ABSTRACT
CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement-mediated attack. Human transgenic CD46 homozygous mice (CD46+/+ ) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46+/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro-inflammatory mediators such as IL-6, TNF, IL-12, and IL-1ß In the presence of M-CSF or GM-CSF, CD46 signaling enhances monocyte-macrophage differentiation. Additionally, CD46+/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46+/+ peritoneal macrophages aggravated septic responses in wild-type mice, but the depletion of macrophages partially alleviated septic reactions in CD46+/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages.
Subject(s)
Disease Susceptibility , Macrophages/immunology , Macrophages/metabolism , Membrane Cofactor Protein/genetics , Meningococcal Infections/genetics , Meningococcal Infections/immunology , Neisseria meningitidis/immunology , Sepsis , Adoptive Transfer , Alternative Splicing , Animals , Apoptosis/genetics , Apoptosis/immunology , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation , Genetic Predisposition to Disease , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunophenotyping , Inflammation Mediators/metabolism , Lipopolysaccharides/immunology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Membrane Cofactor Protein/metabolism , Meningococcal Infections/microbiology , Meningococcal Infections/mortality , Mice , Mice, Transgenic , Phagocytosis/immunology , Phenotype , Signal TransductionABSTRACT
UNLABELLED: Neisseria meningitidis colonizes the nasopharyngeal mucosa of healthy populations asymptomatically, although the bacterial surface is rich in motifs that activate the host innate immunity. What determines the tolerant host response to this bacterium in asymptomatic carriers is poorly understood. We demonstrated that the conserved meningococcal surface protein NhhA orchestrates monocyte (Mo) differentiation specifically into macrophage-like cells with a CD200R(hi) phenotype (NhhA-Mφ). In response to meningococcal stimulation, NhhA-Mφ failed to produce proinflammatory mediators. Instead, they upregulated interleukin-10 (IL-10) and Th2/regulatory T cell (Treg)-attracting chemokines, such as CCL17, CCL18, and CCL22. Moreover, NhhA-Mφ were highly efficient in eliminating bacteria. The in vivo validity of these findings was corroborated using a murine model challenged with N. meningitidis systematically or intranasally. The NhhA-modulated immune response protected mice from septic shock; Mo/Mφ depletion abolished this protective effect. Intranasal administration of NhhA induced an anti-inflammatory response, which was associated with N. meningitidis persistence at the nasopharynx. In vitro studies demonstrated that NhhA-triggered Mo differentiation occurred upon engaged Toll-like receptor 1 (TLR1)/TLR2 signaling and extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) activation and required endogenously produced IL-10 and tumor necrosis factor alpha (TNF-α). Our findings reveal a strategy that might be adopted by N. meningitidis to maintain asymptomatic nasopharyngeal colonization. IMPORTANCE: Neisseria meningitidis is an opportunistic human-specific pathogen that colonizes the nasopharyngeal mucosa asymptomatically in approximately 10% of individuals. Very little is known about how this bacterium evades immune activation during the carriage stage. Here, we observed that N. meningitidis, via the conserved surface protein NhhA, skewed monocyte differentiation into macrophages with a CD200R(hi) phenotype. Both in vivo and in vitro data demonstrated that these macrophages, upon meningococcal infection, played an important role in forming a homeostatic immune microenvironment through their capacity to eliminate invading bacteria and to generate anti-inflammatory mediators. This work provides novel insight into the mechanisms underlying the commensal persistence of N. meningitidis.
Subject(s)
Adhesins, Bacterial/metabolism , Carrier State/microbiology , Homeostasis , Macrophages/immunology , Meningococcal Infections/microbiology , Nasopharynx/microbiology , Neisseria meningitidis/physiology , Adhesins, Bacterial/genetics , Animals , Cell Differentiation , Chemokine CCL17/genetics , Chemokine CCL22/genetics , Chemokines, CC/genetics , Disease Models, Animal , Humans , Immunity, Innate , Interleukin-10/genetics , Interleukin-10/immunology , Meningococcal Infections/immunology , Mice , Monocytes/immunology , Monocytes/physiology , Neisseria meningitidis/genetics , Signal Transduction , Symbiosis , T-Lymphocytes, Regulatory/immunology , Toll-Like Receptor 2/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Neisseria meningitidis is an opportunistic human pathogen that usually colonizes the nasopharyngeal mucosa asymptomatically. Upon invasion into the blood and central nervous system, this bacterium triggers a fulminant inflammatory reaction with the manifestations of septicemia and meningitis, causing high morbidity and mortality. To reveal the bacterial adaptations to specific and dynamic host environments, we performed a comprehensive proteomic survey of N. meningitidis isolated from the nasal mucosa, CSF and blood of a mouse disease model. We could identify 51 proteins whose expression pattern has been changed during infection, many of which have not yet been characterized. The abundance of proteins was markedly lower in the bacteria isolated from the nasal mucosa compared to the bacteria from the blood and CSF, indicating that initiating adhesion is the harshest challenge for meningococci. The high abundance of the glutamate dehydrogenase (GdhA) and Opa1800 proteins in all bacterial isolates suggests their essential role in bacterial survival in vivo. To evaluate the biological relevance of our proteomic findings, four candidate proteins from representative functional groups, such as the bacterial chaperone GroEL, IMP dehydrogenase GuaB, and membrane proteins PilQ and NMC0101, were selected and their impact on bacterial fitness was investigated by mutagenesis assays. This study provides an integrated picture of bacterial niche-specific adaptations during consecutive infection processes.
