MDContactCom: a tool to identify differences of protein molecular dynamics from two MD simulation trajectories in terms of interresidue contacts.

SUMMARY: Comparing results from multiple MD simulations performed under different conditions is essential during the initial stages of analysis. We propose a tool called MD Contact Comparison (MDContactCom) that compares residue-residue contact fluctuations of two MD trajectories, quantifies the differences, identifies sites that exhibit large differences and visualizes those sites on the protein structure. Using this method, it is possible to identify sites affected by varying simulation conditions and reveal the path of propagation of the effect even when differences between the 3D structure of the molecule and the fluctuation RMSF of each residue is unclear. MDContactCom can monitor differences in complex protein dynamics between two MD trajectories and identify candidate sites to be analyzed in more detail. As such, MDContactCom is a versatile software package for analyzing most MD simulations. AVAILABILITY AND IMPLEMENTATION: MDContactCom is freely available for download on GitLab. The software is implemented in Python3. https://gitlab.com/chiemotono/mdcontactcom. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Sprouty-Related Ena/Vasodilator-Stimulated Phosphoprotein Homology 1-Domain-Containing Protein-2 Critically Regulates Influenza A Virus-Induced Pneumonia.

OBJECTIVES: Influenza A virus causes acute respiratory infections that induce annual epidemics and occasional pandemics. Although a number of studies indicated that the virus-induced intracellular signaling events are important in combating influenza virus infection, the mechanism how specific molecule plays a critical role among various intracellular signaling events remains unknown. Raf/MEK/extracellular signal-regulated kinase cascade is one of the key signaling pathways during influenza virus infection, and the Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein has recently been identified as a negative regulator of Raf-dependent extracellular signal-regulated kinase activation. Here, we examined the role of Raf/MEK/extracellular signal-regulated kinase cascade through sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein in influenza A viral infection because the expression of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein was significantly enhanced in human influenza viral-induced pneumonia autopsy samples. DESIGN: Prospective animal trial. SETTING: Research laboratory. SUBJECTS: Wild-type and sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice inoculated with influenza A. INTERVENTIONS: Wild-type or sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice were infected by intranasal inoculation of influenza A (A/PR/8). An equal volume of phosphate-buffered saline was inoculated intranasally into mock-infected mice. MEASUREMENTS AND MAIN RESULTS: Influenza A infection of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice led to higher mortality with greater viral load, excessive inflammation, and enhanced cytokine production than wild-type mice. Administration of MEK inhibitor, U0126, improved mortality and reduced both viral load and cytokine levels. Furthermore, bone marrow chimeras indicated that influenza A-induced lung pathology was most severe when sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 expression was lacking in nonimmune cell populations. Furthermore, microarray analysis revealed knockdown of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 led to enhanced phosphatidylinositol 3-kinase signaling pathway, resulting that viral clearance was regulated by sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 expression through the phosphatidylinositol 3-kinase signaling pathway in murine lung epithelial cells. CONCLUSIONS: These data support an important function of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 in controlling influenza virus-induced pneumonia and viral replication. Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 may be a novel therapeutic target for controlling the immune response against influenza influenza A virus infection.

The Effect of Conformational Flexibility on Binding Free Energy Estimation between Kinases and Their Inhibitors.

Accurate prediction of binding affinities of drug candidates to their targets remains challenging because of protein flexibility in solution. Conformational flexibility of the ATP-binding site in the CDK2 and ERK2 kinases was identified using molecular dynamics simulations. The binding free energy (ΔG) of twenty-four ATP-competitive inhibitors toward these kinases was assessed using an alchemical free energy perturbation method, MP-CAFEE. However, large calculation errors of 2-3 kcal/mol were observed using this method, where the free energy simulation starts from a single equilibrated conformation. Here, we developed a new ΔG computation method, where the starting structure was set to multiconformations to cover flexibility. The calculation accuracy was successfully improved, especially for larger molecular size compounds, leading to reliable prediction of a broader range of drug candidates. The present study demonstrates that conformational flexibility of interactions between a compound and the glycine-rich loop in the kinases is a key factor in ΔG estimation.

