Involvement Of Mitogen-activated Protein Kinase Research Articles

Thalidomide inhibits fibronectin production in TGF-β1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway

Keloids are characterized by the vigorously continuous production of extracellular matrix protein and aberrant cytokine activity in the dermis. There is a growing body of evidence that thalidomide, α-N-phthalimidoglutarimide, has anti-fibrotic properties. The aims were to examine possible therapeutic effects of thalidomide on fibronectin expression in transforming growth factor-β1 (TGF-β1)-treated normal fibroblasts (NFs) and keloid-derived fibroblasts (KFs) and the underlying mechanism of action, especially the involvement of mitogen-activated protein kinase (MAPKs) and Sma- and Mad-related family (Smads) pathways. In surgically removed human tissues, TGF-β1 and fibronectin immunoreactivity was high in keloid tissue, but barely detectable in normal tissue. TGF-β1 induced significant fibronectin expression in NFs and KFs and the effect was inhibited by pretreatment with thalidomide. TGF-β1 also induced phosphorylation of MAPKs (ERK1/2, p38, and JNK) and Smad2/3 and pretreatment with PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) inhibited TGF-β1-induced fibronectin expression. Furthermore, pretreatment with thalidomide inhibited the TGF-β1-induced phosphorylation of p38 and Smad3, but not that of ERK1/2, JNK, and Smad2. In addition, thalidomide pretreatment inhibited the TGF-β-induced DNA binding activity of AP-1 and Smad3/4, caused fibronectin degradation by increasing the activity of matrix metalloproteinase 9, and decreased production of TGF-β1 and fibronectin and the number of fibroblasts in an in vivo keloid model. These results show that thalidomide has an antifibrotic effect on keloid fibroblasts that is caused by suppression of TGF-β1-induced p38 and Smad3 signaling. Our findings indicate that thalidomide may be a potential candidate drug for the treatment and prevention of keloids.

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase-9 (MMP-9), and a major role for 15-(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK, whereas lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15-HETE also promoted phosphorylation of MAPK-activated protein kinase-2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15-HETE upregulate MMP-9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP-9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria.

A Possible Mechanism of Cisplatin-Induced Tumor Necrosis Factor (TNF)-α Production in Murine Macrophages

Cisplatin has been used for the treatment of various solid cancers or sarcomas; however, it can induce severe adverse effects. Among these adverse effects, nephrotoxicity, which has the potential to be a dose-limiting factor of this agent, develops due to the secretion of tumor necrosis factor-α (TNF-α) from macrophages; however, the precise mechanisms are still unclear. To elucidate possible mechanisms, we investigated the involvement of mitogen-activated protein kinases (MAPK) and reactive oxygen species (ROS) in cisplatin-induced TNF-α mRNA expression and protein production in the mouse macrophage-like cell line, RAW 264. Cisplatin (1 μM) significantly increased TNF-α mRNA expression and protein production. Extracellular-regulated kinase (ERK) and p38 MAPK, but not c-Jun N-terminal kinase (JNK), phosphorylation increased in response to cisplatin. Although an ERK inhibitor (PD98059) suppressed both cisplatin-induced TNF-α mRNA expression and its protein production, a p38 MAPK inhibitor (SB203580) decreased TNF-α protein production only. A JNK inhibitor (SP600125) had no effect on cisplatin-induced TNF-α mRNA expression. Furthermore, a scavenger of ROS, N,N’-dimethylthiourea, suppressed both ERK activation and TNF-α mRNA expression. These results suggest that the phosphorylation of ERK by ROS is involved in cisplatin-induced TNF-α mRNA expression and that the signaling pathway of p38 MAPK is related to TNF-α protein production.

Interleukin-6-induced S100B secretion is inhibited by haloperidol and risperidone

Although inflammation may be a physiological defense process, imbalanced neuroinflammation has been associated with the pathophysiology of brain disorders, including major depression and schizophrenia. Activated glia releases a variety of pro-inflammatory cytokines that contribute to neuronal dysfunction. Elevated levels of S100B, a glia derived protein, have been observed in the serum and CSF of schizophrenic patients suggesting a glial role in the disease. We evaluated whether S100B secretion (in C6 glioma cells and hippocampal slices in Wistar rats) could be directly modulated by the main inflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-8) altered in schizophrenia, as well as the possible involvement of mitogen-activated protein kinase (MAPK) pathways in these responses. We also investigated the effects of typical and atypical antipsychotic drugs on glial cytokine-induced S100B release. Our results suggest that S100B secretion is increased by pro-inflammatory cytokines via MAPK and that oxidative stress may be a component of this modulation. These results reinforce the idea that the S100B protein is involved in the inflammatory response observed in many brain diseases, including schizophrenia. Moreover the antipsychotics, haloperidol and risperidone, were able to inhibit the secretion of S100B following IL-6 stimulation in C6 glioma cells.

