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Chuenmao Yang - One of the best experts on this subject based on the ideXlab platform.
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cooperation of tlr2 with myd88 pi3k and rac1 in lipoteichoic acid induced cpla2 cox 2 dependent airway inflammatory responses
American Journal of Pathology, 2010Co-Authors: Ita Lee, Chihchung Lin, Chiangwen Lee, Weihsuan Tung, Shyiwu Wang, Jwuching Shu, Chuenmao YangAbstract:Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA2 and COX-2 expression and PGE2 or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (Helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA2 and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE2 or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA2 (AACOCF3) and COX-2 (NS-398) or transfection with cPLA2 siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-κB translocation or cPLA2 phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE2 could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-κB, and ultimately induces cPLA2/COX-2–dependent PGE2 and IL-6 generation.
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involvement of mapks and nf κb in tumor necrosis factor α induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts
Arthritis & Rheumatism, 2010Co-Authors: Shuefen Luo, Hsilung Hsieh, Chihchung Lin, Rouyi Fang, Peiling Chi, Lider Hsiao, Jongshyan Wang, Chuenmao YangAbstract:Objective To investigate the roles of MAPKs and NF-κB in tumor necrosis factor α (TNFα)–induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). Methods Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-κB in TNFα-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase–polymerase chain reaction, and gene promoter assay. NF-κB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results TNFα-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-κB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-κB (Helenalin) or by transfection with their respective shRNA. TNFα-stimulated translocation of NF-κB into the nucleus and NF-κB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFα in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with Helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFα or by anti–VCAM-1 antibody before the addition of lymphocytes. Conclusion In RASFs, TNFα-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-κB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.
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tumor necrosis factor α induces mmp 9 expression via p42 p44 mapk jnk and nuclear factor κb in a549 cells
Toxicology and Applied Pharmacology, 2008Co-Authors: Chihchung Lin, Hsilung Hsieh, Hsiaowei Tseng, Chiangwen Lee, Chingyi Cheng, Chuenmao YangAbstract:Abstract Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-α (TNF-α) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-α in human A549 cells remain unclear. Here, we showed that TNF-α induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-κB (Helenalin), and transfection with dominant negative mutants of ERK2 (ΔERK) and JNK (ΔJNK), and siRNAs for MEK1, p42 and JNK2. TNF-α-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of ΔERK and ΔJNK. Furthermore, the involvement of NF-κB in TNF-α-induced MMP-9 production was consistent with that TNF-α-stimulated degradation of IκB-α and translocation of NF-κB into the nucleus which were blocked by Helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-κB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-α in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by Helenalin, U0126, or SP600125. In contrast, TNF-α-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-κB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-α-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-κB are essential for TNF-α-induced MMP-9 gene expression.
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interleukin 1β induces mmp 9 expression via p42 p44 mapk p38 mapk jnk and nuclear factor κb signaling pathways in human tracheal smooth muscle cells
Journal of Cellular Physiology, 2007Co-Authors: Kaochih Liang, Chihchung Lin, Chiangwen Lee, Weining Lin, Shuefen Luo, Chuenmao YangAbstract:Matrix metalloproteinases (MMPs) are responsible for degradation of extracellular matrix and play important roles in cell migration, proliferation, and tissue remodeling related to airway inflammation. Interleukin-1beta (IL-1beta) has been shown to induce MMP-9 production in many cell types and contribute to airway inflammatory responses. However, the mechanisms underlying MMP-9 expression induced by IL-1beta in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we investigated the roles of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB pathways for IL-1beta-induced MMP-9 production in HTSMCs. IL-1beta induced production of MMP-9 protein and mRNA in a time- and concentration-dependent manner determined by zymographic, Western blotting, and RT-PCR analyses, which was attenuated by inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK (SP600125), and NF-kappaB (Helenalin), and transfection with dominant negative mutants of MEK1/2, p38 and JNK, respectively. IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was attenuated by pretreatment with U0126, SB202190, SP600125, or transfection with these dominant negative mutants of MEK, ERK, p38 and JNK, respectively. Furthermore, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by Helenalin. Finally, the reporter gene assay revealed that MAPKs and NF-kappaB are required for IL-1beta-induced MMP-9 luciferase activity in HTSMCs. MMP-9 promoter activity was enhanced by IL-1beta in HTSMCs transfected with MMP-9-Luc, which was inhibited by Helenalin, U0126, SB202190, and SP600125. Taken together, the transcription factor NF-kappaB, p42/p44 MAPK, p38 MAPK, and JNK that are involved in MMP-9 expression in HTSMCs exposed to IL-1beta have now been identified.