Extended template-based modeling and evaluation method using consensus of binding mode of GPCRs for virtual screening.

G-protein-coupled receptors (GPCRs) are a pharmaceutically important protein family because they mediate numerous physiological functions. The crystal structures of several GPCR subtypes have been determined recently, encouraging efforts to apply structure-based virtual screening (SBVS) along with ligand-based virtual screening (LBVS) to improve the hit rate of active ligands from large chemical libraries. Three-dimensional models are also necessary for GPCR targets whose structures are unknown. Current challenges include the selection of structural templates from available structurally known GPCRs to use for accurate modeling and understanding the diversity of sites recognizing distinct ligands. We have developed and validated an extended template-based modeling and evaluation method for SBVS. Models were generated using a fragmental template procedure in addition to typical template-based modeling methods. The reliability of the models was evaluated using a virtual screening test with known active ligands and decoys and the consensus of the binding mode using the protein-ligand interaction fingerprint (PLIF) derived from the results of docking simulations. This novel workflow was applied to three targets with known structures (human dopamine receptor 3, human histamine H1 receptor, and human delta opioid receptor) and to a target with an unknown structure (human serotonin 2A receptor). In each case, model structures having high ligand selectivity with consensus binding mode were generated.

Galectin-9 prolongs the survival of septic mice by expanding Tim-3-expressing natural killer T cells and PDCA-1+ CD11c+ macrophages.

INTRODUCTION: Galectin-9 ameliorates various inflammatory conditions including autoimmune diseases by regulating T cell and macrophage/dendritic cell (DC) functions. However, the effect of galectin-9 on polymicrobial sepsis has not been assessed. METHODS: We induced polymicrobial sepsis by cecal ligation and puncture (CLP) in mice. The survival rate was compared between galectin-9- and PBS-treated CLP mice. An ELISA was used to compare the levels of various cytokines in the plasma and culture supernatants. Fluorescence-activated cell sorting analysis was further performed to compare the frequencies of subpopulations of spleen cells. RESULTS: Galectin-9 exhibited a protective effect in polymicrobial sepsis as demonstrated in galetin-9 transgenic mice and therapeutic galectin-9 administration. In contrast, such effect was not observed in nude mice, indicating the involvement of T cells in galectin-9-mediated survival prolongation. Galectin-9 decreased TNFα, IL-6, IL-10 and, high mobility group box 1 (HMGB1) and increased IL-15 and IL-17 plasma and spleen levels. Galectin-9 increased the frequencies of natural killer T (NKT) cells and PDCA-1+ CD11c+ macrophages (pDC-like macrophages) but did not change the frequency of CD4 or CD8 T cells, γδT cells or conventional DC. As expected, galectin-9 decreased the frequency of Tim-3+ CD4 T cells, most likely Th1 and Th17 cells. Intriguingly, many spleen NK1.1+ NKT cells and pDC-like macrophages expressed Tim-3. Galectin-9 increased the frequency of Tim-3-expressing NK1.1+ NKT cells and pDC-like macrophages. Galectin-9 further increased IL-17+ NK1.1+ NKT cells. CONCLUSION: These data suggest that galectin-9 exerts therapeutic effects on polymicrobial sepsis, possibly by expanding NKT cells and pDC-like macrophages and by modulating the production of early and late proinflammatory cytokines.

SAHG, a comprehensive database of predicted structures of all human proteins.