Involvement of PI3 kinase and MAP kinase in IGF-I and insulin-induced ovarian steroidogenesis in common carp Cyprinus carpio

Previously, we observed that in vitro steroidogenesis in intact ovarian follicles of common carp Cyprinus carpio can alone be induced by recombinant human insulin-like growth factor (IGF-I) and bovine insulin (b-insulin) and this induction was gonadotropin-independent. To investigate early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during ovarian steroidogenesis was examined. IGF-I and b-insulin induced testosterone and 17β-estradiol production in carp ovarian theca and granulosa cells in short-term coincubation and this induction was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase from 30min onwards with a maximum at 90min. In this study, we found the involvement of mitogen-activated protein kinase (MAP kinase) in the regulation of IGF-I- and b-insulin-induced steroidogenesis in carp ovary. An antagonist of mitogen-activated protein kinase kinase1/2 (MEK1/2) markedly attenuated IGF-I- and b-insulin-induced steroid production. Cells treated with IGF-I and b-insulin stimulated ERK1/2-dependent phosphorylation of extracellular signal regulated protein kinase1/2 (ERKs1/2) in a time-dependent manner, which was significantly attenuated in presence of MEK1/2 inhibitor. PI3 kinase inhibitors strongly attenuated phosphorylation and activation of MAP kinase, which was increased during IGF-I and b-insulin-induced steroidogenesis. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes the MAP kinase during IGF-I- and b-insulin-induced steroidogenesis in C. carpio ovarian follicles.

Mitogen-Activated Protein Kinase Pathways are Involved in the Upregulation of Calcitonin Gene-Related Peptide of Rat Trigeminal Ganglion After Organ Culture

The trigeminal ganglion (TG) can express and release calcitonin gene-related peptide (CGRP), an important neuropeptide that plays a crucial role in migraine attack and cluster headache. Activation of rat TG increases CGRP expression. However, the regulatory mechanism of CGRP expression in TG neurons remains to be explored. This study aims to evaluate the involvement of mitogen-activated protein kinase (MAPK) pathways in CGRP upregulation after rat TG organ culture. Rat TG was cultured alone for 24 h or cultured in combination with MAPK inhibitors, tumor necrosis factor α (TNF-α), or interleukin 1β (IL-1β) for 24 h. CGRP protein was determined using immunohistochemistry. The mRNA levels of CGRP, TNF-α, and IL-1β were analyzed through real-time quantitative polymerase chain reaction. MAPK phosphorylation was detected via western blot. After rat TG organ culture, the expressions of CGRP, TNF-α, and IL-1β were upregulated at 24 h. The phosphorylation of extracellular signal-regulated kinases (ERK1/2), P38, and c-jun N-terminal kinases (JNK) significantly increased at 30 min compared with fresh rat TG. In addition, both CGRP expression and phosphorylation of ERK1/2, P38, and JNK were enhanced obviously after rat TG treatment with TNF-α or IL-1β compared with fresh rat TG. However, they decreased markedly after rat TG pretreatment with PD98059 (ERK1/2 inhibitor), SB203580 (P38 inhibitor), or SP600125 (JNK inhibitor) compared with rat TG co-culture with TNF-α or IL-1β. In conclusion, the elevated CGRP expression after rat TG organ culture can be regulated via MAPK pathways. The findings provide insight into the molecular mechanisms and experimental evidence for therapeutic targets of migraine.

Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways

BackgroundAstrocytes are an integral component of the blood–brain barrier (BBB) which may be compromised by ischemic or traumatic brain injury. In response to trauma, astrocytes increase expression of the endopeptidase matrix metalloproteinase (MMP)-9. Compromise of the BBB leads to the infiltration of fluid and blood-derived proteins including albumin into the brain parenchyma. Albumin has been previously shown to activate astrocytes and induce the production of inflammatory mediators. The effect of albumin on MMP-9 activation in astrocytes is not known. We investigated the molecular mechanisms underlying the production of MMP-9 by albumin in astrocytes.MethodsPrimary enriched astrocyte cultures were used to investigate the effects of exposure to albumin on the release of MMP-9. MMP-9 expression was analyzed by zymography. The involvement of mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) and the TGF-β receptor-dependent pathways were investigated using pharmacological inhibitors. The production of ROS was observed by dichlorodihydrofluorescein diacetate fluorescence. The level of the MMP-9 inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 produced by astrocytes was measured by ELISA.ResultsWe found that albumin induces a time-dependent release of MMP-9 via the activation of p38 MAPK and extracellular signal regulated kinase, but not Jun kinase. Albumin-induced MMP-9 production also involves ROS production upstream of the MAPK pathways. However, albumin-induced increase in MMP-9 is independent of the TGF-β receptor, previously described as a receptor for albumin. Albumin also induces an increase in TIMP-1 via an undetermined mechanism.ConclusionsThese results link albumin (acting through ROS and the p38 MAPK) to the activation of MMP-9 in astrocytes. Numerous studies identify a role for MMP-9 in the mechanisms of compromise of the BBB, epileptogenesis, or synaptic remodeling after ischemia or traumatic brain injury. The increase in MMP-9 produced by albumin further implicates both astrocytes and albumin in the acute and long-term complications of acute CNS insults, including cerebral edema and epilepsy.

