sequester TRAF2 in detergent-soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor-interacting protein (RIP1) and I,B, kinase (IKK) signalosome to the TNFR1,TRADD complex and inhibited NF,B activation after TNF, stimuli. In addition, we found that HSP70,TRAF2 interaction can promote TNF,-induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNF,-induced activation of NF,B and JNK through interaction with TRAF2, contributing to the pro-apoptotic roles of HSP70 in TNF,-induced apoptosis of human colon cancer cells. [source] Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-,-treated human aortic endothelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2001Yung-Hsiang Chen Abstract Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-, (TNF-,)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 ,g/ml), the TNF-,-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2,±,3.2% and 80.0,±,2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 ,g/ml), 84.5,±,1.9%, 78.8,±,1.2%, 58.9,±,0.4%, 58.7,±,0.9%, and 57.4,±,0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 ,g/ml) or Sal B (5 ,g/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-,-stimulated HAECs (45.7,±,2.5% and 55.8,±,1.2%, respectively). SME or Sal B significantly inhibited TNF-,-induced activation of nuclear factor kappa B (NF-,B) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 82:512,521, 2001. © 2001 Wiley-Liss, Inc. [source] Dopamine D1 and D3 receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via NMDA receptor phosphorylationJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Hongyuan Jiao Abstract Development of drug addiction involves complex molecular changes in the CNS. The mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse. We previously showed that dopamine D1 and D3 receptors play different roles in regulating cocaine-induced MAPK activation. Although there are functional and physical interactions between dopamine and glutamate receptors, little is known regarding the involvement of D1 and D3 receptors in modulating glutamate-induced MAPK activation and underlying mechanisms. In this study, we show that D1 and D3 receptors play opposite roles in regulating N -methyl- d -aspartate (NMDA) -induced activation of extracellular signal-regulated kinase (ERK) in the caudate putamen (CPu). D3 receptors also inhibit NMDA-induced activation of the c-Jun N-terminal kinase and p38 kinase in the CPu. NMDA-induced activation of the NMDA-receptor R1 subunit (NR1), Ca2+/calmodulin-dependent protein kinase II and the cAMP-response element binding protein (CREB), and cocaine-induced CREB activation in the CPu are also oppositely regulated by dopamine D1 and D3 receptors. Finally, the blockade of NMDA-receptor reduces cocaine-induced ERK activation, and inhibits phosphorylation of NR1, Ca2+/calmodulin-dependent protein kinase II, and CREB, while inhibiting ERK activation attenuates cocaine-induced CREB phosphorylation in the CPu. These results suggest that dopamine D1 and D3 receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via phosphorylation of NR1. [source] Alcohol and Mitochondria in Cardiac Apoptosis: Mechanisms and VisualizationALCOHOLISM, Issue 5 2005György Hajnóczky Apoptosis of myocytes is likely to contribute to a variety of heart conditions and could also be important in the development of alcoholic heart disease. A fundamental pathway to apoptosis is through mitochondrial membrane permeabilization and release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. The authors' results show that prolonged exposure of cultured cardiac cells to ethanol (35 mM for 48 hr) promotes Ca2+ -induced activation of the mitochondrial permeability transition pore (PTP). PTP-dependent mitochondrial membrane permeabilization is followed by release of cytochrome c and execution of apoptosis. The authors propose that chronic ethanol exposure, in combination with other stress signals, may allow for activation of the PTP by physiological calcium oscillations, providing a trigger for cardiac apoptosis during chronic alcohol abuse. Coincidence of apoptosis promoting factors occurs in only a small fraction of myocytes, but because of the absence of regeneration, even a modest increase in the rate of cell death may contribute to a decrease in cardiac contractility. Detection of apoptotic changes that are present in only a few myocytes at a certain time in the heart is not feasible with most of the apoptotic assays. Fluorescence imaging is a powerful technology to visualize changes that are confined to a minor fraction of cells in a tissue, and the use of multiphoton excitation permits imaging in situ deep in the wall of the intact heart. This article discusses potential mechanisms of the effect of alcohol on mitochondrial membrane permeabilization and visualization of