Extracellular signal-regulated kinase induces phosphorylation of IκBα in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>-infected gastric epithelial AGS cells

In Helicobacter pylori (H. pylori)-induced gastric ulceration, NF-κB regulates the expression of inflammatory genes. NF-κB is activated by phsophorylation of its endogenous inhibitor, IκBα. The possible involvement of mitogenactivated protein kinase (MAPK) on NF-κB activation has been suggested in various cells. Present study aims to investigate whether H. pylori in a Korean isolate induces phosphorylation of IkBα and whether H. pylori-induced phosphorylation of IkBα is mediated by MAPK in gastric epithelial AGS cells. AGS cells were treated with MAPK inhibitors (U0126 for extracellular signal-regulated kinase, SB203580 for p38 kinase, SP600125 for c-Jun NH2-terminal protein kinases) and stimulated with H. pylori. As a result, H. pylori increased phospho-specific IκBα accompanied with the decrease in control IκBα. H. pylori-induced phosphorylation of IκBα was inhibited by treatment of U0126, but not by SB203580 or SP600125. In conclusion, extracellular signal-regulated kinase induces phosphorylation of IκBα in H. pylori-infected AGS cells. Received 4 August 2006; accepted 21 August 2006     

Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-κB and MAPK activation,and acetylation of histone H3

Although inflammation acts as host defense mechanism against infection or injury and is primarily a self limiting process, inadequate resolution of inflammatory responses leads to various chronic disorders. This work aimed to elucidate the anti-inflammatory effects of 2-methoxy-4-vinylphenol (2M4VP) isolated from pine needles in LPS-stimulated RAW264.7 cells. Some key pro-inflammatory mediators including nitric oxide (NO), prostaglandins (PGE₂), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied by sandwich ELISA and western blot. In addition, suppression of NF-κB and MAPK activation, and histone acetylation was studied by western blot analysis and immunostaining. 2M4VP dosedependently inhibited NO and PGE₂ production and also blocked LPS-induced iNOS and COX-2 expression. In addition, 2M4VP potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation and the phosphorylation of MAPKs such as p38, ERK1/2, and JNK. Also, 2M4VP inhibited hyper-acetylation of histone H3 (Lys9/Lys14) induced by LPS. Taken together, our results suggest that 2M4VP, a naturally occurring phenolic compound, exert potent anti-inflammatory effects by inhibiting LPS-induced NO, PGE₂, iNOS, and COX-2 in RAW264.7 cells. These effects are mediated by suppression of NF-κB and MAPK activation and histone acetylation.     

HMGB1 increases RAGE expression in vascular smooth muscle cells via ERK and p-38 MAPK-dependent pathways

The increased expression of receptors for advanced glycation end-product (RAGE) is known as a key player in the progression of vascular remodeling. However, the precise signal pathways regulating RAGE expression in vascular smooth muscle cells (VSMCs) in the injured vasculatures are unclear. Given the importance of mitogen-activated protein kinase (MAPK) signaling in cell proliferation, we investigated the importance of MAPK signaling in high-mobility group box 1 (HMGB1)-induced RAGE expression in VSMCs. In HMGB1 (100 ng/ml)-stimulated human VSMCs, the expression of RAGE mRNA and protein was increased in association with an increase in AGE-induced VSMC proliferation. The HMGB1-induced RAGE expression was attenuated in cells pretreated with inhibitors for ERK (PD98059, 10 μM) and p38 MAPK (SB203580, 10 μM) as well as in cells deficient in ERK and p38 MAPK using siRNAs, but not in cells deficient of JNK signaling. In cells stimulated with HMGB1, the phosphorylation of ERK, JNK, and p38 MAPK was increased. This increase in ERK and p38 MAPK phosphorylation was inhibited by p38 MAPK and ERK inhibitors, respectively, but not by JNK inhibitor. Moreover, AGE-induced VSMC proliferation in HMGB1-stimulated cells was attenuated in cells treated with ERK and p38 MAPK inhibitors. Taken together, our results indicate that ERK and p38 MAPK signaling are involved in RAGE expression in HMGB1-stimulated VSMCs. Thus, the ERK/p38 MAPK-RAGE signaling axis in VSMCs was suggested as a potential therapeutic target for vascular remodeling in the injured vasculatures.     

