The PPARalpha-leukotriene B4 pathway to inflammation control.

Inflammation is a local immune response to 'foreign' molecules, infection and injury. Leukotriene B4, a potent chemotactic agent that initiates, coordinates, sustains and amplifies the inflammatory response, is shown to be an activating ligand for the transcription factor PPARalpha. Because PPARalpha regulates the oxidative degradation of fatty acids and their derivatives, like this lipid mediator, a feedback mechanism is proposed that controls the duration of an inflammatory response and the clearance of leukotriene B4 in the liver. Thus PPARalpha offers a new route to the development of anti- or pro-inflammatory reagents.

Arbuscular mycorrhiza-specific signaling in rice transcends the common symbiosis signaling pathway.

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

Critical Role of the GM-CSF Signaling Pathway in Macrophage Pro-Repair Activities.

OBJECTIVE: Macrophages play a critical role in intestinal wound repair. However, the molecular pathways that regulate macrophage wound repair activities remain poorly understood. The aim of this study was to evaluate the role of GM-CSF receptor signaling in the wound repair activities of macrophages. METHODS: Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of Crohn's disease (CD) patients. In vitro models were used to study the repair activities of macrophages. RESULTS: We provide evidence that GM-CSF receptor signaling is required for murine macrophages to promote epithelial repair. In addition, we demonstrate that the deficient repair properties of macrophages from CD patients with active disease can be recovered via GM-CSF therapy. CONCLUSION: Our data support a critical role of the GM-CSF signaling pathway in the pro-repair activities of mouse and human macrophages. © 2014 S. Karger AG, Basel.

Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSITIVE1-dependent pathway

Expression of AtPHO1;H10, a member of the Arabidopsis (Arabidopsis thaliana) PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE1 (COI1). The induction of AtPHO1;H10 expression by wounding and dehydration was dependent on COI1 and was comparable in both the wild type and the OPDA reductase 3-deficient (opr3) mutant. In contrast, induction of AtPHO1;H10 expression by exogenous abscisic acid (ABA) was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA-insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, whereas a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in coregulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration

Role of retinoic acid signaling pathway in endoderm antero-posterior patterning

Summary : During vertebrate embryonic development, the endoderm gives rise to the digestive tract and associated organs such as thyroid, lung, liver and pancreas. Earlier studies have shown that extracellular signals coming from the lateral plate mesoderm pattern the endoderm along the antero-posterior axis specifying different organ primordia. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. In this study, we focused on the role of the retinoic acid (RA) signaling pathway in the regionalization of the future gut tube along the main body axis. We show that the RA-synthesizing enzyme Raldh2 is expressed in mesoderm close to the endoderm during gastrulation and during somitogenesis. During the same period, all retinoic acid receptors (RARs), which directly activate gene transcription, are expressed in endoderm suggesting that endoderm can be responsive to RA. Activation or inhibition of RA signaling was achieved by adding RA or RAR inhibitors tither on beads or in the medium to cultured chick embryos. Branchial arch (BA) endoderm markers were shifted posteriorly upon depletion of RA at gastrulation, but were not shifted after this stage. Conversely, exposure to exogenous RA repressed the most-anterior BA markers and shifted more posterior BA markers anteriorly. This suggests that graded levels of RA activity in the foregut define gene boundaries and expression levels. The posterior foregut and midget markers Pdxl and CdxA require RA for their expression, but elevated RA does not shift their expression domain along the antero-posterior axis. In addition, we investigated if RA signaling pathway interacts with other signaling pathways to pattern the endoderm. Although both RA and FGFs block anterior foregut marker expression, our experiments suggest that FGF signaling does not depend on RA in anterior endoderm. To validate our chick data in mammalians and evaluate whether RA acts directly on endoderm, we have further generated a conditional loss-of-function system in the mouse, which is still under examination.

Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway.

Apoptosis, differentiation, and proliferation are cellular responses which play a pivotal role in wound healing. During this process PPARbeta translates inflammatory signals into prompt keratinocyte responses. We show herein that PPARbeta modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARbeta also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARbeta protects keratinocytes against apoptosis and may contribute to the process of skin wound closure.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Indirect hydrogen monitoring by chemi-ionisation following an associative ionisation pathway after metastable helium atoms production by electron impact ionisation: Protonation and deuteration of carrier gas

Gas chromatography (GC) is an analytical tool very useful to investigate the composition of gaseous mixtures. However, hydrogen (H2) detection after a GC separation is only possible with a Thermal Conductivity Detector (TCD), a Helium Ionisation Detector (HID) or expensive Atomic Emission Detector (AED). Recently, indirect H2 detection by GC coupled to mass spectrometry (MS) was demonstrated but the mechanism of carrier gas protonation remained unclear. With electron impact as ionisation source of MS and helium (He) as GC carrier gas, H2 is not ionised according the expected Penning ionisation neither according to the Associative ionisation. Rearrangement ionisation (RI) was found to be the main channel for H2 and D2 ionisation under GC-MS conditions used in most of laboratories using GC-MS, leading to the formation of [He−H]+ and [He−D]+ ions.

Bone regeneration and stem cells.