mitochondria-dependent apoptosis in cardiac muscle. [source] Rapamycin inhibits cholangiocyte regeneration by blocking interleukin-6,induced activation of signal transducer and activator of transcription 3 after liver transplantationLIVER TRANSPLANTATION, Issue 2 2010Li-Ping Chen Cholangiocyte proliferation is necessary for biliary recovery from cold ischemia and reperfusion injury (CIRI), but there are few studies on its intracellular mechanism. In this process, the role of rapamycin, a new immunosuppressant used in liver transplantation, is still unknown. In order to determine whether rapamycin can depress cholangiocyte regeneration by inhibiting signal transducer and activator of transcription 3 (STAT3) activation, rapamycin (0.05 mg/kg) was administered to rats for 3 days before orthotopic liver transplantation. The results indicated that cholangiocytes responded to extended cold preservation (12 hours) with severe bile duct injures, marked activation of the interleukin-6 (IL-6)/STAT3 signal pathway, and increased expression of cyclin D1 until 7 days after transplantation, and this was followed by compensatory cholangiocyte regeneration. However, rapamycin treatment inhibited STAT3 activation and resulted in decreased cholangiocyte proliferation and delayed biliary recovery after liver transplantation. On the other hand, rapamycin showed no effect on the expression of IL-6. We conclude that the IL-6/STAT3 signal pathway is involved in initiating cholangiocytes to regenerate and repair CIRI. Rapamycin represses cholangiocyte regeneration by inhibiting STAT3 activation, which might have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation. Insights gained from this study will be helpful in designing therapy using rapamycin in clinical patients after liver transplantation. Liver Transpl, 2010. © 2010 AASLD. [source] Mechanism of low CO2 -induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942MOLECULAR MICROBIOLOGY, Issue 1 2008Takashi Nishimura Summary The cmp operon of the cyanobacterium Synechococcus elongatus strain PCC 7942, encoding the subunits of the ABC-type bicarbonate transporter, is activated under CO2 -limited growth conditions in a manner dependent on CmpR, a LysR family transcription factor of CbbR subfamily. The 0.7 kb long regulatory region of the operon carried a single promoter, which responded to CO2 limitation. Using the luxAB reporter system, three cis -acting elements involved in the low-CO2 activation of transcription, each consisting of a pair of LysR recognition signatures overlapping at their ends, were identified in the regulatory region. CmpR was shown to bind to the regulatory region, yielding several DNA,protein complexes in gel shift assays. Addition of ribulose-1,5-bisphosphate (> 1 mM) or 2-phosphoglycolate (> 10 ,M) enhanced the binding of CmpR in a concentration-dependent manner, promoting formation of large DNA,protein complexes. Given the involvement of O2 in adaptive responses of cyanobacteria to low-CO2 conditions, our results suggest that 2-phosphoglycolate, which is produced by oxygenation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of ribulose-1,5-bisphosphate under CO2 -limited conditions, acts as the co-inducer in the activation of the cmp operon by CmpR. [source] Carbocisteine inhibits oxidant-induced apoptosis in cultured human airway epithelial cellsRESPIROLOGY, Issue 7 2009Motoki YOSHIDA ABSTRACT Background and objective: Increased oxidant levels have been associated with exacerbations of COPD, and L-carbocisteine, a mucolytic agent, reduces the frequency of exacerbations. The mechanisms underlying the inhibitory effects of L-carbocisteine on oxidant-induced COPD exacerbations were examined in an in vitro study of human airway epithelial cells. Methods: In order to examine the antioxidant effects of L-carbocisteine, human tracheal epithelial cells were treated with L-carbocisteine and exposed to hydrogen peroxide (H2O2). Cell apoptosis was assessed using a cell death detection ELISA, and the pathways leading to cell apoptosis were examined by measurement of caspase-3 and caspase-9 by western blot analysis with fluorescent detection. Results: The proportion of apoptotic cells in human tracheal epithelium was increased in a concentration- and time-dependent manner, following exposure to H2O2. Treatment with L-carbocisteine reduced the proportion of apoptotic cells. In contrast, H2O2 did not increase the concentration of LDH in supernatants of epithelial cells. Exposure to H2O2 activated caspase-3 and caspase-9, and L-carbocisteine inhibited the H2O2 -induced activation of these caspases. L-carbocisteine activated Akt phosphorylation, which modulates caspase activation, and the inhibitors of Akt, LY294002 and wortmannin, significantly reversed the inhibitory effects of L-carbocisteine