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bk induced cox 2 expression via pkc δ dependent activation of p42 p44 mapk and nf κb in astrocytes
Cellular Signalling, 2007Co-Authors: Hsilung Hsieh, Huihsin Wang, Meijie Jou, Maohsiung Yen, Peter J Parker, Chuenmao YangAbstract:Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by Helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.
Thomas J. Schmidt - One of the best experts on this subject based on the ideXlab platform.
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Helenalin acetate a natural sesquiterpene lactone with anti inflammatory and anti cancer activity disrupts the cooperation of ccaat box enhancer binding protein β c ebpβ and co activator p300
Journal of Biological Chemistry, 2016Co-Authors: Anke Jakobs, Thomas J. Schmidt, Simone Steinmann, Sarah Marie Henrich, Karlheinz KlempnauerAbstract:Recent work has demonstrated pro-oncogenic functions of the transcription factor CCAAT box/enhancer-binding protein β (C/EBPβ) in various tumors, implicating C/EBPβ as an interesting target for the development of small-molecule inhibitors. We have previously discovered that the sesquiterpene lactone Helenalin acetate, a natural compound known to inhibit NF-κB, is a potent C/EBPβ inhibitor. We have now examined the inhibitory mechanism of Helenalin acetate in more detail. We demonstrate that Helenalin acetate is a significantly more potent inhibitor of C/EBPβ than of NF-κB. Our work shows that Helenalin acetate inhibits C/EBPβ by binding to the N-terminal part of C/EBPβ, thereby disrupting the cooperation of C/EBPβ with the co-activator p300. C/EBPβ is expressed in several isoforms from alternative translational start codons. We have previously demonstrated that Helenalin acetate selectively inhibits only the full-length (liver-enriched activating protein* (LAP*)) isoform but not the slightly shorter (LAP) isoform. Consistent with this, Helenalin acetate binds to the LAP* but not to the LAP isoform, explaining why its inhibitory activity is selective for LAP*. Although Helenalin acetate contains reactive groups that are able to interact covalently with cysteine residues, as exemplified by its effect on NF-κB, the inhibition of C/EBPβ by Helenalin acetate is not due to irreversible reaction with cysteine residues of C/EBPβ. In summary, Helenalin acetate is the first highly active small-molecule C/EBPβ inhibitor that inhibits C/EBPβ by a direct binding mechanism. Its selectivity for the LAP* isoform also makes Helenalin acetate an interesting tool to dissect the functions of the LAP* and LAP isoforms.
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natural sesquiterpene lactones as inhibitors of myb dependent gene expression structure activity relationships
European Journal of Medicinal Chemistry, 2013Co-Authors: Caroline Schomburg, Karlheinz Klempnauer, Wolfgang Schuehly, Fernando B Da Costa, Thomas J. SchmidtAbstract:Abstract c-myb is a proto-oncogene encoding a transcription factor which is highly expressed in hematopoietic progenitor cells. It regulates the expression of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-myb expression is known to be involved in the development of human tumors, especially certain types of leukemia and breast and colon cancer. The c-Myb protein has thus been identified as an interesting therapeutic target. We recently discovered that some sesquiterpene lactones suppress Myb-dependent gene expression which is a new mechanism for these natural products' potential anti-cancer activity. We developed a test system to screen compounds for inhibitory activity on Myb-inducible reporter gene activation. Using this system we have now investigated 60 sesquiterpene lactones for their capacity to inhibit c-Myb-dependent gene activation. The IC50 values were in a range between 0.7 and >30 μM. The furanoheliangolide goyazensolide and the pseudoguaianolide Helenalin acetate (IC50 = 0.6 and 0.7 μM, respectively) represent the most active inhibitors of c-Myb dependent gene expression found up to present. Control measurements for cell viability (MTS assay) proved that the observed activity on c-Myb dependent gene expression is not a function of cytotoxicity/unspecific cell damage. Structure–activity relationships were investigated by a QSAR approach based on flexible alignment of the most active compounds and a common pharmacophore model. These investigations resulted in a QSAR model which indicates that the potency of inhibitory activity on c-Myb-dependent transcription does not only depend on the presence of reactive Michael-acceptor features but also on their optimal spatial arrangement in the molecule.