Subject(s)
Adaptation, Physiological , Meningococcal Infections/microbiology , Neisseria meningitidis/physiology , Animals , Bacteremia/microbiology , Blood/microbiology , Carrier State/microbiology , Cerebrospinal Fluid/microbiology , DNA Mutational Analysis , Disease Models, Animal , Meningitis, Bacterial/microbiology , Mice , Nasal Mucosa/microbiology , Neisseria meningitidis/chemistry , Neisseria meningitidis/isolation & purification , Proteome/analysis , Virulence Factors/geneticsABSTRACT
Activation of macrophages by Toll-like receptors (TLRs) and functionally related proteins is essential for host defense and innate immunity. TLRs recognize a wide variety of pathogen-associated molecules. Here, we demonstrate that the meningococcal outer membrane protein NhhA has immunostimulatory functions and triggers release of proinflammatory cytokines from macrophages. NhhA-induced cytokine release was found to proceed via two distinct pathways in RAW 264.7 macrophages. Interleukin-6 (IL-6) secretion was dependent on activation of TLR4 and required the TLR signaling adaptor protein MyD88. In contrast, release of tumor necrosis factor (TNF) was TLR4 and MyD88 independent. Both pathways involved NF-κB-dependent gene regulation. Using a PCR-based screen, we could identify additional targets of NhhA-dependent gene activation such as the cytokines and growth factors IL-1α, IL-1ß, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In human monocyte-derived macrophages, G-CSF, GM-CSF, and IL-6 were found to be major targets of NhhA-dependent gene regulation. NhhA induced transcription of IL-6 and G-CSF mRNA via TLR4-dependent pathways, whereas GM-CSF transcription was induced via TLR4-independent pathways. These data provide new insights into the role of NhhA in host-pathogen interaction.
Subject(s)
Adhesins, Bacterial/physiology , Cytokines/biosynthesis , Macrophages/immunology , Membrane Glycoproteins/immunology , NF-kappa B/metabolism , Neisseria meningitidis/immunology , Toll-Like Receptor 4/genetics , Cells, Cultured , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Interleukin-6/biosynthesis , Macrophages/metabolism , Membrane Glycoproteins/genetics , Monocytes/immunology , Signal Transduction , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolismABSTRACT
Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvß3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.
Subject(s)
Meningitis, Meningococcal/metabolism , Meningitis, Meningococcal/microbiology , Neisseria meningitidis/drug effects , Neisseria meningitidis/pathogenicity , Nitric Oxide/metabolism , Thyroid Hormones/pharmacology , Animals , Cell Line , Gene Expression Regulation, Enzymologic/drug effects , Humans , Integrin alphaVbeta3/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Intracellular Space/microbiology , Meningitis, Meningococcal/pathology , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , Survival AnalysisABSTRACT
It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5' and 3'-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3'-region, there is a dramatic gain of long-range chromatin loops impinging on the 5'-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.
Subject(s)
Chromatin/metabolism , RNA, Untranslated/metabolism , Repressor Proteins/metabolism , Transcriptional Activation , Alleles , Animals , Binding Sites , CCCTC-Binding Factor , Chromatin/genetics , Chromatin Immunoprecipitation , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , Crosses, Genetic , DNA Methylation , DNA Replication , Genomic Imprinting , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Liver/cytology , Liver/metabolism , Mice , Mice, Transgenic , Protein Interaction Mapping , RNA, Long Noncoding , RNA, Untranslated/genetics , Repressor Proteins/genetics , Structure-Activity Relationship , Time FactorsABSTRACT
Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.
