ABSTRACT
Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.
Subject(s)
Flagella/metabolism , Peptides, Cyclic/biosynthesis , Plant Growth Regulators/biosynthesis , Plant Roots/microbiology , Pseudomonas fluorescens/metabolism , Antibiosis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Beta vulgaris/growth & development , Beta vulgaris/microbiology , DNA Transposable Elements , Flagella/genetics , Gene Expression , Movement , Peptides, Cyclic/metabolism , Plant Growth Regulators/metabolism , Plant Roots/growth & development , Pseudomonas fluorescens/genetics , Pythium/drug effects , Pythium/growth & development , Pythium/pathogenicity , Seedlings/growth & development , Seedlings/microbiology , Symbiosis , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
Ty1 retrotransposons of the yeast Saccharomyces cerevisiae are activated by different kinds of stress. Here we show that Ty1 transcription is stimulated under severe adenine starvation conditions. The Bas1 transcriptional activator, responsible for the induction of genes of the de novo AMP biosynthesis pathway (ADE) in the absence of adenine, is not involved in this response. Activation occurs mainly on Ty1 elements, whose expression is normally repressed by chromatin and is suppressed in a hta1-htb1Delta mutant that alters chromatin structure. Activation is also abolished in a snf2Delta mutant. Several regions of the Ty1 promoter are necessary to achieve full activation, suggesting that full integrity of the promoter sequences might be important for activation. Together, these observations are consistent with a model in which the activation mechanism involves chromatin remodeling at Ty1 promoters. The consequence of Ty1 transcriptional activation in response to adenine starvation is an increase in Ty1 cDNA levels and a relief of Ty1 dormancy. The retrotransposition of four native Ty1 elements increases in proportion to their increase in transcription. Implications for the regulation of Ty1 mobility by changes in Ty1 mRNA levels are discussed.
Subject(s)
Adenine/metabolism , Retroelements/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transcription, Genetic/genetics , Adenosine Monophosphate/biosynthesis , Adenosine Triphosphatases , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Mutation/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/deficiency , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.
Subject(s)
Aldehyde Oxidoreductases/deficiency , Aldehyde Oxidoreductases/metabolism , Pancreas/embryology , Tretinoin/metabolism , Aldehyde Oxidoreductases/genetics , Animals , Cell Differentiation/drug effects , Endoderm/cytology , Endoderm/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Deletion , Gene Expression Regulation, Developmental , Hedgehog Proteins , Heterozygote , Homeodomain Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Mesoderm/cytology , Mesoderm/drug effects , Mesoderm/metabolism , Metalloproteins/metabolism , Mice , Mice, Knockout , Mice, Mutant Strains , Pancreas/cytology , RNA, Messenger/metabolism , Trans-Activators/deficiency , Trans-Activators/metabolism , Transcription Factors/metabolism , Transgenes , Tretinoin/administration & dosage , Tretinoin/pharmacology , beta-Galactosidase/metabolismABSTRACT
Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.
Subject(s)
Asthma/etiology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Trans-Activators/deficiency , Trans-Activators/genetics , Acetylcysteine/pharmacology , Animals , Antioxidants/metabolism , Asthma/metabolism , Asthma/pathology , Base Sequence , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Chemokine CCL11 , Chemokines, CC/metabolism , DNA, Complementary/genetics , Gene Expression Regulation , Lung/immunology , Lung/metabolism , Lung/pathology , Male , Mice , Mice, Knockout , NF-E2-Related Factor 2 , NF-kappa B/metabolism , Ovalbumin/immunology , Oxidation-Reduction , Oxidative Stress , Th2 Cells/immunologyABSTRACT
Signal transducers and activators of transcription (STATs) are a family of transcription factors linked to class I cytokine receptors. In the present study, we investigated whether their distribution in the hypothalamus reflects the feedback regulation by growth hormone and what role they might play in the functioning of target neurones. We demonstrate that each of the seven known STATs has a distinct distribution in the hypothalamus. Notably, the STAT5 proteins, that are important in growth hormone (GH) and prolactin signalling in peripheral tissues, were expressed in somatostatin neurones of the periventricular nucleus and dopamine neurones of the arcuate nucleus. Because somatostatin neurones are regulated by feedback from circulating GH, we investigated the importance of STAT5 in these neurones. We demonstrate that STAT5b protein expression, similar to somatostatin mRNA, is sexually dimorphic in the periventricular nucleus of rats and mice. Furthermore, chronic infusion of male dwarf rats with GH increased the expression of STAT5b, while a single injection of GH into similar rats induced the phosphorylation of STAT5 proteins. The cellular abundance of somatostatin mRNA in STAT5b-deficient mice was significantly reduced in the periventricular nucleus, effectively reducing the sexually dimorphic expression. These results are consistent with the hypothesis that STAT5 proteins are involved in the feedback regulation of somatostatin neurones by GH, and that these neurones may respond to patterned GH secretion to reinforce sexual dimorphism in the GH axis.
