Clusterin stimulates the chemotactic migration of macrophages through a pertussis toxin sensitive G-protein-coupled receptor and Gβγ-dependent pathways

Clusterin induces the expression of various chemotactic cytokines including tumor necrosis factor-α (TNF-α) in macrophages and is involved in the cell migration. According to the results of this study, clusterin induced the directional migration (chemotaxis) of macrophages based on a checkerboard analysis. The chemotactic activity of clusterin was prevented by pretreatment with pertussis toxin (PTX), indicating that the G_αi/o-protein coupled receptor (GPCR) was involved in the chemotactic response of clusterin. Clusterin-stimulated chemotaxis was abrogated in a dose-dependent manner by pretreatment with gallein (a G_βγ inhibitor), indicating the involvement of G_βγ released from the GPCR. In addition, inhibitors of phospholipase C (PLC, U73122) and phosphoinositide 3-kinase (PI3K, LY294002), the key targets of G_βγ binding and activation, suppressed chemotactic migration by clusterin. The phosphorylation of Akt induced by clusterin was blocked by pretreatment with gallein or LY294002 but not with U73122, indicating that G_βγ released from the PTX-sensitive G_i protein complex activated PLC and PI3K/Akt signaling pathways separately. The activation of cellular MAP kinases was essential in that their inhibitors blocked clusterin-induced chemotaxis, and G_βγ was required for the activation of MAP kinases because gallein reduced their phosphorylations induced by clusterin. In addition, the inflammation-induced migration of macrophages was greatly reduced in clusterin-deficient mice based on a thioglycollate-induced peritonitis model system. These results suggest that clusterin stimulates the chemotactic migration of macrophages through a PTX-sensitive GPCR and G_βγ-dependent pathways and describe a novel role of clusterin as a chemoattractant of monocytes/macrophages, suggesting that clusterin may serve as a molecular bridge between inflammation and its remodeling of related tissue by recruiting immune cells.     

TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.     

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.     

β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages

Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.     

Degraded λ-carrageenan activates NF-κB and AP-1 pathways in macrophages and enhances LPS-induced TNF-α secretion through AP-1

Background

Carrageenan (CGN), a high molecular weight sulfated polysaccharide, is a traditional ingredient used in food industry. Its degraded forms have been identified as potential carcinogens, although the mechanism remains unclear.

Methods

The effects of degraded λ-carrageenan (λ-dCGN) on murine RAW264.7 cells and human THP-1-derived macrophage cells were investigated by studying its actions on tumor necrosis factor alpha (TNF-α) secretion, Toll-like receptor 4 (TLR4) expression, and activation of nuclear factor-κb (NF-κB) and activation protein-1 (AP-1) pathways.

Results

We found that λ-dCGN was much stronger than native λ-CGN in the activation of macrophages to secrete TNF-α. Treatment of RAW264.7 cells with λ-dCGN resulted in the upregulation of TLR4, CD14 and MD-2 expressions, but it did not increase the binding of lipopolysacchride (LPS) with macrophages. Meanwhile, λ-dCGN treatment activated NF-κB via B-cell lymphoma/leukemia 10 (Bcl10) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation. In addition, λ-dCGN induced extracellular signal-regulated kinases/1/2/mitogen-activated protein kinases (ERK1/2/MAPK) and AP-1 activation. Interestingly, pretreatment of RAW264.7 cells with λ-dCGN markedly enhanced LPS-stimulated TNF-α secretion. This pretreatment resulted in the enhanced phosphorylation of ERK1/2 and c-Jun N-terminal kinase (JNK) and intensified activation of AP-1.

Conclusions

λ-dCGN induced an inflammatory reaction via both NF-κB and AP-1, and enhanced the inflammatory effect of LPS through AP-1 activation.

General significance

The study demonstrated the role of λ-dCGN to induce the inflammatory reaction and to aggravate the effect of LPS on macrophages, suggesting that λ-dCGN produced during food processing and gastric digestion may be a safety concern.     

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.     