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anti trypanosomal activity of Helenalin and some structurally related sesquiterpene lactones
Planta Medica, 2002Co-Authors: Thomas J. Schmidt, Reto Brun, Ga Nter Willuhn, Sami A KhalidAbstract:The anti-trypanosomal activity of six sesquiterpene lactones (Helenalin, mexicanin I, 11alpha,13-dihydroHelenalin acetate, chamissonolide, ivalin and isoalantolactone) against the African Trypanosoma brucei rhodesiense and American T. cruzi was investigated. All tested compounds were found active towards both parasites, the former being generally more sensitive. Helenalin was the most active compound in the series with IC50 values of 0.051 and 0.695 microM against T. brucei rhodesiense and T. cruzi, respectively. The low IC50 value for T. b. rhodesiense indicates that Helenalin type compounds may be interesting candidates for further evaluation.
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inhibitory effects of Helenalin and related compounds on 5 lipoxygenase and leukotriene c4 synthase in human blood cells
Biochemical Pharmacology, 2001Co-Authors: Susanne Tornhamre, Thomas J. Schmidt, Barbro Nasmanglaser, Inger Ericsson, Jan Ake LindgrenAbstract:Abstract The sesquiterpene lactone Helenalin, which can be isolated from several plant species of the Asteraceae family, is a potent anti-inflammatory and antineoplastic agent. In agreement, alcohol extracts of these plants are used for local external treatment of inflammatory conditions. Since leukotrienes are important mediators in inflammatory processes, the inhibitory effects of Helenalin and some derivatives on leukotriene (LT) biosynthesis were studied. Treatment of human platelets with Helenalin provoked irreversible inhibition of LTC 4 synthase in a concentration- and time-dependent manner with an ic 50 of 12 μM after a 60 min preincubation. 11α,13-DihydroHelenalin acetate was less potent. Interestingly, individual donors could be divided into two distinct groups with respect to the efficacy of Helenalin to suppress platelet LTC 4 synthase. In human granulocytes, Helenalin inhibited both the 5-lipoxygenase ( ic 50 9 μM after 60 min preincubation) and LTC 4 synthase in a concentration- and time-dependent fashion. In contrast, the drug was without effect on LTA 4 hydrolase. The GSH-containing adducts (2β- (S -glutathionyl)-2,3-dihydroHelenalin and 2β- (S -glutathionyl)-2,3,11α,13-tetra hydroHelenalin acetate) did not significantly inhibit LTC 4 synthase. The present results indicate a mechanism for the anti-inflammatory effect of Helenalin and related compounds.