Involvement of mitogen-activated protein kinases and peroxisome proliferator-activated receptor γ in monosodium urate crystal-induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts

To investigate whether monosodium urate (MSU) crystals could induce the production of VCAM-1 (vascular cell adhesion molecule1) in human synovial cells and its possible signaling pathways, human synovial cells isolated from synovial tissue explants were stimulated with various doses of MSU crystals for different time intervals. Expression of VCAM-1 was evaluated with Western blotting. To explore the underlying mechanisms, VCAM-1 protein expression was also evaluated after activation of several signaling molecules including mitogen-activated protein kinases (MAPKs) and peroxisome proliferator-activated receptorγ (PPARγ) were blocked. Exposure of synovial cells to MSU crystals induced VCAM-1 expression in culture medium in a dose- and time-dependent manner, reaching a plateau at 1000µM and 24h. Inhibition of the activation of MAPKs and PPARγ could block this increase. The present results demonstrated that MSU crystals could induce VCAM-1 expression. MAPKs and PPARγ signaling pathways regulated the induced VCAM-1 expression.

Formation of New High Density Glycogen-Microtubule Structures Is Induced by Cardiac Steroids

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.

Aggravation of ADAMTS and Matrix Metalloproteinase Production and Role of ERK1/2 Pathway in the Interaction of Osteoarthritic Subchondral Bone Osteoblasts and Articular Cartilage Chondrocytes — Possible Pathogenic Role in Osteoarthritis

Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMP), play key roles in development of osteoarthritis (OA). We investigated if crosstalk between subchondral bone osteoblasts (SBO) and articular cartilage chondrocytes (ACC) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13, and also tested the possible involvement of mitogen-activated protein kinase (MAPK) signaling pathway during this process. ACC and SBO were isolated from normal and OA patients. An in vitro coculture model was developed to study the regulation of ADAMTS and MMP under normal and OA joint crosstalk conditions. The MAPK-ERK inhibitor PD98059 was applied to delineate the involvement of specific pathways during this interaction process. Indirect coculture of OA SBO with normal ACC resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3, and MMP-9 in ACC, whereas coculture of OA ACC led to increased MMP-1 and MMP-2 expression in normal SBO. Upregulation of ADAMTS and MMP under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway, and addition of the MAPK-ERK inhibitor PD98059 reversed the overexpression of ADAMTS and MMP in cocultures. These results add to the evidence that in human OA, altered bidirectional signals between SBO and ACC significantly influence the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMP. We have demonstrated for the first time that this altered crosstalk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

The Involvement of Mitogen-Activated Protein Kinases in the 1^|^alpha;,25-Dihydroxy-Cholecalciferol-Induced Inhibition of Adipocyte Differentiation In Vitro

The present study was undertaken to investigate the mechanism by which 1α, 25-dihydroxy-cholecalciferol [1α,25-(OH)₂-VD₃] modulates the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes. Treatment with 1α,25-(OH)₂-VD₃ in the presence of insulin, dexamethasone and 3-isobutyl-1-methyl-xanthine significantly inhibited the triacylglycerol accumulation, and mRNA expressions of adipocytokines (adiponectin and tumor necrosis factor-α) and plasminogen activator inhibitor-1 in the pico-nanomolar concentration range, indicating that 1α,25-(OH)₂-VD₃ under physiological conditions inhibits the differentiation of 3T3-L1 cells. 1α,25-(OH)₂-VD₃ potently reduced the mRNA and/or protein expressions of CCAAT-enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), and the nuclear translocation of PPARγ. Furthermore, it inhibited the mRNA expression and phosphorylation of extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases. These results indicate that 1α,25-(OH)₂-VD₃ can be an inhibitor of adipocyte differentiation, and suggest, in addition to C/EBPα and PPARγ, an important role of ERK in mediating 1α,25-(OH)₂-VD₃-induced alteration in adipocyte differentiation.

