Immunopharmacology and InflammationEupatolide inhibits lipopolysaccharide-induced COX-2 and iNOS expression in RAW264.7 cells by inducing proteasomal degradation of TRAF6
Introduction
Inflammation is a central feature of many pathophysiological conditions in response to tissue injury and host defenses against invading microbes. Proinflammatory cells, mainly activated macrophages, mediate most of the cellular and molecular pathophysiology of inflammation by producing cytokines and other pro-inflammatory molecules, including prostaglandins, enzymes, and free radicals such as NO (Laskin and Pendino, 1995). Activated macrophages express iNOS, which catalyzes the oxidative deamination of L-arginine to produce large amounts of NO (Xie and Nathan, 1994). The aberrant release of NO can provoke deleterious consequences such as septic shock and inflammatory diseases (Zamora et al., 2000). COX exists as two isoforms, COX-1 and COX-2, and is the rate-limiting enzyme in the synthesis of dienoic eicosanoids such as PGE2. COX-2 is induced by proinflammatory stimuli, including bacterial LPS, and produces pro-inflammatory prostaglandins at the inflammatory site (Akira and Takeda, 2004).
LPS triggers several inflammatory reactions by binding to its specific receptor, toll-like receptor 4 (TLR4), in various cells including macrophages. Ligation of LPS to TLR4 leads to signal activation via downstream signaling factors, which include the adaptors myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-associated kinases (IRAKs), TRAF6 and transforming growth factor-β-activated kinase-1 (TAK1), and thereafter activates NF-κB and the MAPKs pathways (Akira & Takeda, 2004, Akira et al., 2001). TRAF6 is an essential component for the LPS-induced activation of signaling pathways. TRAF6 is a RING-finger-dependent E3 ubiquitin ligase which is itself activated by ubiquitination through a Lys63-ubiquitinated mechanism (Akira & Takeda, 2004, Akira et al., 2001, Deng et al., 2000).
NF-κB and MAPKs signaling pathways are key regulators in the expression of inflammatory mediators including COX-2 and iNOS (Pouliot et al., 1997, Zhang & Ghosh, 2001). In unstimulated cells, the dimeric form of NF-κB is trapped in the cytoplasm by physical association with an inhibitory protein, IκB (Baeuerle & Baltimore, 1988, Baldwin, 1996). Many stimuli, including LPS, cytokines, and viruses, activate NF-κB via several signal transduction pathways, leading to the phosphorylation of IκB by a multisubunit IκB kinase (IKK). Phosphorylated IκB becomes rapidly ubiquitinated and degraded by the proteasome complex (Scherer et al., 1995). Following IκB degradation, NF-κB is translocated to the nucleus. Activation of MAPKs contributes to the production of inflammatory mediators such as COX-2 and iNOS in activated macrophages (Ajizian et al., 1999, Carter et al., 1999), and specific MAPKs inhibitors suppress COX-2 and iNOS expression (Chan & Riches, 1998, Chen et al., 1999).
In the present study, as part of our continuing search to identify anti-inflammatory compounds from natural products, we have identified eupatolide as a potent inhibitor of iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells. We describe for the first time that eupatolide is a novel anti-inflammatory natural compound that blocks LPS-induced NF-κB and MAPKs pathways by inducing proteasomal degradation of TRAF6.
Section snippets
Cell culture and chemicals
RAW264.7 cells and human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's Modified Essential Medium supplemented with penicillin (100 units/ml) -streptomycin (100 μg/ml) and 10% heat-inactivated fetal bovine serum. U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), MG132 (carbobenzoxy-Leu-Leu-leucinal) were purchased from Calbiochem
Eupatolide inhibits production of NO and PGE2 in LPS-stimulated RAW264.7 cells
To investigate the anti-inflammatory effect of eupatolide, we first determined whether eupatolide inhibits LPS-induced production of NO and PGE2. RAW264.7 cells were stimulated with 1 μg/ml of LPS for 24 h in the presence of increasing concentrations of eupatolide, and the amount of NO and PGE2 in the culture supernatant was measured (Fig. 2). Eupatolide inhibited the LPS-induced production of NO and PGE2 with an IC50 value of 2.2 ± 0.15 μM and 3.1 ± 0.24 μM, respectively, concentrations that did not
Discussion
Inula britannica (compositae) is a traditional medicinal plant used to treat bronchitis, digestive disorders, and inflammation, in China, Korea, and Japan (Bensky and Gamble, 1993). Despite its various pharmacological activities, the molecular mechanism has not been sufficiently explained. Here, we identified eupatolide, a sesquiterpene lactone, as an inhibitor of COX-2 and iNOS expression, and investigated how this compound suppressed COX-2 and iNOS expression.