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glutathione adducts of Helenalin and 11 alpha 13 dihydroHelenalin acetate inhibit glutathione s transferase from horse liver
Planta Medica, 2000Co-Authors: Thomas J. SchmidtAbstract:The 2-mono- and 2,13-bis-glutathionyl adducts of Helenalin and the 2-monoglutathionyl adduct of 11 alpha,13-dihydroHelenalin acetate were previously shown to be formed by spontaneous Michael addition at physiological pH. In living cells, glutathione (GSH) conjugation of many types of electrophilic agents is catalysed by a family of GSH S-transferase enzymes (GST). The capability of a glutathione S-transferase from horse liver to catalyze the reaction of Helenalin and other helenanolides with GSH was investigated. The enzyme did not accelerate GSH conjugation of Helenalin, 11 alpha,13-dihydroHelenalin, or 2-deacetyl-6-deoxychamissonolide. The GSH-adducts, formed by spontaneous reaction, were found to be inhibitors of this enzyme. Free Helenalin, a potent inhibitor of many enzymes containing free sulfhydryl groups, did not show any inhibitory activity on GST. It was thus demonstrated that GSH-adducts of sesquiterpene lactones possess their own specific biological activity. Two further enzymes using GSH as substrate, glutathione reductase and glyoxalase I, were not influenced by free Helenalin or its GSH-adducts.
Irmgard Merfort - One of the best experts on this subject based on the ideXlab platform.
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surface plasmon resonance analysis of nuclear factor κb protein interactions with the sesquiterpene lactone Helenalin
Analytical Biochemistry, 2010Co-Authors: Berthold Buchele, Irmgard Merfort, Waltraud Zugmaier, Oleg Lunov, Tatiana Syrovets, Thomas SimmetAbstract:Sesquiterpene lactones such as Helenalin have generally been considered as highly promising compounds for the treatment of inflammatory disorders. Although sesquiterpene lactones are known to inhibit signaling through transcription factor nuclear factor-kappaB (NF-kappaB), the nature of their molecular targets remains controversial. To characterize the interactions of Helenalin with putative target proteins, a surface plasmon resonance-based method was developed and validated to analyze the interactions of Helenalin with the NF-kappaB protein p65/RelA, with recombinant IkappaB kinases (IKKs) alpha and beta, and with the intracellular antioxidant glutathione, all immobilized on sensor chips. At pH 7.4, Helenalin is interacting with RelA (K(D)=4.8microM), yet it failed to bind either IKKalpha or IKKbeta. When DNA with NF-kappaB binding sites was immobilized on sensor chips, the binding of RelA was inhibited by Helenalin with an IC(50) of 5.0microM. At pH 8.0, Helenalin was also able to interact with reduced, but not oxidized, glutathione with a K(D) of 24microM, but no significant interaction was observed at pH 7.4. Thus, with this optimized method, we showed that the sesquiterpene lactone Helenalin interacts with the NF-kappaB protein RelA but not with IKKalpha or IKKbeta. Moreover, at physiological pH, Helenalin does not interact with glutathione to any significant extent.
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skin penetration behaviour of sesquiterpene lactones from different arnica preparations using a validated gc msd method
Journal of Pharmaceutical and Biomedical Analysis, 2007Co-Authors: Steffen Wagner, Irmgard MerfortAbstract:Abstract Preparations of Arnica montana L. are widely used for the topical treatment of inflammatory diseases. The anti-inflammatory activity is mainly attributed to their sesquiterpene lactones (SLs) from the Helenalin and 11α,13-dihydroHelenalin type. To study the penetration kinetics of SLs in Arnica preparations, a stripping method with adhesive tape and pig skin as a model was used. For the determination of SLs in the stripped layers of the stratum corneum (SC), a gas chromatography/mass spectrometry method was developed and validated. Thereby the amount of Helenalin derivatives was calculated as Helenalin isobutyrate, and 11α,13-dihydroHelenalin derivatives as 11α,13-dihydroHelenalin methacrylate. This GC-MSD method is suitable also to determine low amounts of SLs in Arnica preparations. The penetration behaviour of one gel preparation and two ointment preparations was investigated. The SLs of all preparations show a comparable penetration in and a permeation through the stratum corneum, the uppermost part of the skin. Interestingly, the gel preparation showed a decrease of the penetration rate over 4 h, whereas the penetration rate of ointments kept constant over time. Moreover, we could demonstrate that the totally penetrated amount of SLs only depends on the kind of the formulation and of the SLs-content in the formulation but not on the SLs composition or on the used extraction agent.
