Toll-interacting protein modulates colitis susceptibility in mice.

BACKGROUND: The intestinal epithelium accommodates with a myriad of commensals to maintain immunological homeostasis, but the underlying mechanisms regulating epithelial responsiveness to flora-derived signals remain poorly understood. Herein, we sought to determine the role of the Toll/interleukin (IL)-1 receptor regulator Toll-interacting protein (Tollip) in intestinal homeostasis. METHODS: Colitis susceptibility was determined after oral dextran sulfate sodium (DSS) administration or by breeding Tollip on an IL-10 background. The intestinal flora was depleted with 4 antibiotics before DSS exposure to assess its contribution in colitis onset. Bone marrow chimeras were generated to identify the cellular compartment, whereby Tollip may negatively regulate intestinal inflammation in response to DSS. Tollip-dependent epithelial barrier functions were studied in vitro by using Tollip-knockdown in Caco-2 cells and in vivo by immunohistochemistry and fluorescein isothiocyanate-labeled dextran gavage. RESULTS: Genetic ablation of Tollip did not lead to spontaneous intestinal inflammatory disorders. However, Tollip deficiency aggravated spontaneous disease onset in IL-10 mice and increased susceptibility to DSS colitis. Increased colitis severity in Tollip-deficient mice was not improved by bacterial flora depletion using broad-spectrum antibiotics. In addition, DSS exposure of bone marrow chimeric mice revealed a protective role for Tollip in nonhematopoietic cells. Knockdown of Tollip in epithelial cells led to exaggerated NFκ-B activity and proinflammatory cytokine secretion. Finally, DSS-treated Tollip mice showed enhanced intestinal permeability and increased epithelial apoptosis when compared with wild-type controls, a finding that coincided with tight junction alterations on injury. CONCLUSION: Overall, our data show an essential role for Tollip on colitis susceptibility in mice.

HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality.

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7-37) or the related GLP-I(7-36)amide. These hormones have specific beta-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep beta-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the beta-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.

Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.

OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC

Angiogenesis, the process of generating new blood vessels, is essential to embryonic development, organ formation, tissue regeneration and remodeling, reproduction and wound healing. Also, it plays an important role in many pathological conditions, including chronic inflammation and cancer. Angiogenesis is regulated by a complex interplay of growth factors, inflammatory mediators, adhesion molecules, morphogens and guidance molecules. Transcription factor SOX18 is transiently expressed in nascent endothelial cells during embryonic development and postnatal angiogenesis, but little is known about signaling pathways controlling its expression. The aim of this study was to investigate whether pro-angiogenic molecules and pharmacological inhibitors of angiogenesis modulate SOX18 expression in endothelial cells. Therefore, we treated human umbilical vein endothelial cells (HUVEC) with angiogenic factors, extracellular matrix proteins, inflammatory cytokines and nonsteroidal anti-inflammatory drugs (NSAID) and monitored SOX18 expression. We have observed that the angiogenic factor VEGF and the inflammatory cytokine TNF increase, while the NSAID ibuprofen and NS398 decrease the SOX18 protein level. These results for the first time demonstrate that SOX18 expression is modulated by factors and drugs known to positively or negatively regulate angiogenesis. This opens the possibility of pharmacological manipulation of SOX18 gene expression in endothelial cells to stimulate or inhibit angiogenesis.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

E2F1 mediates DNA damage and apoptosis through HCF-1 and the MLL family of histone methyltransferases.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

Transcriptional regulation of RNA polymerase III in mammalian cells

L'ARN Polymérase III (Pol III) transcrit un ensemble de petits ARN non traduits impliqués dans des processus cellulaires tels que la biosynthèse des protéines, la maturation des ARNs ou le contrôle transcriptionnel. De ce fait, la Pol III joue un rôle important dans la régulation de la croissance et la prolifération cellulaire. L'initiation de la transcription par la Pol III nécessite l'interaction entre des facteurs de transcription et le complexe de la Pol III lui-même. Un sous- complexe de la Pol III, composé de 3 sous-unités, HsRPC3, HsRPC6 et HsRPC7 sert d'intermédiaire dans cette interaction. Dans cette étude, nous avons caractérisé une nouvelle sous-unité de la Pol III, HsRPC7-Like, homologue à HsRPC7. Nous avons montré que ces deux homologues se trouvent spécifiquement chez les vertébrés. Ils proviennent d'un ancêtre commun qui, après duplication il y a 600 millions d'années, a donné naissance à ces deux paralogues. Dans les cellules humaines, deux formes de Pol III coexistent : l'une contientt HsRPC7, l'autre HsRPC7-Like. Nous avons localisé, à l'échelle du génome entier, la présence de ces deux formes de Pol III dans des cellules humaines et dans le foie de souris. Les deux sous-unités ont démontré des caractéristiques identiques, suggérant qu'elles possèdent des fonctions similaires. Cependant, nous avons analysé les motifs d'expression des gènes codant pour RPC7 et RPC7-Like dans des lignées cellulaires dans des conditions variées telles que la concentration de sérum et la densité cellulaire, ainsi que les motifs d'expression dans le foie de souris et des cellules d'hépatocarcinome de souris. Nos résultats suggèrent que l'expression de ces deux sous-untiés varie en fonction de l'activité de prolifération de la cellule. - RNA polymerase III (Pol III) transcribes a set of genes coding for short untranslated RNAs involved in essential cellular processes as for example protein biosynthesis, RNA maturation, and transcriptional control. Thereby Pol III plays an important role in regulating cell growth and proliferation. Initiation of Pol III transcription requires interactions between transcription factors and the Pol III core complex. A Pol III sub-complex composed of three subunits, HsRPC3, HsRPC6, and HsRPC7 mediates this interaction. In this study, we have characterized a new Pol III subunit, HsRPC7-Like, an homologue of HsRPC7. We have shown that these two homologues are specific to vertebrates and originate from an ancestor gene that duplicated 600 mio years ago to give birth to two paralogues. In human cells, two forms of Pol III coexist, one containing HsRPC7 and the other HsRPC7-Like. We have localized, genome-wide, these two Pol III forms in human cells and mouse liver. Both subunits were found on all types of Pol III genes, suggesting that they share similar function. However, we analysed the expression patterns of the RPC7 and RPC7-Like coding genes under various conditions of serum concentration and cell density in different cell lines, as well as expression patterns in mouse liver and mouse hepatocarcinoma cells. Our results suggest that the expression of these two subunits varies with the proliferation rate of the cell.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Toward Protein Biomarkers for Allergy: CD4+ T Cell Proteomics in Allergic and Nonallergic Subjects Sampled in and out of Pollen Season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

Extracellular matrix molecules enhance the neurotrophic effect of schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions.

Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti β1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.