on H2O2 -induced cell apoptosis. Conclusions: These findings suggest that in human airway epithelium, L-carbocisteine may inhibit cell damage induced by H2O2 through the activation of Akt phosphorylation. L-carbocisteine may have antioxidant effects, as well as mucolytic activity, in inflamed airways. [source] Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G,iGDP and G,,THE JOURNAL OF PHYSIOLOGY, Issue 14 2009Moran Rubinstein G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of G,, following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although G,i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed G,i3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed G,,. Here we show that this regulation is exerted by the ,inactive' GDP-bound G,i3GDP and involves the formation of G,i3,, heterotrimers, by a mechanism distinct from mere sequestration of G,,,away' from the channel. The regulation of basal and G,,-evoked current was produced by the ,constitutively inactive' mutant of G,i3, G,i3G203A, which strongly binds G,,, but not by the ,constitutively active' mutant, G,i3Q204L, or by G,,-scavenging proteins. Furthermore, regulation by G,i3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified G,, enhanced the binding of G,i3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ,classical' G,, effector, showing low Ibasal and strong G,,-dependent activation. Expression of G,i3G203A did not affect either Ibasal or G,,-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by G,,. Expression of G,i3GDP reduced Ibasal and restored the ability of G,, to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both G,i3GDP and G,,, while GIRK2 is a G,,-effector insensitive to G,i3GDP. [source] Ca2+ -activated myosin-ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbitTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005N. Gueguen Mitochondrial respiration rates and their regulation by ADP, AMP and creatine, were studied at different free Ca2+ concentrations (0.1 versus 0.4 ,m) on permeabilized fibre bundles of rabbit skeletal muscles differing in their myosin heavy chain profiles. Four fibre bundle types were obtained: pure types I and IIx, and mixed types IIax (approximately 50% IIa and 50% IIx fibres) and IIb+ (60% IIb fibres, plus IIx and IIa). At rest, pure type I fibres displayed a much higher apparent Km for ADP (212 ,m) than IIx fibres (8 ,m). Within the IIax and IIb+ mixed fibre bundle types, two KADPm values were observed (70 ,m and 5 ,m). Comparison between pure IIx and mixed types indicates that the intermediate Km of 70 ,m most probably corresponds to the mitochondrial affinity for ADP in IIa fibres, the lowest Km for ADP (5 ,m) corresponding to IIx and IIb types. Activation of mitochondrial creatine and adenylate kinase reactions stimulated mitochondrial respiration only in type I and IIax fibre bundles, indicating an efficient coupling between both kinases and ADP rephosphorylation in type I and, likely, IIa fibres, since no effect was observed in pure IIx fibres. Following Ca2+ -induced activation of myosin-ATPase, an increase in mitochondrial sensitivity to ADP of 45% and 250% was observed in type IIax and I bundles, respectively, an effect mostly prevented by addition of vanadate, an inhibitor of myosin-ATPase. Ca2+ -induced activation of myosin-ATPase also prevented the stimulation of respiration rates by creatine and AMP in I and IIax bundles. In addition to differential regulation of mitochondrial respiration and energy transfer systems at rest in I and IIa versus IIx and IIb muscle fibres, our results indicate a regulation of phosphotransfer systems by Ca2+ via the stimulation of myosin-ATPases in type I and IIa fibres of rabbit muscles. [source] MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytesARTHRITIS & RHEUMATISM, Issue 5 2010Nahid Akhtar Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin-1, (IL-1,) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR),based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription,PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-,B was evaluated using specific inhibitors. Results In IL-1,,stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3,-untranslated region (3,-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3,-UTR of human MMP-13 mRNA and inhibited the IL-1,,induced expression of MMP-13 protein in chondrocytes. NF-,B and MAPK activation down-regulated the expression of miR-27b. Conclusion Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1,,induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes. [source] Inhibition of NF-,B signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2010Hiroshi Okamoto Objective Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and is characterized mainly by symmetric polyarticular joint disorders. The