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new sesquiterpene lactones from arnica tincture prepared from fresh flowerheads of arnica montana
Planta Medica, 2005Co-Authors: Olha Kos, Maja T Lindenmeyer, Aurelia Tubaro, Silvio Sosa, Irmgard MerfortAbstract:Investigation of an ethanolic extract prepared from fresh Arnica montana flowers afforded three new 1,5- trans-guaianolides, of which 11alpha,13-dihydro-2-O-tigloylflorilenalin and the respective 2-O-isovaleryl derivative are reported for the first time. Additionally, three new and one known 2beta-ethoxy-2,3-dihydroHelenalin esters were isolated. GC/MS studies of the extract after a two year storage at 4 degrees C demonstrated that the latter were artefacts that had been formed by addition of ethanol to the cyclopentenone structure of Helenalin. Formation of these adducts gave compounds possessing an inhibitory activity comparable to that of 11alpha,13-dihydroHelenalin derivatives in the NF-kappaB EMSA and the IL-8 ELISA in vitro assays as well as in the in vivo croton oil-induced mouse ear edema test for one adduct, namely 2beta-ethoxy-6-O-acetyl-2,3-dihydroHelenalin. As expected, 6-O-(2-methylbutyryl)- and 6-O-methacryloyl-Helenalin exhibited a stronger activity in the NF-kappaB EMSA and IL-8 ELISA. Sesquiterpene lactones seem to be the most important NF-kappaB inhibiting compounds in the Arnica extract. Bioguided fractionation using the luciferase reporter gene assay resulted in the isolation of only moderately active compounds, such as 6-acetoxy-2,2-dimethylchroman-4-one and 10-acetoxy-8,9-epoxythymol isobutyrate.
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skin penetration studies of arnica preparations and of their sesquiterpene lactones
Planta Medica, 2004Co-Authors: Steffen Wagner, Andreas Suter, Irmgard MerfortAbstract:Alcoholic preparations of Amica montana are widely used fOT the top.ical treatment of various inflammatory diseases. Sesquiterpene lactones (SLs) are mainly responsible fOT their anti-inflammatory activity. Here we have studied the penetration kinetics of Amica tinctures prepared from dried Amica flowers originating from different chemotypes as weIl as of their respective dom inating SLs, Helenalin isobutyrate and lla.13-dihydroHelenalin acetate. Same alcoholic preparations of fresh Amica flowers and an Amica fresh plant gel were also included in the,study. We used the stripping method with adhesive tape and pig skin as a model and 'determined the quantity of SLs in the stripped layers ofthe stratum comeum (SC). Thus, we observed the penetration into and permeation through this uppermost part of the skin. Whereas isolated SLs permeate through the SC only in a very small amount, permeation of SLs was much higher when they
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the anti inflammatory sesquiterpene lactone Helenalin inhibits the transcription factor nf κb by directly targeting p65
Journal of Biological Chemistry, 1998Co-Authors: Guido Lys, Heike L Pahl, Thomas J. Schmidt, Alexander Knorre, Irmgard MerfortAbstract:The sesquiterpene lactone Helenalin is a potent anti-inflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that Helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-κB, a central mediator of the human immune response. These drugs must target a central step in NF-κB pathway, since they inhibit NF-κB induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IκB, the inhibitory subunit of NF-κB. These data contradicted our report that IκB is not detectable in Helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IκB-released, nuclear NF-κB in Helenalin-treated, tumor necrosis factor-α stimulated cells. These data show that neither IκB degradation nor NF-κB nuclear translocation are inhibited by Helenalin. Rather, we provide evidence that Helenalin selectively alkylates the p65 subunit of NF-κB. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-κB.
Hsilung Hsieh - One of the best experts on this subject based on the ideXlab platform.