Effect of Zanthoxylum schinifolium on TNF-α-induced vascular inflammation in human umbilical vein endothelial cells

Pro-inflammatory cytokines induce the injury of endothelial cells in response to increases of adhesion molecules, leading to vascular inflammation and the development of atherosclerosis. In this study, we evaluated an ethanol extract of Zanthoxylum schinifolium (EZS) to determine if it inhibits the expressions of cellular adhesion molecules in human umbilical vein endothelial cells (HUVEC). When pretreatment of HUVEC with EZS, EZS suppressed the expression levels of cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin induced by TNF-α. The adhesion of HL-60 cells to TNF-α-induced endothelial cells was decreased significantly in a concentration-dependent manner. Furthermore, TNF-α-induced MCP-1 and IL-8 mRNA expression levels were also attenuated by pretreatment with EZS. In addition, EZS suppressed TNF-α-induced production of reactive oxygen species (ROS). EZS inhibited NF-κB activation and IκB-α phosphorylation induced by TNF-α, subsequent degradation of IκB-α. Finally, EZS inhibited TNF-α-induced p38 MAPK and c-Jun N-terminal kinase (JNK) phosphorylation. Taken together, these results demonstrate that EZS suppresses vascular inflammatory process, which may be closely related to the inhibition of ROS, JNK, p38 MAPK and NF-κB activation in HUVEC.     

Expression of TNF-α and IL-6 in HMC-1 cells treated with bisphenol A is attenuated by plant-originating glycoprotein (75 kDa) by blocking p38 MAPK

Bisphenol A (BPA) is known as an estrogen-mimic environmental hormone which has the ability to indirectly stimulate the production of allergic inflammation-related cytokines. Cudrania tricuspidata Bureau (CTB) has been used in Korean folk medicine for a long time. In order to determine the inhibitory effect of a glycoprotein (CTB glycoprotein, 75 kDa) isolated from CTB fruits on the activities of allergic inflammation-related cytokines (TNF-α and IL-6) caused by BPA, we evaluated the activities of protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor (NF)-κB, and inflammation-related cytokine (TNF-α and IL-6) in the BPA-induced HMC-1 cells using immunoblot analysis and RT-PCR. The results obtained from this study revealed that CTB glycoprotein (100 μg/ml) inhibits the translocation of PKC from cytosol to the membrane, the phosphorylation of p38 MAPK, the activation of NF-κB, and the expression levels of TNF-α and IL-6. Taken together, the results in this study suggest that CTB glycoprotein inhibits the expression of allergic inflammation-related cytokines (TNF-α and IL-6) by blocking NF-κB and p38 kinase in BPA-induced HMC-1 cells.     

<Emphasis Type="Italic">Atractylodes japonica</Emphasis> Koidzumi inhibits the production of proinflammatory cytokines through inhibition of the NF-κB/IκB signal pathway in HMC-1 human mast cells

The rhizome of Atractylodes japonica Koidzumi (AJK) has been used in traditional medicine for treatment of arthritis, bronchitis and respiratory infectious disease, whereas its effects on inflammatory reactions have not been unknown recently. In this study, the effects of AJK on allergic inflammation and its signaling were investigated in the induced human mast cells and animal model. This study showed that ethanol extract of AJK interestingly suppressed the production and mRNA expression of TNF-α, IL-6 and IL-8, as important inflammatory cytokines. Furthermore, AJK inhibited the nuclear translocation of nuclear factor (NF)-κB through inhibition of the phosphorylation of IB-κ, which was additionally elucidated by NF-κB promoter-mediated luciferase activity. In addition, the phosphorylation of ERK was increased in pretreatment with AJK, whereas there was no change in JNK and p38 MAPK. However, AJK showed no effects on anti-DNP IgE-mediated in vivo PCA reaction and histamine release, as key events of mast cell-mediated immediate allergic reactions. These results suggest that AJK might be involved in not early-phase but transition to late-phase reactions of allergic inflammation and could modulate through other signal pathways. Taken together, AJK could be used as a treatment for mast cell mediated late-phase/chronic allergic inflammatory reactions.     