Most proteins from higher organisms are known to be multi-domain proteins and contain substantial numbers of intrinsically disordered (ID) regions. To analyse such protein sequences, those from human for instance, we developed a special protein-structure-prediction pipeline and accumulated the products in the Structure Atlas of Human Genome (SAHG) database at http://bird.cbrc.jp/sahg. With the pipeline, human proteins were examined by local alignment methods (BLAST, PSI-BLAST and Smith-Waterman profile-profile alignment), global-local alignment methods (FORTE) and prediction tools for ID regions (POODLE-S) and homology modeling (MODELLER). Conformational changes of protein models upon ligand-binding were predicted by simultaneous modeling using templates of apo and holo forms. When there were no suitable templates for holo forms and the apo models were accurate, we prepared holo models using prediction methods for ligand-binding (eF-seek) and conformational change (the elastic network model and the linear response theory). Models are displayed as animated images. As of July 2010, SAHG contains 42,581 protein-domain models in approximately 24,900 unique human protein sequences from the RefSeq database. Annotation of models with functional information and links to other databases such as EzCatDB, InterPro or HPRD are also provided to facilitate understanding the protein structure-function relationships.

Spred-2 deficiency exacerbates acetaminophen-induced hepatotoxicity in mice.

MAPKs are involved in acetaminophen (APAP)-hepatotoxicity, but the regulatory mechanism remains unknown. Here, we explored the role of Spred-2 that negatively regulates Ras/ERK pathway in APAP-hepatotoxicity. Spred-2 knockout (KO) mice demonstrated exacerbated liver injury, an event that was associated with increased numbers of CD4(+) T, CD8(+) T and NK cells in the liver compared to the control. Levels of CXCL9/CXCL10 that attract and activate these cells were increased in Spred-2 KO-liver. Kupffer cells isolated from Spred-2 KO mice after APAP challenge expressed higher levels of CXCL9/CXCL10 than those from the control. Upon stimulation with APAP or IFNγ, naïve Kupffer cells from Spred-2 KO mice expressed higher levels of CXCL9/CXCL10. NK cell-depletion attenuated APAP-hepatotoxicity with lowered hepatic IFNγ and decreased numbers of not only NK cells but also CD4(+) T and CD8(+) T cells in the liver. These results suggest that Spred-2 negatively regulates APAP-hepatotoxicity under the control of Kupffer cells and NK cells.

Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics.

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Characterization of five Indonesian mangoes using gas chromatography-mass spectrometry-based metabolic profiling and sensory evaluation.

Indonesia is one of the world's leading mango producers and grows many cultivars. However, only a few cultivars have been commercialized, perhaps because of limited information on consumer preferences that meet the market demands. Here, non-targeted gas chromatography-mass spectrometry (GC-MS)-based metabolome analysis was used to characterize five Indonesian mango cultivars considering their taste characteristics. A total of 95 components (47 annotated and 48 unknown metabolites) were identified. Cultivars with a higher general impression score (Arumanis 143 and Gedong) in principal component analysis (PCA) accumulated more sugars and sweetening components, such as glycine and lyxose. Meanwhile, cultivars with a lower general impression score in PCA (Lalijiwo and Cengkir Indramayu) accumulated more aspartic acid, isocitric acid, and citric acid, which increase sourness; methionine, which is a precursor of sulfur-containing volatile aroma components; and phenylalanine, which contributes to bitterness. Furthermore, orthogonal projection to latent structures discriminant analysis revealed that nicotinic acid, glutamic acid, aspartic acid, glycine, and ribose characterized higher or lower general impression cultivars. In addition, metabolic profiling of eight mango cultivars, including five Indonesian and three overseas cultivars, suggested that taste was more influential than differences in cultivars, production areas, and cultivation conditions by its hydrophilic primary metabolomics. These findings will serve as fundamental data for future mango industry development considering the association between the unique taste of each cultivar and its metabolites as well as the consumer preferences for Indonesian mango.

Machine learning to estimate the local quality of protein crystal structures.

Low-resolution electron density maps can pose a major obstacle in the determination and use of protein structures. Herein, we describe a novel method, called quality assessment based on an electron density map (QAEmap), which evaluates local protein structures determined by X-ray crystallography and could be applied to correct structural errors using low-resolution maps. QAEmap uses a three-dimensional deep convolutional neural network with electron density maps and their corresponding coordinates as input and predicts the correlation between the local structure and putative high-resolution experimental electron density map. This correlation could be used as a metric to modify the structure. Further, we propose that this method may be applied to evaluate ligand binding, which can be difficult to determine at low resolution.