?  Introduction ?  Bone fracture healing and healing problems ?  Biomaterial scaffolds and tissue engineering in bone formation -  Bone tissue engineering -  Biomaterial scaffolds -  Synthetic scaffolds -  Micro- and nanostructural properties of scaffolds -  Conclusion ?  Mesenchymal stem cells and osteogenesis -  Bone tissue -  Origin of osteoblasts -  Isolation and characterization of bone marrow derived MSC -  In vitro differentiation of MSC into osteoblast lineage cells -  In vivo differentiation of MSC into bone -  Factors and pathways controlling osteoblast differentiation of hMSC -  Defining the relationship between osteoblast and adipocyte differentiation from MSC -  MSC and sex hormones -  Effect of aging on osteoblastogenesis -  Conclusion ?  Embryonic, foetal and adult stem cells in osteogenesis -  Cell-based therapies for bone -  Specific features of bone cells needed to be advantageous for clinical use -  Development of therapeutic biological agents -  Clinical application concerns -  Conclusion ?  Platelet-rich plasma (PRP), growth factors and osteogenesis -  PRP effects in vitro on the cells involved in bone repair -  PRP effects on osteoblasts -  PRP effects on osteoclasts -  PRP effects on endothelial cells -  PRP effects in vivo on experimental animals -  The clinical use of PRP for bone repair -  Non-union -  Distraction osteogenesis -  Spinal fusion -  Foot and ankle surgery -  Total knee arthroplasty -  Odontostomatology and maxillofacial surgery -  Conclusion ?  Molecular control of osteogenesis -  TGF-β signalling -  FGF signalling -  IGF signalling -  PDGF signalling -  MAPK signalling pathway -  Wnt signalling pathway -  Hedgehog signalling -  Notch signalling -  Ephrin signalling -  Transcription factors regulating osteoblast differentiation -  Conclusion ?  Summary This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Targeting the JNK Signaling Pathway Potentiates the Antiproliferative Efficacy of Rapamycin in LS174T Colon Cancer Cells.

Background. Targeting the mTOR signaling pathway with rapamycin in cancer therapy has been less successful than expected due in part to the removal of a negative feedback loop resulting in the over-activation of the PI3K/Akt signaling pathway. As the c-Jun N-terminal kinase (JNK) signaling pathway has been found to be a functional target of PI3K, we investigate the role of JNK in the anticancer efficacy of rapamycin.Materials and Methods. The colon cancer cell line LS174T was treated with rapamycin and JNK phosphorylation was analyzed by Western Blot. Overexpression of a constitutively negative mutant of JNK in LS174T cells or treatment of LS174T cells with the JNK inhibitor SP600125 were used to determine the role of JNK in rapamycin-mediated tumor growth inhibition.Results. Treatment of LS174T cells with rapamycin resulted in the phosphorylation of JNK as observed by Western Blot. The expression of a negative mutant of JNK in LS174T cells or treatment of LS174T cells with SP600125 enhanced the antiproliferative effects of rapamycin. In addition, in vivo, the antitumor activity of rapamycin was potentiated on LS174T tumor xenografts that expressed the dominant negative mutant of JNK.Conclusions. Taken together, these results show that rapamycin-induced JNK phosphorylation and activation reduces the antitumor efficacy of rapamycin in LS174T cells. (C) 2011 Elsevier Inc. All rights reserved.

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors.

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Major Histocompatibility Class II Pathway Is Not Required for the Development of Nonalcoholic Fatty Liver Disease in Mice.

Single-nucleotide polymorphisms within major histocompatibility class II (MHC II) genes have been associated with an increased risk of drug-induced liver injury. However, it has never been addressed whether the MHC II pathway plays an important role in the development of nonalcoholic fatty liver disease, the most common form of liver disease. We used a mouse model that has a complete knockdown of genes in the MHC II pathway (MHCII(Δ/Δ)). Firstly we studied the effect of high-fat diet-induced hepatic inflammation in these mice. Secondly we studied the development of carbon-tetra-chloride- (CCl4-) induced hepatic cirrhosis. After the high-fat diet, both groups developed obesity and hepatic steatosis with a similar degree of hepatic inflammation, suggesting no impact of the knockdown of MHC II on high-fat diet-induced inflammation in mice. In the second study, we confirmed that the CCl4 injection significantly upregulated the MHC II genes in wild-type mice. The CCl4 treatment significantly induced genes related to the fibrosis formation in wild-type mice, whereas this was lower in MHCII(Δ/Δ) mice. The liver histology, however, showed no detectable difference between groups, suggesting that the MHC II pathway is not required for the development of hepatic fibrosis induced by CCl4.

Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury.

BACKGROUND: After peripheral nerve injury, spontaneous ectopic activity arising from the peripheral axons plays an important role in inducing central sensitization and neuropathic pain. Recent evidence indicates that activation of spinal cord microglia also contributes to the development of neuropathic pain. In particular, activation of p38 mitogen-activated protein kinase (MAPK) in spinal microglia is required for the development of mechanical allodynia. However, activity-dependent activation of microglia after nerve injury has not been fully addressed. To determine whether spontaneous activity from C- or A-fibers is required for microglial activation, we used resiniferatoxin (RTX) to block the conduction of transient receptor potential vanilloid subtype 1 (TRPV1) positive fibers (mostly C- and Adelta-fibers) and bupivacaine microspheres to block all fibers of the sciatic nerve in rats before spared nerve injury (SNI), and observed spinal microglial changes 2 days later. RESULTS: SNI induced robust mechanical allodynia and p38 activation in spinal microglia. SNI also induced marked cell proliferation in the spinal cord, and all the proliferating cells (BrdU+) were microglia (Iba1+). Bupivacaine induced a complete sensory and motor blockade and also significantly inhibited p38 activation and microglial proliferation in the spinal cord. In contrast, and although it produced an efficient nociceptive block, RTX failed to inhibit p38 activation and microglial proliferation in the spinal cord. CONCLUSION: (1) Blocking peripheral input in TRPV1-positive fibers (presumably C-fibers) is not enough to prevent nerve injury-induced spinal microglial activation. (2) Peripheral input from large myelinated fibers is important for microglial activation. (3) Microglial activation is associated with mechanical allodynia.

Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.