Subject(s)
DNA-Binding Proteins/physiology , Growth Hormone/physiology , Hypothalamus/metabolism , Neurons/metabolism , Somatostatin/metabolism , Trans-Activators/physiology , Animals , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Disease Models, Animal , Dwarfism, Pituitary/metabolism , Feedback, Physiological/physiology , Female , Growth Hormone/deficiency , Hypothalamus/cytology , Male , Mice , Mice, Knockout , Midline Thalamic Nuclei/cytology , Midline Thalamic Nuclei/metabolism , Milk Proteins/genetics , Rats , Rats, Mutant Strains , STAT5 Transcription Factor , Sex Characteristics , Signal Transduction/genetics , Signal Transduction/physiology , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin. Dietary administration of broccoli seeds to nrf2(+/+) and nrf2(-/-) mice produced a approximately 1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small intestine of nrf2(+/+) mice but not nrf2(-/-) mice. An aqueous broccoli seed extract was prepared for treatment of cultured cells that contained ITC at approximately 600 mumol/L, composed of 61% 3-methylsulfinylpropyl ITC, 30% sulforaphane, 4% allyl ITC, and 4% 3-butenyl ITC. This extract induced GSTA1/2, GSTA3, NQO1, and GCLC between 3-fold and 10-fold in mouse Hepa-1c1c7 and rat liver RL-34 cells. The broccoli seed extract affected increases in GSTA3, GSTM1, and NQO1 proteins in nrf2(+/+) mouse embryonic fibroblasts but not in nrf2(-/-) mouse embryonic fibroblasts. These experiments show that broccoli seeds are effective at inducing antioxidant and detoxication proteins, both in vivo and ex vivo, in an Nrf2-dependent manner.
Subject(s)
Brassica/chemistry , DNA-Binding Proteins/physiology , Glutamate-Cysteine Ligase/biosynthesis , Glutathione Transferase/biosynthesis , Isothiocyanates/pharmacology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Trans-Activators/physiology , Animals , Cell Line , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Diet , Enzyme Induction/drug effects , Gene Expression/drug effects , Glucosinolates/analysis , Glucosinolates/metabolism , Intestine, Small/enzymology , Isothiocyanates/administration & dosage , Isothiocyanates/analysis , Liver/enzymology , Mice , Mice, Knockout , Mutation , NF-E2-Related Factor 2 , Plant Extracts/chemistry , Plant Extracts/pharmacology , Seeds/chemistry , Stomach/enzymology , Trans-Activators/deficiency , Trans-Activators/genetics , TransfectionABSTRACT
Mice with mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein-6 (LRP6) have a smaller and severely disorganized dorsal thalamus and lack thalamocortical projections. Using molecular markers, we showed that most dorsal thalamic and epithalamic neurons were missing, and most of the major dorsal thalamic nuclei were not identifiable. However, the ventral thalamus was essentially unaffected, although the dorsal thalamic defect leads to rostral displacement of portions of the ventral thalamus. Analysis of younger embryos showed that epithalamic and dorsal thalamic neurons were not produced at early stages of development, whereas ventral thalamic neurons were still produced. These defects were accompanied by improper formation of the boundary between dorsal and ventral thalamus, the zona limitans interthalamica (ZLI). Furthermore, the expression of an early marker of posterior forebrain development that marks the compartment from the midbrain-hindbrain junction to the ZLI (including the future dorsal thalamus, pretectum, and midbrain) was disrupted, supporting the idea that diencephalic development is abnormal from very early in embryogenesis. This study provides compelling in vivo evidence that thalamic development requires normal activity of the LRP6-mediated canonical Wnt signaling pathway.