pathologic processes are mediated by a number of cytokines, chemokines, cell adhesion molecules, and matrix metalloproteinases. The expression of most of these molecules is controlled at the transcriptional level. In addition, activation of NF-,B is involved in RA pathogenesis. This study was performed to explore the role of a novel serine/threonine kinase inhibitor, fasudil, in the control of the NF-,B activation pathway and to investigate the therapeutic effects of fasudil on arthritis development in a rat model of RA. Methods Fibroblast-like synoviocytes (FLS) from RA patients and human endothelial cells (ECs) were established and maintained. To study the role of fasudil on cytokine expression, various cytokines expressed in the RA FLS and human ECs were measured by enzyme-linked immunosorbent assay following stimulation of the cells with interleukin-1, (IL-1,) in the presence of various concentrations of fasudil. The role of fasudil on NF-,B activation was studied using a reporter gene assay, Western blotting of I,B,, immunofluorescence analysis of the p65 subunit of NF-,B, and electrophoretic mobility shift assay. The in vivo effects of fasudil on arthritis were studied in a rat adjuvant-induced arthritis (AIA) model. Results Fasudil inhibited cytokine expression in RA FLS and human ECs and also inhibited the activation of ECs, in a dose-dependent manner. Fasudil inhibited IL-1,,induced activation of NF-,B independent of the inhibition of I,B, degradation and nuclear translocation of NF-,B, and inhibited IL-1,,induced DNA binding of NF-,B. Finally, in vivo, fasudil ameliorated arthritis in rats with AIA, without any adverse effects. Conclusion Serine/threonine kinase inhibitor fasudil inhibits the development of arthritis in a rat model of RA, and also inhibits the NF-,B signaling required for binding of NF-,B to specific DNA sequences through, for example, the phosphorylation of p65, suggesting that a specific target of fasudil might be a novel NF-,B kinase. Thus, fasudil serves as a novel strategy for the treatment of RA. [source] Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and AktARTHRITIS & RHEUMATISM, Issue 3 2009Maha David-Raoudi Objective To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS). Methods The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1, (IL-1,). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by real-time polymerase chain reaction analysis. The effect of CS and IL-1, on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography. Results CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1, increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1,,induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1,,induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1,,treated FLS than in FLS treated with IL-1, alone. Conclusion CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1,,enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint. [source] CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in miceARTHRITIS & RHEUMATISM, Issue 1 2009Paula Kolar Objective CD4+CD25+ regulatory T cells (known as Treg cells) suppress unwanted and autoreactive T cell responses. Treg cells express the costimulatory molecule CTLA-4 intracellularly, but the mechanisms by which Treg cells exploit CTLA-4 signaling remain unclear. The present study was undertaken to investigate the role of CTLA-4 in controlling the homeostasis and suppressive function of Treg cells. Methods Murine Treg cells were analyzed by flow cytometry for coexpression of CTLA-4 and typical Treg cell,expressed molecules, and the influence of CTLA-4 on T cell proliferation, suppression, and apoptosis was investigated by in vitro assays. To analyze the importance of CTLA-4 in Treg cell,mediated suppression in vivo, wild-type Treg cells were transferred into CTLA-4,deficient mice displaying lymphoproliferation, and survival was monitored over time. Results A strong correlation between expression of forkhead box P3 and ex vivo expression of CTLA-4 in Treg cells was observed. Inhibition of CTLA-4 signaling in Treg cells during in vitro stimulation increased cell cycling and led to enhanced activation-induced cell death (AICD), which was mediated by CD95/CD95 ligand,induced activation of caspases. Blockade of CTLA-4 signaling resulted in impairment of the suppressive capacity of Treg cells. Despite these effects, high amounts of Treg cells persisted in CTLA-4,deficient mice. Results of transfer experiments in CTLA-4,deficient mice showed that the mice had a significantly prolonged lifespan when CTLA-4,competent Treg cells were injected. Conclusion Expression of CTLA-4 on Treg cells serves to control T cell proliferation, to confer resistance against AICD, and to maintain the suppressive function of Treg cells. [source] | |||||||||||||