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involvement of mapks and nf κb in tumor necrosis factor α induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts
Arthritis & Rheumatism, 2010Co-Authors: Shuefen Luo, Hsilung Hsieh, Chihchung Lin, Rouyi Fang, Peiling Chi, Lider Hsiao, Jongshyan Wang, Chuenmao YangAbstract:Objective To investigate the roles of MAPKs and NF-κB in tumor necrosis factor α (TNFα)–induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). Methods Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-κB in TNFα-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase–polymerase chain reaction, and gene promoter assay. NF-κB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results TNFα-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-κB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-κB (Helenalin) or by transfection with their respective shRNA. TNFα-stimulated translocation of NF-κB into the nucleus and NF-κB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFα in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with Helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFα or by anti–VCAM-1 antibody before the addition of lymphocytes. Conclusion In RASFs, TNFα-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-κB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.
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tumor necrosis factor α induces mmp 9 expression via p42 p44 mapk jnk and nuclear factor κb in a549 cells
Toxicology and Applied Pharmacology, 2008Co-Authors: Chihchung Lin, Hsilung Hsieh, Hsiaowei Tseng, Chiangwen Lee, Chingyi Cheng, Chuenmao YangAbstract:Abstract Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-α (TNF-α) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-α in human A549 cells remain unclear. Here, we showed that TNF-α induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-κB (Helenalin), and transfection with dominant negative mutants of ERK2 (ΔERK) and JNK (ΔJNK), and siRNAs for MEK1, p42 and JNK2. TNF-α-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of ΔERK and ΔJNK. Furthermore, the involvement of NF-κB in TNF-α-induced MMP-9 production was consistent with that TNF-α-stimulated degradation of IκB-α and translocation of NF-κB into the nucleus which were blocked by Helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-κB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-α in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by Helenalin, U0126, or SP600125. In contrast, TNF-α-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-κB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-α-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-κB are essential for TNF-α-induced MMP-9 gene expression.
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bk induced cox 2 expression via pkc δ dependent activation of p42 p44 mapk and nf κb in astrocytes
Cellular Signalling, 2007Co-Authors: Hsilung Hsieh, Huihsin Wang, Meijie Jou, Maohsiung Yen, Peter J Parker, Chuenmao YangAbstract:Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by Helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.
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involvement of p42 p44 mapk p38 mapk jnk and nuclear factor kappa b in interleukin 1beta induced matrix metalloproteinase 9 expression in rat brain astrocytes
Journal of Neurochemistry, 2004Co-Authors: Hsilung Hsieh, Meijie Jou, Chuenmao YangAbstract:Matrix metalloproteinase (MMP)-9 expression induced by interleukin-1beta (IL-1beta) was investigated in rat brain astrocyte-1 (RBA-1). Here we report that the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) pathways participate in the induction of MMP-9 expression by IL-1beta. Zymographic, western blotting, and RT-PCR analyses showed that IL-1beta increased expression of MMP-9 mRNA and protein, which were inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). In accordance with these findings, IL-1beta stimulated phosphorylation of p42/p44 MAPK, p38, and c-Jun N-terminal kinase (JNK), which was attenuated by U0126, SB202190, or SP600125, respectively. Furthermore, this up-regulation of MMP-9 mRNA and protein was blocked by a specific NF-kappaB inhibitor Helenalin. Consistently, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of inhibitory kappa B-alpha (IkappaB-alpha) was revealed by western blotting and immunofluorescence staining, which was blocked by Helenalin, but not by U0126, SB202190, or SP600125. Taken together, these results suggest that in RBA-1 cells, activation of p42/p44 MAPK, p38, JNK and NF-kappaB pathways is essential for IL-1beta-induced MMP-9 gene expression via transcription and translation processes. An increased understanding of the signal transduction pathways involved in IL-1beta-induced MMP-9 expression on RBA-1 may be of potential therapeutic value in the treatment of inflammatory disease.
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cooperation of tlr2 with myd88 pi3k and rac1 in lipoteichoic acid induced cpla2 cox 2 dependent airway inflammatory responses
American Journal of Pathology, 2010Co-Authors: Ita Lee, Chihchung Lin, Chiangwen Lee, Weihsuan Tung, Shyiwu Wang, Jwuching Shu, Chuenmao YangAbstract:Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA2 and COX-2 expression and PGE2 or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (Helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA2 and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE2 or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA2 (AACOCF3) and COX-2 (NS-398) or transfection with cPLA2 siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-κB translocation or cPLA2 phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE2 could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-κB, and ultimately induces cPLA2/COX-2–dependent PGE2 and IL-6 generation.