Subject(s)
Apoptosis , Bacterial Outer Membrane Proteins/physiology , Macrophages/physiology , Meningococcal Infections/pathology , Neisseria meningitidis/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/physiology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/pharmacology , Cell Lineage , Macrophages/drug effects , Macrophages/metabolism , Meningococcal Infections/immunology , Mice , Monocytes/cytology , Monocytes/physiology , Neisseria meningitidis/genetics , Nitric Oxide/metabolism , Nitric Oxide/physiology , Protein Binding , Signal Transduction/genetics , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts.
Subject(s)
Gene Expression Regulation, Developmental , Genomic Imprinting/genetics , Germ Cells/growth & development , Germ Cells/metabolism , Alleles , Animals , Cells, Cultured , Embryonic Stem Cells , Female , Male , Mice , Mice, Inbred C57BL , RNA, Long Noncoding , RNA, UntranslatedABSTRACT
Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.
Subject(s)
Chromosomes/chemistry , Cloning, Molecular/methods , Epigenesis, Genetic/physiology , Gene Expression Regulation/genetics , Animals , Animals, Newborn , Binding Sites , CCCTC-Binding Factor , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Embryonic Stem Cells , Genomic Imprinting/physiology , Liver/metabolism , Mice , Mice, Transgenic , Models, Biological , Nucleic Acid Conformation , Oligonucleotide Array Sequence Analysis/methods , RNA, Long Noncoding , RNA, Untranslated/genetics , Repressor Proteins/metabolism , Trans-ActivatorsABSTRACT
In the dipteran Chironomus tentans, actin binds to hrp65, a nuclear protein associated with mRNP complexes. Disruption of the actin-hrp65 interaction in vivo by the competing peptide 65-2CTS reduces transcription drastically, which suggests that the actin-hrp65 interaction is required for transcription. We show that the inhibitory effect of the 65-2CTS peptide on transcription is counteracted by trichostatin A, a drug that inhibits histone deacetylation. We also show that actin and hrp65 are associated in vivo with p2D10, an evolutionarily conserved protein with histone acetyltransferase activity that acts on histone H3. p2D10 is recruited to class II genes in a transcription-dependent manner. We show, using the Balbiani ring genes of C. tentans as a model system, that p2D10 is cotranscriptionally associated with the growing pre-mRNA. We also show that experimental disruption of the actin-hrp65 interaction by the 65-2CTS peptide in vivo results in the release of p2D10 from the transcribed genes, reduced histone H3 acetylation, and a lower level of transcription activity. Furthermore, antibodies against p2D10 inhibit run-on elongation. Our results suggest that actin, hrp65, and p2D10 are parts of a positive feedback mechanism that contributes to maintaining the active transcription state of a gene by recruiting HATs at the RNA level.
Subject(s)
Actins/metabolism , Chromatin/metabolism , RNA Precursors/physiology , RNA, Messenger/physiology , Transcription, Genetic/physiology , Acetyltransferases/metabolism , Animals , Antibodies/pharmacology , Chironomidae/genetics , Chironomidae/growth & development , DNA Polymerase III/metabolism , Down-Regulation/genetics , Down-Regulation/immunology , Histone Acetyltransferases , Insect Proteins/metabolism , Nuclear Proteins/metabolism , RNA Polymerase II/metabolism , RNA-Binding Proteins , Transcription Factors, TFIII/immunology , Transcription Factors, TFIII/metabolismABSTRACT
Neutrophils from patients with chronic myeloid leukaemia (CML) have an aberrant expression of leukotriene (LT)C4 synthase. In order to learn more about the regulation of this abnormality, LTC4 synthase mRNA expression was determined by reverse transcription polymerase chain reaction. A digoxigenin (DIG)-labelled LTC4 synthase RNA was synthesized and incubated in cytosolic extracts from CML neutrophils, normal neutrophils and eosinophils. LTC4 synthase mRNA was detected in total but not cytoplasmic RNA from normal neutrophils. In contrast, LTC4 synthase mRNA was found in the cytoplasm of CML neutrophils and in normal eosinophils, which also express the enzyme. The DIG-labelled LTC4 synthase RNA was, as opposed to normal neutrophils, degraded in cytosolic extracts from CML neutrophils. The degradation was time dependent and cell concentration dependent. Degradation was also seen in eosinophils, indicating that degradation of LTC4 synthase RNA was correlated to the expression of the protein. This study showed that the difference in expression of LTC4 synthase in normal and CML neutrophils was not because of a total lack of LTC4 synthase mRNA in normal neutrophils. However normal neutrophils lack, in contrast to CML neutrophils, LTC4 synthase mRNA in the cytoplasm. This discrepancy is not caused by a stabilized LTC4 synthase RNA in the cytosol of CML neutrophils. Instead an abnormal degradation of LTC4 synthase RNA was found in the cytosol of CML neutrophils.