Tenascin‐c renders a proangiogenic phenotype in macrophage via annexin II

Tenascin‐c is an extracellular matrix glycoprotein, the expression of which relates to the progression of atherosclerosis, myocardial infarction and heart failure. Annexin II acts as a cell surface receptor of tenascin‐c. This study aimed to delineate the role of tenascin‐c and annexin II in macrophages presented in atherosclerotic plaque. Animal models with atherosclerotic lesions were established using ApoE‐KO mice fed with high‐cholesterol diet. The expression of tenascin‐c and annexin II in atherosclerotic lesions was determined by qRT‐PCR, Western blot and immunohistochemistry analysis. Raw 264.7 macrophages and human primary macrophages were exposed to 5, 10 and 15 μg/ml tenascin‐c for 12 hrs. Cell migration as well as the proangiogenic ability of macrophages was examined. Additionally, annexin II expression was delineated in raw 264.7 macrophages under normal condition (20% O₂) for 12 hrs or hypoxic condition (1% O₂) for 6–12 hrs. The expression of tenascin‐c and annexin II was markedly augmented in lesion aorta. Tenascin‐c positively regulated macrophage migration, which was dependent on the expression of annexin II in macrophages. VEGF release from macrophages and endothelial tube induction by macrophage were boosted by tenascin‐c and attenuated by annexin II blocking. Furthermore, tenascin‐c activated Akt/NF‐κB and ERK signalling through annexin II. Lastly, hypoxia conditioning remarkably facilitates annexin II expression in macrophages through hypoxia‐inducible factor (HIF)‐1α but not HIF‐2α. In conclusion, tenascin‐c promoted macrophage migration and VEGF expression through annexin II, the expression of which was modulated by HIF‐1α.     

Cross-linking of SIGNR1 activates JNK and induces TNF-α production in RAW264.7 cells that express SIGNR1

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-α, and the JNK inhibitor inhibited the antibody-induced TNF-α production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-α production in macrophage-like RAW264.7 cells.     

The ubiquitin-like protein MNSFbeta regulates ERK-MAPK cascade

MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.     

Honokiol: an effective inhibitor of tumor necrosis factor-α-induced up-regulation of inflammatory cytokine and chemokine production in human synovial fibroblasts

In this study, we investigated the mechanisms underlying the anti-inflammatory effects of honokiol in tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis synovial fibroblasts (RASFs). RASFs pre-treated with honokiol (0-20 μM) were stimulated with TNF-α (20 ng/ml). The levels of prostaglandin E2 (PGE2), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. In addition, protein expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated Akt, nuclear factor kappa B (NFκB), and extracellular signal-regulated kinase (ERK)1/2 were determined by western blot. The expression of NFκB-p65 was assessed by immunocytochemical analysis. TNF-α treatment significantly up-regulated the levels of PGE2, NO, sICAM-1, TGF-β1, MCP-1, and MIP-1α in the supernatants of RASFs, increased the protein expression of COX-2, iNOS, and induced phosphorylation of Akt, IκB-α, NFκB, and ERK1/2 in RASFs. TNF-α-induced expression of these molecules was inhibited in a dose-dependent manner by pre-treatment with honokiol. The inhibitory effect of honokiol on NFκB-p65 activity was also confirmed by immunocytochemical analysis. In conclusion, honokiol is a potential inhibitor of TNF-α-induced expression of inflammatory factors in RASFs, which holds promise as a potential anti-inflammatory drug.     

Role of c-Jun N-terminal protein kinase 1/2 (JNK1/2) in macrophage-mediated MMP-9 production in response to Moraxella catarrhalis lipooligosaccharide (LOS)

Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Recent reports have provided strong evidence for the presence of high levels of matrix metalloproteinase (MMPs) in effusion fluids from children suffering with OM, however, the precise mechanisms by which MMPs are generated are currently unknown. We hypothesized that MMPs are secreted from macrophages in the presence of M. catarrhalis lipooligosaccharide (LOS). In this report, we demonstrate that in vitro stimulation of murine macrophage RAW 264.7 cells with LOS leads to secretion of MMP-9 as determined by ELISA and zymogram assays. We have also shown that inhibition of ERK1/2 and p38 kinase completely blocked LOS induced MMP-9 production. In contrast, inhibition of JNK1/2 by the specific inhibitor SP600125 actually increased the level of expression and production of MMP-9 at both mRNA and protein levels, respectively by almost five fold. This latter result was confirmed by knocking down JNK1/2 using siRNA. Similar results have been observed in murine bone marrow derived macrophages in vitro. In contrast to and in parallel with the LOS-induced increased levels of MMP-9 in the presence of SP600125, we found a corresponding dose-dependent inhibition of TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) secretion. Results of subsequent in vitro studies provided evidence that when JNK1/2 was inhibited prior to stimulation with LOS, it significantly increased both the extent of macrophage cell migration and invasion compared to control cells or cells treated with LOS alone. The results of these studies contribute to an increased understanding of the underlying pathophysiology of OM with effusion in children.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