Role of mitogen-activated protein kinase family in serum-induced leukaemia inhibitory factor and interleukin-6 secretion by bone marrow stromal cells

1: In the haematopoietic microenvironment, bone marrow stromal cells play an important role in regulating haematopoiesis by expressing various cytokines, including leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6). However, the intracellular signal that regulates cytokine secretion in bone marrow stromal cells has not been determined. The aim of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) family in serum-induced secretion of LIF and IL-6 by bone marrow stromal cells. 2: Transformed human bone marrow stromal cells (HS-5) were stimulated with foetal calf serum (FCS) to produce LIF and IL-6. FCS also induced activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). 3: Both PD98059 (MAPK/ERK kinase inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated FCS-induced LIF protein production and gene expression. SB203580 decreased IL-6 production and gene expression, but PD98059 had no effect on IL-6 production and gene expression. 4: Expression of a dominant-negative mutant form of JNK1 that blocked FCS-induced JNK activity had no effect on protein production and gene expression of these cytokines. 5: These findings demonstrate that both ERK and p38 MAPK are involved in FCS-induced LIF secretion, whereas only p38 MAPK is important for IL-6 secretion, and that FCS-induced activation of JNK has no effect on the production of LIF and IL-6. We conclude that, in spite of their similar biological effects, they are differentially regulated at the level of MAPK activity in bone marrow stromal cells.     

Attenuated expression and function of the RECK tumor suppressor under hypoxic conditions is mediated by the MAPK signaling pathways

Downregulation of the tumor suppressor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been reported under hypoxic conditions (Lee et al., 2010); however, the signaling pathways involved in this downregulation have not yet been identified. Hypoxia causes the silencing of RECK mRNA expression, but treatment with inhibitors of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (MAPK) (PD98059, SP600125, and SB203580 respectively) or their dominant negative mutants recovered RECK suppression induced by hypoxia as analyzed with semiquantitative RT-PCR analysis and a RECK promoter luciferase assay. Hypoxia increased phosphorylation of ERK1/2, JNK and p38 MAPKs. The activities of matrix metalloproteinase (MMP)-9 and MMP-2 were increased under hypoxic conditions but treatment with PD98059, SP600125 and SB203580 inhibited their activation in cancer cells, as seen by zymography. Moreover, treatment with the inhibitors blocked cancer cell migration induced by hypoxia in H-Ras transformed MCF10A mammary cells. RECK suppression under hypoxic conditions was inversely related to HIF-1α expression; however, treatment with PD98059, SP600125 and SB203580 did not influence binding of HIF-1α to the reverse hypoxia responsive element site of the RECK promoter in a DNA precipitation assay. These results suggest that the ERK, JNK and p38 MAPK signaling pathways are indirectly involved in RECK suppression but are not involved in the binding activity of HIF-1α to the reverse hypoxia responsive element site on the RECK promoter under hypoxic conditions.     

Anti-inflammatory effects of pitavastatin in interleukin-1β-induced SW982 human synovial cells

The present study shows the basis for the anti-inflammatory effects of pitavastatin in interleukin (IL)-1β-induced human synovial cells. The SW982 cells were pretreated with pitavastatin at different concentrations (5 μM and 10 μM), followed by IL-1β (10 ng/mL) stimulation. The results showed that pitavastatin inhibited the expression of inflammatory mediators IL-6 and IL-8. Furthermore, pitavastatin inhibited the phosphorylation of p38, extracellular signal-related kinase (ERK), c-jun N-terminal kinase (JNK) and protein kinase B (Akt). It also suppressed the degradation of I kappa B alpha and blocked p65 translocation into the nucleus. These findings suggest that the mechanism underlying the inhibitory effects of pitavastatin on IL-1β-induced IL-6 and IL-8 release might be mediated by the suppression of mitogen-activated protein kinase (MAPK), Akt, and nuclear factor-κB (NF-κB) signaling pathways. These results may also indicate that pitavastatin may be potentially utilized as an effective therapeutic agent for the treatment of osteoarthritis.     