Subject(s)
Glutathione Transferase/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Neutrophils/enzymology , RNA Stability , Cytosol/enzymology , Gene Expression , Glutathione Transferase/biosynthesis , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolismABSTRACT
Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.
Subject(s)
Anti-Bacterial Agents/pharmacology , Genetic Markers/genetics , Macrolides/pharmacology , Pichia/drug effects , Pichia/genetics , Transformation, Genetic/genetics , Acetyl-CoA Carboxylase/genetics , Blotting, Southern , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , Polymerase Chain ReactionABSTRACT
A human cDNA library was screened for proteins interacting with the deafness locus putative guanine nucleotide exchange factor (DelGEF) using a yeast two-hybrid system. A protein with a predicted size of 9 kDa was identified as a binding partner, this protein was designated DelGEF interacting protein 1 (DelGIP1). The interaction between DelGEF and DelGIP1 was verified by co-immunoprecipitation of a DelGEF-DelGIP1 complex from cell lysates. Highly conserved homologues of DelGIP1 were identified in higher and lower eukaryotes by database searching. The human DelGIP1 gene is ubiquitously expressed as judged by human multiple tissue Northern blot analysis. DelGEF was recently shown to interact with Sec5, a protein involved in secretion, and to regulate secretion of proteoglycans. Downregulation of endogenous DelGIP1 in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGIP1 in the secretion process.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/physiology , Guanine Nucleotide Exchange Factors/metabolism , Amino Acid Sequence , Carrier Proteins/metabolism , Evolution, Molecular , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Precipitin Tests , Proteoglycans/metabolism , RNA, Messenger/metabolism , Sequence Alignment , Tissue Distribution , Two-Hybrid System Techniques , Vesicular Transport ProteinsABSTRACT
OBJECTIVE: Leukotriene (LT) C(4) synthase (LTC(4)S) is the key enzyme in the biosynthesis of LTC(4), which has been reported to stimulate the growth of human myeloid progenitor cells and is specifically overproduced in chronic myeloid leukemia (CML). The aim of this study was to clarify the expression of LTC(4)S during normal and leukemic myelopoiesis and to investigate the correlation between abnormal LTC(4)S expression in CML myeloid cells and the activity of the disease-specific tyrosine kinase p210 BCR-ABL. MATERIALS AND METHODS: Immature and mature myeloid cell subpopulations were isolated with magnetic cell sorting from healthy volunteer bone marrow (n = 11) and CML patient peripheral blood (n = 8), respectively. The cells were subjected to analysis of LTC(4)S protein expression and activity. Expression of LTC(4)S was investigated in CD16(+) neutrophils from CML patients before and after 1 month of medication with imatinib mesylate (STI571), which is a specific inhibitor of p210 BCR-ABL. RESULTS: Among normal cells, the highest enzyme activity was observed in the most immature, CD34(+) progenitor cell-enriched and CD15(+) myelocyte-enriched fractions. Subsequently, LTC(4)S activity decreased with increasing maturity, with only negligible amounts of LTC(4) produced in CD16(+) neutrophils. LTC(4)S was expressed at the protein level in the immature myeloid cell fractions but not in CD16(+) cells. In CML cells, LTC(4)S activity and expression were consistently elevated. Thus, the CML CD34(+) and CD15(+) cell fractions, as well as the CD11b(+) myelocyte/metamyelocyte-enriched fractions, produced 6 to 10 times as much LTC(4) as the corresponding normal cells. Again, enzyme expression was highest in the most immature cells, although evident LTC(4)S expression and activity remained in CML CD16(+) neutrophils. Interestingly, treatment of five CML patients with imatinib mesylate down-regulated the abnormal neutrophil LTC(4)S expression and activity. CONCLUSIONS: Expression of LTC(4)S in immature myelopoid cells is in line with a role for this enzyme in myelopoiesis. In addition, consistent overexpression of LTC(4)S in CML and the correlation to p210 BCR-ABL activity suggests that LTC(4)S may be involved in leukemic pathogenesis.
Subject(s)
Gene Expression Regulation, Leukemic , Glutathione Transferase/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Myeloid Cells/enzymology , Myelopoiesis , Myelopoiesis/physiology , Antigens, CD34 , Benzamides , Case-Control Studies , Cell Differentiation/drug effects , Gene Expression Regulation, Leukemic/drug effects , Glutathione Transferase/physiology , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Lewis X Antigen , Myeloid Cells/cytology , Myelopoiesis/drug effects , Neutrophils/enzymology , Neutrophils/pathology , Piperazines/pharmacology , Piperazines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Receptors, IgGABSTRACT
In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two-hybrid system. The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.