Rapid LC-MS/MS Determination of Hesperidin in Fermented Tea Prepared from Unripe Satsuma Mandarin (Citrus unshiu) Fruits and Third-crop Green Tea (Camellia sinensis) Leaves.

For improving quality control in the fermented tea production process and advancing the corresponding food labeling with function claims, a rapid and robust hesperidin analysis method using LC-MS/MS with the sample dilution approach was developed by following internationally accepted criteria of the Association of Official Analytical Chemists (AOAC). The linear correlation coefficient (r2) of the regression line was 0.9997 in the concentration range of 0.025 - 2.5 mg/L. The matrix effect evaluated using regression line slope values was negligible. The recovery rate of 100.7% indicated improved trueness. The performance of the newly developed method in determining the hesperidin content of fermented tea samples did not significantly vary from that of a well-established, conventional method. The HorRat values of intra- and inter-laboratory reproducibility studies were both within the acceptable range, indicating sufficient accuracy of the newly developed method according to the AOAC criteria.

Serine 298 Phosphorylation in Linker 2 of UHRF1 Regulates Ligand-Binding Property of Its Tandem Tudor Domain.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential factor for the maintenance of mammalian DNA methylation and harbors several reader modules for recognizing epigenetic marks. The tandem Tudor domain (TTD) of UHRF1 has a peptide-binding groove that functions as a binding platform for intra- or intermolecular interactions. Besides the groove interacting with unphosphorylated linker 2 and spacer of UHRF1, it also interacts with di/tri-methylated histone H3 at Lys9 and DNA ligase 1 (LIG1) at Lys126. Here we focus on the phosphorylation of Ser298 in linker 2, which was implied to regulate the ligand-binding property of the TTD. Although the protein expression level of UHRF1 is unchanged throughout the cell cycle, Ser298 phosphorylated form of UHRF1 is notably increased in the G2/M phase, which is revealed by immunoprecipitation followed by Western blotting. Molecularly, while unphosphorylated linker 2 covers the peptide-binding groove to prevent access of other interactors, small-angle X-ray scattering, thermal stability assay and molecular dynamics simulation revealed that the phosphate group of Ser298 dissociates linker 2 from the peptide-binding groove of the TTD to permit the other interactors to access to the groove. Our data reveal a mechanism in which Ser298 phosphorylation in linker 2 triggers a change of the TTD's structure and may affect multiple functions of UHRF1 by facilitating associations with LIG1 at DNA replication sites and histone H3K9me2/me3 at heterochromatic regions.

Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel.

Microfluidic devices have emerged as a new cell culture tool, which can mimic the structure and physiology of living human organs. However, no standardized culture method for a microfluidic device has yet been established. Here, we describe the effects of various conditions on cell proliferation in a microchannel with a depth smaller than 100 µm. Primary endothelial cell proliferation was suppressed with a decrease in the culture medium volume per cell culture area. Moreover, cell growth was compared with or without medium flow, and the optimum culture condition was determined to be 1 µL/h flow in a 65-µm-deep microchannel. In addition, glucose consumption was greater under fluidic conditions than under static conditions, and the ability of tumor (HeLa) cells to convert glucose into lactate appeared to be higher in a static culture than that in a fluidic culture. Overall, our results will serve as a useful guide for designing a microfluidic cell culture platform in a channel smaller than 100 µm.

Spred2 Deficiency Exacerbates D-Galactosamine/Lipopolysaccharide -induced Acute Liver Injury in Mice via Increased Production of TNFα.