Flavonol-rich RVHxR from Rhus verniciflua Stokes and its major compound fisetin inhibits inflammation-related cytokines and angiogenic factor in rheumatoid arthritic fibroblast-like synovial cells and in vivo models

Rheumatoid arthritis (RA) is an aggressive inflammatory disease in which cytokines/chemokines are thought to recruit leukocytes and induce angiogenesis. The aim of this study is to investigate the effect of flavonol-rich residual layer of hexane fraction from Rhus verniciflua Stokes (RVHxR) and its major compound fisetin on inflammatory cytokine/chemokine production and angiogenic factor in IL-1β-stimulated RA fibroblast-like synovial cells (FLS) and inflammatory in vivo models. Flavonol-rich RVHxR and its major compound fisetin significantly inhibited IL-1β-induced FLS proliferation in a dose-dependent manner. Flavonol-rich RVHxR and fisetin significantly decreased IL-1β-induced inflammatory cytokines (TNF-α, interleukin (IL)-6)/chemokines (IL-8, monocyte chemoattractant protein (MCP)-1), and vascular endothelial growth factor (VEGF) of RA FLS. Flavonol-rich RVHxR dose dependently diminished the phophorylation of extracellular signal regulated kinase (ERK) and phospho-Jun NH(₂)-terminal kinase (JNK), and its down regulation induced by RVHxR at nontoxic concentrations, while activated the phosphorylation of p38 MAPK in IL-1β-stimulated RA FLS. The p38 specific inhibitor SB203580 cotreatment with RVHxR effectively increased the expression of VEGF and blocked the phosphorylation of p38 MAPK in IL-1β-stimulated RA FLS, confirming a critical role of p38 MAPK pathway in angiogenesis inhibition. In experimental inflammation-related models, flavonol-rich RVHxR and fisetin have shown significant anti-inflammatory activities on vascular permeability, leukocyte migration and cellular immunity. Also, flavonol-rich RVHxR and fisetin treatments significantly reduced the incidence and severity of collagen-induced arthritis model. These results suggest that RVHxR and its major compound fisetin have shown potent suppressive effects on some inflammatory cytokines/chemokines and angiogenic factor in IL-1β-stimulated RA FLS and inflammatory in vivo models. We believe that flavonol-rich RVHxR is a potential therapeutic agent in the treatment of inflammatory and angiogenesis related diseases.     