Subject(s)
Intercellular Signaling Peptides and Proteins/physiology , Receptors, LDL/physiology , Thalamus/embryology , Animals , Cytoskeletal Proteins/physiology , Diencephalon/abnormalities , Diencephalon/embryology , Gestational Age , Hedgehog Proteins , LDL-Receptor Related Proteins , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , Mice, Knockout , Morphogenesis , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/physiology , Receptors, LDL/deficiency , Receptors, LDL/genetics , Signal Transduction/physiology , Thalamic Nuclei/abnormalities , Thalamic Nuclei/embryology , Thalamus/abnormalities , Trans-Activators/analysis , Trans-Activators/deficiency , Trans-Activators/physiology , Wnt Proteins , Wnt-5a Protein , beta CateninABSTRACT
The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.
Subject(s)
Cytoskeletal Proteins/metabolism , Lung/embryology , Trans-Activators/metabolism , Animals , Base Sequence , Cell Differentiation , Cell Division , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , DNA, Complementary/genetics , Doxycycline/pharmacology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Deletion , Immunohistochemistry , In Situ Hybridization , Integrases/genetics , Lung/cytology , Lung/metabolism , Mice , Mice, Mutant Strains , Mice, Transgenic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Trans-Activators/deficiency , Trans-Activators/genetics , Viral Proteins/genetics , beta CateninABSTRACT
In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.
Subject(s)
DNA-Binding Proteins/physiology , Interleukin-12/metabolism , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/physiology , Signal Transduction/immunology , Th1 Cells/cytology , Th1 Cells/immunology , Trans-Activators/physiology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Division/genetics , Cell Division/immunology , Cell Line , Clone Cells , Cytoplasm/immunology , Cytoplasm/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/immunology , Interleukin-12/physiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Peptide Fragments/immunology , Peptide Fragments/metabolism , Phosphorylation , Receptors, Interleukin/metabolism , Receptors, Interleukin-12 , STAT3 Transcription Factor , STAT4 Transcription Factor , Signal Transduction/genetics , Th1 Cells/metabolism , Trans-Activators/deficiency , Trans-Activators/genetics , Trans-Activators/metabolism , Tyrosine/metabolism , Tyrosine/physiologyABSTRACT
There is growing interest in the potential health benefits of tea, and a recent report described the potent antimutagenic activity of white tea in comparison with green tea against several heterocyclic amines, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [Mutat. Res. 495 (2001) 61]. We compared the inhibitory effects of white and green teas with sulindac, a nonsteroidal anti-inflammatory agent, in two different mouse models of intestinal tumorigenesis. In the Apc(min) mouse, white and green teas given at human-relevant concentrations (1.5% w/v, 2-min brew), and sulindac (80 ppm in the drinking water), each suppressed polyp formation by approximately 50%, and the combination of white tea plus sulindac was more effective than either treatment alone (P=0.05). Mice expressing an N-terminally truncated, oncogenic version of beta-catenin (A 33(delta N beta-cat) mutant mice) developed colonic aberrant crypt foci (ACF) spontaneously, but PhIP treatment increased the incidence and number of ACF per colon. In the normal-looking intestinal mucosa of Apc(min) and A 33(delta N beta-cat) mice, white tea plus sulindac treatment markedly attenuated the expression of beta-catenin protein, and this was recapitulated in vitro in cells transiently transfected with beta-catenin plus Tcf-4 and treated with tea or the major tea polyphenol epigallocatechin-3-gallate (EGCG). Expression of a beta-catenin/Tcf reporter was inhibited by EGCG in the transfected cells, and the beta-catenin/Tcf target genes cyclin D1 and c-jun were downregulated in vivo by tea plus sulindac treatment. Collectively, the data support a chemopreventive role for tea and sulindac against intermediate and late stages of colon cancer, via effects on the beta-catenin/Tcf signaling pathway.