脓肿分枝杆菌经Toll样受体2介导的JNK/ERK信号通路诱导巨噬细胞表达肿瘤坏死因子α和白细胞介素8

为探讨脓肿分枝杆菌脓肿亚种和马赛亚种经Toll样受体2(Toll-like receptor 2,TLR2)介导的c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)诱导THP-1巨噬细胞内肿瘤坏死因子α(tumor necrosis factor α,TNF-α)和白细胞介素8(interleukin 8,IL-8)表达的相关分子机制,本研究将脓肿分枝杆菌脓肿亚种和马赛亚种感染THP-1巨噬细胞,细菌与巨噬细胞最佳感染之比为感染复数(multiplicity of infection,MOI)＝3,用荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测THP-1巨噬细胞感染两细菌亚种6 h后的胞内TNF-α和IL-8 mRNA水平,以及分别阻断TLR2、JNK 和ERK信号蛋白后TNF-α和IL-8 mRNA水平的变化。结果显示,脓肿分枝杆菌脓肿亚种和马赛亚种作用于THP-1巨噬细胞6 h后,均可诱导细胞内TNF-α和IL-8 mRNA水平显著上调,差异有统计学意义(P＜0.05);分别阻断TLR2、JNK和ERK信号蛋白,脓肿亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平出现明显抑制,差异有统计学意义(P＜0.05);分别阻断TLR2和JNK信号蛋白,马赛亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平均出现明显抑制,差异有统计学意义(P＜0.05);而阻断ERK信号蛋白后,马赛亚种组仅见IL-8 mRNA水平明显抑制,差异有统计学意义(P＜0.05),而TNF-α mRNA水平未见明显变化,差异无统计学意义(P＞0.05)。本研究提示,脓肿分枝杆菌脓肿亚种和马赛亚种均可作用于TLR2,诱导THP-1细胞内TNF-α和IL-8 mRNA水平上调,脓肿亚种可经JNK和ERK信号蛋白诱导TNF-α mRNA上调,马赛亚种可经JNK信号蛋白诱导TNF-α mRNA上调;脓肿亚种和马赛亚种诱导IL-8 mRNA上调可能与JNK和ERK信号蛋白相关。     

Activation of phosphatidylinositol 3-kinase is required for tumor necrosis factor-α-induced upregulation of matrix metalloproteinase-9: Its direct inhibition by quercetin

Matrix metalloproteinases (MMPs) are involved in various skin disorders, including photoaging, dermatitis, and tumorigenesis. Tumor necrosis factor (TNF)-α is a key proinflammatory cytokine that acts to provoke inflammation, proliferation, and tumorigenesis. The present study investigated the possible inhibitory effects of red wine polyphenols on TNF-α-induced upregulation of MMP-9 and on the migratory phenotype of JB6 P+ mouse epidermal (JB6 P+) cells. Red wine extract (RWE) and quercetin, which is a major flavonoid present in red wine, inhibited significantly the TNF-α-induced upregulation of MMP-9 and cell migration, whereas resveratrol did not have significant inhibitory effects. The inhibitory effects of RWE and quercetin were mediated by suppression of the phosphorylation of Akt and the transactivation of activator protein-1 and nuclear factor-κB, as determined by Western blotting and luciferase assays, respectively. Aside from Akt, quercetin had no effect on the phosphorylation of other mitogen-activated protein kinases. Direct kinase assay data revealed that RWE and quercetin inhibited phosphatidylinositol 3-kinase (PI3K) activity. The results of direct and cell-based pull-down assays demonstrated that RWE and quercetin bound to PI3K, resulting in the inhibition of PI3K activity. Using chemical inhibitors, it was confirmed that the PI3K-dependent Akt pathway was involved in TNF-α-induced MMP-9 upregulation and migration in JB6 P+ cells. Collectively, these results indicate that TNF-α-induced MMP-9 upregulation and the migratory phenotype are associated with the PI3K/Akt pathway, and that these effects are inhibited strongly by RWE and quercetin.