MAPKs signaling in 5-LOX pathway-associated inflammatory responses

5-Lipoxygenase(5-LOX) is a crucial enzyme catalyzing arachidonic acid to form cysteinyl leukotrienes(Cys LTs, including LTC4, LTD4, LTE4). Cys LTs are potent inflammatory mediators that mediate many pathophysiological responses by activating two distinct G-protein-coupled receptors(CysLT1R and CysLT2R). 5-LOX pathway is implicated in inflammatory pathological processes. Mitogenactivated protein kinases(MAPKs) are important signal transduction pathways of complex cellular processes.Classical MAPK families in mammalian cells include the P38 MAPK, extracellular signal-regulated kinases(ERK)and c-Jun amino N-terminal kinase(JNK). Increasing evidence has suggested that MAPKs signaling acts as key modulator in 5-LOX pathway-related pathological processes. It was reported that P38 MAPK and 5-LOX were dramatically activated to mediate neuronal injury in oxygen-glucose deprivation(OGD)-treated PC12 cells,which could be inhibited by P38 MAPK inhibitor SB203580 and nonselective 5-LOX inhibitor caffeic acid. In primary cultured rat astrocytes, OGD induced increased expression of CysLT2R and aquaporin 4, and ischemic astrocyte injury. Also, OGD increased phosphorylation of ERK and P38 MAPK. These astrocyte responses were attenuated by P38 MAPK inhibitor SB203580, ERK inhibitor U0126, CysLT2R antagonist Bay Cys LT2 and non-selective Cys LTR antagonist Bay u9773, but not by JNK inhibitor SP600125 and CysLT1R antagonist montelukast. These data demonstrate the roles of ERK and P38 MAPK signaling pathways in 5-LOX and CysLT2R-mediated ischemic-like injury in neuron-like PC12 cel s and rat astrocytes. Furthermore, CysLT1R and ERK signaling were found to be involved in regulation of LTD4-stimulated expression of glial fibrillary acidic protein and cell proliferation in astrocytes. ERK inhibitor PD98059 and CysLT1R antagonist montelukast, MK-571 abolished Cys LTs-induced astrocyte proliferation. Vascular endothelial cells play a pivotal role in maintaining brain homeostasis. Endothelial cells undergo a number of pathological changes that can be characterized as uncontrolled cell proliferation, migration and dysfunction in respond to brain insults. In human endothelial cell line EA.hy926, LTD4-induced migration of endothelial cells could be modulated by CysLT1R via phosphorylation of ERK. LTD4 was also reported to enhanced cell proliferation mediated by activating CysLT1R in human umbilical vein endothelial cells(HUVECs), and this is regulated by ERK pathway. ERK inhibitor(U0126, PD98059), CysLT1R antagonist(montelukast, MK571) and dual antagonist(Bay u9773)suppressed LTD4-induced endothelial responses in EA.hy926 cells and HUVECs. Consistent with these results,a study showed that the involvement of ERK/early growth response-1(Egr-1) pathway in CysLT2R-mediated cytokine IL-8 production. ERK inhibitor U0126 and CysLT2R antagonist HAMI3379 inhibited Egr-1 and IL-8 expression as well as IL-8 release in LTC4 and LTD4-stimulated hCys LT2-HEK293 cells. In addition, LTD4 and LTC4 induced monocyte chemoattractant protein-1(MCP-1) by enhanced CysLT1R via phosphorylations of ERK and JNK in human monocytes/macrophages. Pretreatment with the ERK inhibitor PD98059 and JNK inhibitor SP600125 reduced MCP-1 production. CysLT1R antagonist pranlukast inhibited Cys LTs-induced phosphorylation of ERK and JNK as well as production of MCP-1. A better understanding of the roles of MAPKs signaling in 5-LOX pathway-associated pathological processes may open up new avenues for the development of therapeutic strategies targeting inflammatory diseases.     

Up-regulation of BDNF in Astrocytes by TNF-α: A Case for the Neuroprotective Role of Cytokine

Tumor necrosis factor-alpha (TNF-α) is widely known to be involved in physiological and pathophysiological processes of the brain where this proinflammatory cytokine is implicated with regulation of inflammatory and survival components. We report that TNF-α up-regulates exon-IV-bdnf mRNA and brain-derived neurotrophic factor (BDNF) protein in primary astrocytes. The BDNF protein was detectable both in cellular lysate and in the extracellular medium. Activation of NF-κB by TNF-α and inhibition of TNF-α-induced BDNF expression by Δp65 (a dominant-negative mutant) and NEMO-binding domain peptide (an inhibitor of NF-κB) suggests that TNF-α induces BDNF expression through the activation of NF-κB. Similarly, TNF-α induced the activation of C/EBPβ and the expression of BDNF was sensitive to overexpression of ΔC/EBPβ (a dominant-negative mutant) and ETO (an inhibitor of C/EBPβ). Among three MAP kinases, TNF-α-induced BDNF up-regulation was sensitive only to inhibitors of ERK MAP kinase. However, the ERK MAP kinase pathway was coupled to activation of C/EBPβ but not NF-κB. Taken together, this study identifies a novel property of TNF-α in inducing the expression of BDNF via NF-κB and C/EBPβ in astrocytes that may be responsible for neurotrophic activity of the cytokine.     