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

Abstract A48: Osteopontin increases heme oxygenase-1 expression and subsequently induces cell migration in glioma cells.

Abstract Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Gliomas are associated high morbidity and mortality because these tumors are highly invasive into surrounding brain tissue, making complete surgical resection impossible. Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor in a variety of neuronal cell populations. However, the molecular mechanisms and pathologic roles underlying GDNF-induced glioma migration remain unclear. In this study, we found that application of recombinant human GDNF enhances the migration of U87 and U251 cells but not C6 cells. In addition, we found that the expression of matrix metalloproteinase-13 (MMP-13) mRNA, protein and secretion increase in response to GDNF stimulation. The GDNF-induced increase in cell migration was antagonized by MMP-13 neutralizing antibody or silencing MMP-13. We then examined the involvement of mitogen-activated protein kinases (MAPKs) in glioma cell migration induced by GDNF. GDNF-induced MMP-13 expression and glioma migration were attenuated by MEK/ extracellular signal-regulating kinase (ERK) and c-Jun N-terminal protein kinase (JNK) inhibitors, as well as ERK and JNK dominant-negative mutants. Treatment with GDNF induced MEK/ERK and JNK/c-Jun activation and increased AP-1 DNA binding activity in a time-dependent manner. Treatment with AP-1 inhibitors (tanshinone IIA and curcumin) also reduced GDNF-induced glioma cell migration. In migration-prone sublines, cells with greater migration ability had higher GDNF expression. These results indicate that GDNF enhances migration of glioma cells through the increase of MMP-13 production and is mainly regulated by the MEK/ERK and JNK, c-Jun and AP-1 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A48.

Oxidized LDL promotes the mitogenic actions of Chlamydia pneumoniae in vascular smooth muscle cells

The atherogenic actions of Chlamydia pneumoniae (C. pneumoniae), a common respiratory pathogen, are dependent upon a high-cholesterol environment in vivo. It is possible that oxidized low-density lipoprotein (oxLDL) is responsible for promoting the atherogenic effects of C. pneumoniae through a stimulation of cell proliferation. This study determined whether oxLDL can enhance the mitogenic action of C. pneumoniae in vascular smooth muscle cells (VSMCs) and the involvement of mitogen-activated protein kinase (MAPK) pathways and heat shock protein 60 (HSP60) in these mechanisms. Primary rabbit VSMCs were treated with live C. pneumoniae, heat-inactivated C. pneumoniae or infection medium, and subsequently incubated for up to 48 h in the presence or absence of oxLDL. Chlamydia pneumoniae infection alone stimulated cell proliferation and the addition of oxLDL significantly amplified this proliferative effect. This proliferation was accompanied by extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation and an up-regulation of HSP60 expression. Changes in proliferation and HSP60 expression were attenuated by the inhibition of ERK1/2. These results indicate a novel role for oxLDL in promoting the mitogenic actions of C. pneumoniae in the vasculature. ERK1/2 is an important factor in the stress-mediated response and HSP60 up-regulation in VSMC. These data provide mechanistic evidence that C. pneumoniae may stimulate atherogenesis.

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: Involvement of mitogen-activated protein kinases

Aim We have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells. Main methods The levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR. Key findings PDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C. Significance These results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.

Up-regulation of Nucleotide-binding Oligomerization Domain 1 in Inflamed Human Dental Pulp

Introduction The innate immune response is activated by recognition of microbial components through specific pattern recognition receptors including nucleotide-binding oligomerization domain (NOD)-like receptors. However, the regulation of NOD-1 in inflamed human dental pulp remains poorly understood. This study aimed to evaluate the expression of NOD-1 in healthy and inflamed human dental pulps. In addition, the secretion of chemokines induced by NOD-1 and the related signaling pathways were studied. Methods Samples of human dental pulp tissues were obtained from freshly extracted wisdom teeth. The protein localization of NOD-1 in the pulp tissues was detected by immunohistochemistry. In addition, human dental pulp fibroblasts were stimulated with NOD-1 agonist γ-D-glutamylmeso-diaminopimelic acid. Production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was determined by an enzyme-linked immunosorbent assay. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was examined by Western blot analysis, and the association of MAPK signaling with chemokine production was determined. Results The results demonstrated the expression of NOD-1 in normal dental pulp, and up-regulated NOD-1 expression was observed in inflamed dental pulp. On stimulation with NOD-1 agonist, production of IL-8 and MCP-1 was induced in a dose-dependent manner. Moreover, phosphorylation of p38 MAPK and Jun N-terminal kinase (JNK) was enhanced by stimulation of NOD-1. With the treatment of p38 MAPK and JNK inhibitors, the NOD-1–induced IL-8 production was suppressed. Conclusions In response to microbial invasion, the expression of NOD-1 can be regulated in a ligand-inducible manner. NOD-1 might participate in pulp inflammation through chemokine production via MAPK signaling pathways.