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involvement of mapks and nf κb in tumor necrosis factor α induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts
Arthritis & Rheumatism, 2010Co-Authors: Shuefen Luo, Hsilung Hsieh, Chihchung Lin, Rouyi Fang, Peiling Chi, Lider Hsiao, Jongshyan Wang, Chuenmao YangAbstract:Objective To investigate the roles of MAPKs and NF-κB in tumor necrosis factor α (TNFα)–induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). Methods Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-κB in TNFα-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase–polymerase chain reaction, and gene promoter assay. NF-κB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results TNFα-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-κB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-κB (Helenalin) or by transfection with their respective shRNA. TNFα-stimulated translocation of NF-κB into the nucleus and NF-κB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFα in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with Helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFα or by anti–VCAM-1 antibody before the addition of lymphocytes. Conclusion In RASFs, TNFα-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-κB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.
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tumor necrosis factor α induces mmp 9 expression via p42 p44 mapk jnk and nuclear factor κb in a549 cells
Toxicology and Applied Pharmacology, 2008Co-Authors: Chihchung Lin, Hsilung Hsieh, Hsiaowei Tseng, Chiangwen Lee, Chingyi Cheng, Chuenmao YangAbstract:Abstract Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-α (TNF-α) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-α in human A549 cells remain unclear. Here, we showed that TNF-α induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-κB (Helenalin), and transfection with dominant negative mutants of ERK2 (ΔERK) and JNK (ΔJNK), and siRNAs for MEK1, p42 and JNK2. TNF-α-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of ΔERK and ΔJNK. Furthermore, the involvement of NF-κB in TNF-α-induced MMP-9 production was consistent with that TNF-α-stimulated degradation of IκB-α and translocation of NF-κB into the nucleus which were blocked by Helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-κB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-α in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by Helenalin, U0126, or SP600125. In contrast, TNF-α-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-κB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-α-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-κB are essential for TNF-α-induced MMP-9 gene expression.
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interleukin 1β induces mmp 9 expression via p42 p44 mapk p38 mapk jnk and nuclear factor κb signaling pathways in human tracheal smooth muscle cells
Journal of Cellular Physiology, 2007Co-Authors: Kaochih Liang, Chihchung Lin, Chiangwen Lee, Weining Lin, Shuefen Luo, Chuenmao YangAbstract:Matrix metalloproteinases (MMPs) are responsible for degradation of extracellular matrix and play important roles in cell migration, proliferation, and tissue remodeling related to airway inflammation. Interleukin-1beta (IL-1beta) has been shown to induce MMP-9 production in many cell types and contribute to airway inflammatory responses. However, the mechanisms underlying MMP-9 expression induced by IL-1beta in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we investigated the roles of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB pathways for IL-1beta-induced MMP-9 production in HTSMCs. IL-1beta induced production of MMP-9 protein and mRNA in a time- and concentration-dependent manner determined by zymographic, Western blotting, and RT-PCR analyses, which was attenuated by inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK (SP600125), and NF-kappaB (Helenalin), and transfection with dominant negative mutants of MEK1/2, p38 and JNK, respectively. IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was attenuated by pretreatment with U0126, SB202190, SP600125, or transfection with these dominant negative mutants of MEK, ERK, p38 and JNK, respectively. Furthermore, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by Helenalin. Finally, the reporter gene assay revealed that MAPKs and NF-kappaB are required for IL-1beta-induced MMP-9 luciferase activity in HTSMCs. MMP-9 promoter activity was enhanced by IL-1beta in HTSMCs transfected with MMP-9-Luc, which was inhibited by Helenalin, U0126, SB202190, and SP600125. Taken together, the transcription factor NF-kappaB, p42/p44 MAPK, p38 MAPK, and JNK that are involved in MMP-9 expression in HTSMCs exposed to IL-1beta have now been identified.