Acute liver injury (ALI) is characterized by hepatocyte damage and inflammation. In the present study, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences ALI induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Compared to wild-type mice, Spred2-/- mice developed exacerbated liver injury represented by enhanced hepatocyte damage and inflammation. Enhanced ERK activation was observed in Spred2-/--livers, and the MEK/ERK inhibitor U0126 ameliorated ALI. Hepatic tumour necrosis factor α (TNFα) and interleukin (IL)-1ß levels were increased in Spred-2-/--livers, and the neutralization of TNFα dramatically ameliorated ALI, which was associated with decreased levels of endogenous TNFα and IL-1ß. When mice were challenged with D-GalN and TNFα, much severer ALI was observed in Spred2-/- mice with significant increases in endogenous TNFα and IL-1ß in the livers. Immunohistochemically, Kupffer cells were found to produce TNFα, and isolated Kupffer cells from Spred2-/- mice produced significantly higher levels of TNFα than those from wild-type mice after LPS stimulation, which was significantly decreased by U0126. These results suggest that Spred2 negatively regulates D-GalN/LPS-induced ALI under the control of TNFα in Kupffer cells. Spred2 may present a therapeutic target for the treatment of ALI.

Spred2-deficiecy Protects Mice from Polymicrobial Septic Peritonitis by Enhancing Inflammation and Bacterial Clearance.

Sepsis is an infection-induced systemic inflammatory syndrome and a major cause of death for critically ill patients. Here, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences host defense against polymicrobial sepsis (PMS) induced by cecal ligation and puncture (CLP). Compared to wild-type mice, Spred2-/- mice exhibited higher survival rates with increased level of leukocyte infiltration and local chemokine production and reduced plasma and peritoneal bacterial loads after CLP. The MEK inhibitor U0126 significantly reduced LPS-induced chemokine production by Spred2-/- resident macrophages in vitro, and decreased CLP-induced leukocyte infiltration in vivo. Spred2-/- resident macrophages, but not neutrophils or elicited macrophages, exhibited increased phagocytic activity. Interestingly, surface expression of complement receptor 1/2 (CR1/2) was increased in Spred2-/- resident macrophages in response to lipopolysaccharide in a manner dependent on the ERK/MAPK pathway, and blocking CR1/2 in vivo resulted in reduced leukocyte infiltration and increased bacterial loads after CLP. Taken together, our results indicate that Spred2-deficiency protects mice from PMS via increased activation of the ERK/MAPK pathway and subsequent increase in innate immune responses. Thus, inhibiting Spred2 may present a novel means to prevent the development of PMS.

Corrigendum: Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells.

[This corrects the article on p. 2 in vol. 7, PMID: 26834744.].

Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 plays an important role in the initiation and progression of cancer. We previously reported that in 4T1 murine breast cancer, non-tumor stromal cells, including macrophages, were the major source of MCP-1. In the present study, we analyzed the potential mechanisms by which MCP-1 is upregulated in macrophages infiltrating 4T1 tumors. We found that cell-free culture supernatants of 4T1 cells (4T1-sup) markedly upregulated MCP-1 production by peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly upregulated neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. Physicochemical analysis indicated that an approximately 20-30 kDa 4T1 cell product was responsible for the capacity of 4T1-sup to upregulate MCP-1 expression by macrophages. A neutralizing antibody against granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage CSF, almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF potently upregulated MCP-1 production by macrophages. The expression levels of GM-CSF in 4T1 tumors in vivo were higher than other tumors, such as Lewis lung carcinoma. Treatment of mice with anti-GM-CSF antibody significantly reduced the growth of 4T1 tumors at the injection sites but did not reduce MCP-1 production or lung metastasis in tumor-bearing mice. These results indicate that 4T1 cells have the capacity to directly upregulate MCP-1 production by macrophages by releasing GM-CSF; however, other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment.

Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability.

We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate.

Spred-2 deficiency exacerbates lipopolysaccharide-induced acute lung inflammation in mice.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation. METHODS: Wild-type (WT) mice and Spred-2(-/-) mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2(-/-) mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells. RESULTS: LPS-induced acute lung inflammation was significantly exacerbated in Spred-2(-/-) mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2(-/-) mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells. CONCLUSIONS: The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.