ABSTRACT
Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5' flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-gamma resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.
Subject(s)
Matrix Metalloproteinase Inhibitors , Nitric Oxide/physiology , Repressor Proteins/physiology , Signal Transduction , WT1 Proteins/physiology , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/physiology , Enzyme Activation/genetics , Gene Expression Regulation, Enzymologic , Guanylate Cyclase/physiology , Humans , Lung/cytology , Lung/enzymology , Lung/metabolism , Matrix Metalloproteinase 9/biosynthesis , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide Synthase Type II/physiology , Promoter Regions, Genetic , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Binding/genetics , Receptors, Cytoplasmic and Nuclear/physiology , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/enzymology , Respiratory Mucosa/metabolism , Signal Transduction/genetics , Soluble Guanylyl Cyclase , Tumor Necrosis Factor-alpha/physiology , WT1 Proteins/antagonists & inhibitors , WT1 Proteins/biosynthesis , WT1 Proteins/metabolismABSTRACT
We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.
Subject(s)
Epithelial Cells/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Nitric Oxide/biosynthesis , Protein-Tyrosine Kinases/metabolism , Respiratory Mucosa/metabolism , Tumor Necrosis Factor-alpha/physiology , Bronchi/cytology , Bronchi/metabolism , Cell Line , Down-Regulation , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Integrin beta1/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/metabolism , Phosphorylation , Protein-Tyrosine Kinases/genetics , RNA, Small Interfering/genetics , Respiratory Mucosa/cytology , Signal Transduction , Stilbenes/pharmacology , Syk Kinase , Transcription Factor RelA/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Understanding the ecology of drug-resistant pathogens is essential for devising rational programs to preserve the effective lifespan of antimicrobial agents and to abrogate epidemics of drug-resistant organisms. Mathematical models predict that strain fitness is an important determinant of multidrug-resistant Mycobacterium tuberculosis transmission, but the effects of strain diversity have been largely overlooked. Here we compared the impact of resistance mutations on the transmission of isoniazid-resistant M. tuberculosis in San Francisco during a 9-y period. Strains with a KatG S315T or inhA promoter mutation were more likely to spread than strains with other mutations. The impact of these mutations on the transmission of isoniazid-resistant strains was comparable to the effect of other clinical determinants of transmission. Associations were apparent between specific drug resistance mutations and the main M. tuberculosis lineages. Our results show that in addition to host and environmental factors, strain genetic diversity can influence the transmission dynamics of drug-resistant bacteria.
Subject(s)
Antitubercular Agents/therapeutic use , Isoniazid/therapeutic use , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/genetics , Tuberculosis, Multidrug-Resistant/transmission , Alleles , Cell Lineage/genetics , Genetic Variation , Humans , MutationABSTRACT
Mast cells (MC) are important effector cells in allergic disorders. Recently, the role of MC in innate and adaptive immunity is gaining prominence. Nitric oxide is an important signaling molecule and its production in mast cell has been reported widely. However, controversy exists about whether MC produce NO. This review addresses the role of NO in MC biology and the reasons behind the controversy and discusses effects of NO in regulation of MC phenotype and function.
Subject(s)
Hypersensitivity, Immediate/immunology , Mast Cells/immunology , Nitric Oxide/physiology , Animals , Humans , Hypersensitivity, Immediate/metabolism , Mast Cells/metabolism , Mice , Nitric Oxide/metabolism , Protein Processing, Post-Translational , RatsABSTRACT
Mast cells (MC) are abundant in the lung and other peripheral tissue, where they participate in inflammatory processes against bacterial infections. Like other effector cells of the innate immune system, MC interact directly with a wide variety of infectious agents. This interaction results in MC activation and inflammatory mediator release. We demonstrated that MC interact with Mycobacterium tuberculosis, triggering the release of several prestored reagents, such as histamine and beta-hexosaminidase, and de novo synthesized cytokines, such as TNF-alpha and IL-6. A number of M. tuberculosis Ags, ESAT-6, MTSA-10, and MPT-63, have been implicated in MC activation and mediator release. A MC plasmalemmal protein, CD48, was implicated in interactions with mycobacteria because CD48 appeared to aggregate in the MC membrane at sites of bacterial binding and because Abs to CD48 inhibited the MC histamine response to mycobacteria. Cumulatively, these findings suggest that MC, even in the absence of opsonins, can directly recognize M. tuberculosis and its Ags and have the potential to play an active role in mediating the host's innate response to M. tuberculosis infection.