Hydrogen sulfide inhibits cigarette smoke-induced inflammation and injury in alveolar epithelial cells by suppressing PHD2/HIF-1α/MAPK signaling pathway

Chronic obstructive pulmonary fibrosis (COPD) is a chronic and fatal lung disease with few treatment options. Sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), was found to alleviate cigarette smoke (CS)-induced emphysema in mice, however, the underlying mechanisms have not yet been clarified. In this study, we investigated its effects on COPD in a CS-induced mouse model in vivo and in cigarette smoke extract (CSE)-stimulated alveolar epithelial A549 cells in vitro. The results showed that NaHS not only relieved emphysema, but also improved pulmonary function in CS-exposed mice. NaHS significantly increased the expressions of tight junction proteins (i.e., ZO-1, Occludin and claudin-1), and reduced apoptosis and secretion of pro-inflammatory cytokines (i.e., TNF-α, IL-6 and IL-1β) in CS-exposed mouse lungs and CSE-incubated A549 cells, indicating H₂S inhibits CS-induced inflammation, injury and apoptosis in alveolar epithelial cells. NaHS also upregulated prolyl hydroxylase (PHD)2, and suppressed hypoxia-inducible factor (HIF)-1α expression in vivo and in vitro, suggesting H₂S inhibits CS-induced activation of PHD2/HIF-1α axis. Moreover, NaHS inhibited CS-induced phosphorylation of ERK, JNK and p38 MAPK in vivo and in vitro, and treatment with their inhibitors reversed CSE-induced ZO-1 expression and inflammation in A549 cells. These results suggest that NaHS may prevent emphysema via the suppression of PHD2/HIF-1α/MAPK signaling pathway, and subsequently inhibition of inflammation, epithelial cell injury and apoptosis, and may be a novel strategy for the treatment of COPD.     

Inhibitory effects of clotrimazole on TNF-α-induced adhesion molecule expression and angiogenesis

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-α-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-α-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-α-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-κB activation, as CLT inhibited TNF-α-induced ROS generation as well as NF-κB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-α-induced monocyte adhesion was also mimicked by the specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-α have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-α-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-α-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-α-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.     

Fluoride-induced IL-8 release in human epithelial lung cells: relationship to EGF-receptor-, SRC- and MAP-kinase activation

Exposure of human epithelial lung cells to fluorides is known to induce a marked increase in the release of interleukin (IL)-8, a chemokine involved in neutrophil recruitment. In the present study, the involvement of mitogen-activating protein kinases (MAPKs), the role of upstream activation of Src family kinases (SFKs), epidermal growth factor receptor (EGFR) activation and the interrelationships between these pathways in fluoride-induced IL-8 were examined in a human epithelial lung cell line (A549). Sodium fluoride strongly activated MAPK, in particular JNK1/2 and p38. The ERK1/2-inhibitor PD98059, the p38-inhibitor SB202190 and the JNK1/2-inhibitor SP600125 partially inhibited the fluoride-induced IL-8 response. Combinations of these inhibitors reduced the responses nearly to basal levels. Treatment with siRNA against JNK2 also reduced the IL-8 response to fluoride. Furthermore, fluoride activated SFKs, which was abolished by the SFK-inhibitor PP2. PP2 substantially inhibited the increased levels of IL-8, and partially reduced the fluoride-induced activation of ERK1/2, p38 and JNK1/2. Fluoride exposure also led to a phosphorylation of the EGFR, that was partially inhibited by PP2. AG1478, an EGFR-inhibitor, partially reduced the fluoride-induced IL-8 response and the phosphorylation of JNK1/2 and ERK1/2, but less the phosphorylation of p38. The effects of AG1478 were less than that of PP2. In conclusion, our findings suggest that the fluoride-induced IL-8 release involves the combined activation of ERK1/2, JNK1/2 and p38, and that the phosphorylation of these kinases, and in particular JNK1/2 and ERK1/2, partly, is mediated via a SFK-dependent EGFR-linked pathway. SFK-dependent, but EGFR-independent mechanisms seem important, and especially for phosphorylation of p38.