Gastrodin Inhibits Expression of Inducible NO Synthase, Cyclooxygenase-2 and Proinflammatory Cytokines in Cultured LPS-Stimulated Microglia via MAPK Pathways

BackgroundMicroglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS).Methodology/Principal FindingsBV-2 cells were pretreated with gastrodin (30, 40, and 60 µM) for 1 h and then stimulated with LPS (1 µg/ml) for another 4 h. The effects on proinflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), are analysed by double-immunofluorescence labeling and RT-PCR assay. To reveal the mechanisms of action of gastrodin we investigated the involvement of mitogen-activated protein kinases (MAPKs) cascades and their downstream transcription factors, nuclear factor-κB (NF-κB) and cyclic AMP-responsive element (CRE)-binding protein (CREB). Gastrodin significantly reduced the LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and NF-κB. LPS (1 µg/ml, 30 min)-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) and this was inhibited by pretreatment of BV-2 cells with different concentrations of gastrodin (30, 40, and 60 µM). In addition, gastrodin blocked LPS-induced phosphorylation of inhibitor κB-α (IκB-α) (and hence the activation of NF-κB) and of CREB, respectively.Conclusion and ImplicationsThis study indicates that gastrodin significantly attenuate levels of neurotoxic proinflammatory mediators and proinflammatory cytokines by inhibition of the NF-κB signaling pathway and phosphorylation of MAPKs in LPS-stimulated microglial cells. Arising from the above, we suggest that gastrodin has a potential as an anti-inflammatory drug candidate in neurodegenerative diseases.

Expression of galectin-9 by IFN-γ stimulated human nasal polyp fibroblasts through MAPK, PI3K, and JAK/STAT signaling pathways

Galectin-9 exhibited potent and selective eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by the marked presence of inflammatory cells, particularly eosinophils. Thus, galectin-9 may be implicated in the pathogenesis of nasal polyposis. The study was designed to investigate whether interferon-gamma (IFN-γ) can induce the augmentation of galectin-9 expression and induce the expression of galectin-9 in nasal polyps. We examined the correlation between galectin-9 expression and eosinophil infiltration in nasal polyps. In addition, we identified the signaling pathways involved in the elevation of galectin-9 expression in response to IFN-γ. Our data demonstrate that the involvement of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 phosphate kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) may play important roles in the selective recruitment of eosinophils in nasal polyp tissues through the production of galectin-9. These findings suggest that galectin-9 expression is associated with eosinophil infiltration in polyps of patients with nasal polyposis.

ESAT6 differentially inhibits IFN‐γ‐inducible class II transactivator isoforms in both a TLR2‐dependent and ‐independent manner

Mycobacterium tuberculosis (Mtb) downregulates the surface expression of major histocompatibility class II (MHC II) molecules on macrophages via modulating class II transactivator (CIITA) protein of the host cell. This results in decreased effector function of CD4(+) T cells. In macrophages, CIITA is transcribed by the promoters I (pI) and IV (pIV) and the corresponding gene products are referred to as type I and type IV CIITA, respectively. Earlier studies have mainly focused on CIITA transcribed by pIV; however, these studies also showed that type IV CIITA expression was transient and dispensable for MHC II expression. In the present study, we observed that the Mtb 6-kDa, early secreted antigen (ESAT6) inhibited interferon (IFN)-γ-induced type I as well as type IV CIITA, but, interestingly, inhibition of type I CIITA was found to be independent of Toll-like receptor-2 (TLR2), whereas that of type IV was TLR2 dependent. Moreover, we also present evidence to show that ESAT6-mediated inhibition was regulated via remodeling of the chromatin. We found that ESAT6 caused a decrease in the IFN-γ-stimulated methylation of the histone H3K4, as well as in the levels of histone acetylation at the CIITA pI locus in macrophages. We also found the involvement of mitogen-activated protein kinases ERK1/2 and p38 in the regulation of CIITA by ESAT6. In conclusion, our studies suggest that ESAT6 could inhibit the expression of type I and type IV CIITA through different pathways. Furthermore, ESAT6 could signal through putative receptors other than TLR2, and that the inhibition of IFN-γ-stimulated CIITA by ESAT6 was regulated at the chromatin level.

Proinflammatory cytokines, IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.