Subject(s)
Adenomatous Polyposis Coli Protein/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Catechin/analogs & derivatives , Intestinal Neoplasms/prevention & control , Plant Extracts/pharmacology , Polyps/prevention & control , Sulindac/pharmacology , Tea , Adenomatous Polyposis Coli Protein/deficiency , Adenomatous Polyposis Coli Protein/genetics , Animals , Catechin/pharmacology , Cell Line , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Drug Combinations , Imidazoles/toxicity , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Intestinal Neoplasms/chemically induced , Intestinal Neoplasms/metabolism , Luciferases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Polyps/chemically induced , Polyps/metabolism , Trans-Activators/deficiency , Trans-Activators/genetics , Trans-Activators/metabolism , beta CateninABSTRACT
PRL secretion from the anterior pituitary gland is inhibited by dopamine produced in the tuberoinfundibular dopamine neurons of the hypothalamus. The activity of tuberoinfundibular dopamine neurons is stimulated by PRL; thus, PRL regulates its own secretion by a negative feedback mechanism. PRL receptors are expressed on tuberoinfundibular dopamine neurons, but the intracellular signaling pathway is not known. We have observed that mice with a disrupted signal transducer and activator of transcription 5b gene have grossly elevated serum PRL concentrations. Despite this hyperprolactinemia, mRNA levels and immunoreactivity of tyrosine hydroxylase, the key enzyme in dopamine synthesis, were significantly lower in the tuberoinfundibular dopamine neurons of these signal transducer and activator of transcription 5b-deficient mice. Concentrations of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence were also significantly lower in signal transducer and activator of transcription 5b-deficient mice than in wild-type mice. No changes were observed in nonhypothalamic dopaminergic neuronal populations, indicating that the effects were selective to tuberoinfundibular dopamine neurons. These data indicate that in the absence of signal transducer and activator of transcription 5b, PRL signal transduction in tuberoinfundibular dopamine neurons is impaired, and they demonstrate that this transcription factor plays an obligatory and nonredundant role in mediating the negative feedback action of PRL on tuberoinfundibular dopamine neurons.
Subject(s)
DNA-Binding Proteins/physiology , Milk Proteins , Prolactin/metabolism , Trans-Activators/physiology , Transcription Factors/physiology , Animals , DNA-Binding Proteins/deficiency , Dopamine/metabolism , Feedback , Hypothalamus/physiology , Mice , Mice, Inbred Strains , Mice, Knockout/genetics , Neurons/physiology , Prolactin/blood , STAT5 Transcription Factor , Trans-Activators/deficiencyABSTRACT
The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.
Subject(s)
Nitric Oxide/biosynthesis , Trans-Activators/physiology , Animals , Arginase/biosynthesis , Arginine/deficiency , Cells, Cultured , Down-Regulation/immunology , Interleukin-13/physiology , Interleukin-4/physiology , Macrophage Activation , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Macrophages, Peritoneal/parasitology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , STAT6 Transcription Factor , Substrate Specificity/immunology , Toxoplasmosis, Animal/enzymology , Toxoplasmosis, Animal/immunology , Toxoplasmosis, Animal/metabolism , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
IL-4 promotes allergic responses and inhibits the production of proinflammatory cytokines by monocytes and macrophages. The promotion of allergic responses by IL-4 has been shown to be absolutely dependent on the transcription factor STAT6. We report here that the inhibitory effects of IL-4 on the production of TNF-alpha or IL-12 by macrophages had both STAT6-dependent and -independent components, depending on the stimuli. IL-4 failed to inhibit the release of TNF-alpha or IL-12 from STAT6 null macrophages stimulated with LPS alone. However, IL-4 still induced significant inhibition of the production of TNF-alpha and IL-12 from STAT6 null macrophages that were stimulated with the more physiologically relevant combination of LPS and IFN-gamma. These data show that STAT6 is required for the IL-4-mediated inhibition of the production of TNF-alpha and IL-12 stimulated by LPS alone, but that IL-4 also activates distinct, STAT6 independent mechanism(s) that inhibit the IFN-gamma-mediated enhancement of IL-12 and TNF-alpha production.