Eupatolide suppressed the
Acknowledgments
This work was supported by grants from the Regional Research Universities Program/Medical & Biomaterial Research Center and the Vascular System Research Center [No. 2009-0062786] funded by the Korea government [MEST].
References (33)
- et al.
The p38 mitogen-activated protein kinase is required for NF-κB-dependent gene expression. The role of TATA-binding protein (TBP)
J. Biol. Chem.
(1999) - et al.
Potential role of the JNK/SAPK signal transduction pathway in the induction of iNOS by TNF-α
Biochem. Biophys. Res. Commun.
(1998) - et al.
Hsp70 inhibits lipopolysaccharide-induced NF-κB activation by interacting with TRAF6 and inhibiting its ubiquitination
FEBS Lett.
(2006) - et al.
Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitinconjugating enzyme complex and a unique polyubiquitin chain
Cell
(2000) - et al.
The IRAK-1-BCL10-MALT1-TRAF6-TAK1 cascade mediates signaling to NF-κB from Toll-like receptor 4
J. Biol. Chem.
(2006) - et al.
Organization and regulation of mitogen-activated protein kinase signaling pathways
Curr. Opin. Cell Biol.
(1999) - et al.
LPS induction of gene expression in human monocytes
Cell. Signal.
(2001) - et al.
The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL
J. Biol. Chem.
(2008) - et al.
Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response
Eur. J. Pharmacol.
(2007) - et al.
IκB kinases phosphorylate NF-κB p65 subunit on serine 536 in the transactivation domain
J. Biol. Chem.
(1999)
Myeloid differentiation factor 88-dependent transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: role of NF-κB and p38
J. Biol. Chem.
Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-γ
J. Infect. Dis.
Toll-like receptor signaling
Nat. Rev. Immunol.
Toll-like receptors: critical proteins linking innate and acquired immunity
Nat. Immunol.
IκB: a specific inhibitor of the NF-κB transcription factor
Science
The NF-κB and IκB proteins: new discoveries and insights
Annu. Rev. Immunol.
Cited by (43)
Natural product-based antiinflammatory agents
2023, Recent Developments in Anti-Inflammatory TherapyDiterpenoids isolated from the root of Salvia miltiorrhiza and their anti-inflammatory activity
2021, Natural Product ResearchLactones from the pericarps of Litsea japonica and their anti-inflammatory activities
2018, Bioorganic and Medicinal Chemistry LettersCitation Excerpt :RAW264.7 cells were stimulated with 1 μg/mL LPS for 24 h in the presence of compounds 1–18 at various concentrations (3–30 μM), and then, the levels of NO in the culture supernatants were measured using Griess reaction.26 Celastrol was used as a positive control compound with an IC50 value of 1.0 μM.27 According to the result shown in Table 3, compounds 1–10 inhibited LPS-induced NO production with IC50 values in the range of 2.9–14.0 μM, whereas the other compounds did not show significant inhibitory activities in this cell system.
Sesquiterpene Lactones: Structural Diversity and Perspectives as Anti-Inflammatory Molecules
2016, Studies in Natural Products ChemistryCitation Excerpt :Atractylenolide III (1) attenuates phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced p38 MAPK and JNK phosphorylation, NF-κB activation, and IL-6 secretion in mast cells [61]. SM905, an artemisinin (2) derivative, inhibited the phosphorylation of ERK, p-38, and JNK, and decreased TNF-α, IL-1β, and IL-6 production by LPS-stimulated mouse peritoneal macrophage line (RAW 264.7 cells) [103,117,118]. In these cases, the inhibition of MAPKs by SLs results in the reduction of cytokine production.
The genus Inula and their metabolites: From ethnopharmacological to medicinal uses
2014, Journal of Ethnopharmacology
- 1
These authors contributed equally to this work.