scholarly journals Hypoxic induction of gene expression in chronic granulomatous disease- derived B-cell lines: oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 756-761 ◽  
Author(s):  
RH Wenger ◽  
HH Marti ◽  
CC Schuerer-Maly ◽  
I Kvietikova ◽  
C Bauer ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Oxygen Sensing ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Wild Type ◽  
Superoxide Generation ◽  
Cytochrome B558 ◽  
Nadph Oxidase Complex

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild- type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia- activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.

scholarly journals Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis

1996 ◽  
Vol 315 (2) ◽  
pp. 571-575 ◽  
Author(s):  
Colin D. PORTER ◽  
KURIBAYASHI KURIBAYASHI ◽  
Mohamed H. PARKAR ◽  
Dirk ROOS ◽  
Christine KINNON
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Binding Domain ◽  
Granulomatous Disease ◽  
Cytochrome B558 ◽  
Stable Association ◽  
P22 Phox ◽  
Fad Binding

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

scholarly journals Genomic structure, chromosomal localization, start of transcription, and tissue expression of the human p40-phox, a new component of the nicotinamide adenine dinucleotide phosphate-oxidase complex

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2714-2721 ◽  
Author(s):  
S Zhan ◽  
N Vazquez ◽  
S Zhan ◽  
FB Wientjes ◽  
ML Budarf ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Genomic Structure ◽  
Hybrid Cell ◽  
Cell Lineage ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Single Copy ◽  
Preliminary Evidence ◽  
Nicotinamide Adenine

p40-phox is a newly isolated cytosolic component of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase that copurifies with p67-phox. Although its function is not well defined, preliminary evidence indicates that it is a component of the cytosolic complex. We report the characterization of the human p40-phox gene, which is single copy and spans approximately 18 kb with 10 exons. Based on fluorescent in situ hybridization (FISH) studies and analysis of somatic hybrid cell lines, the chromosomal location of p40-phox is human chromosome 22q13.1. The start of transcription has been mapped to bp -156. The expression of p40-phox message is restricted to hematopoietic cells. In addition to identifying the mRNA transcript on Northern blot analysis in cells known to express components of the NADPH-oxidase, polymorphonuclear leukocytes, monocytes, B lymphoblastoid cell lines, and eosinophils, p40-phox is also expressed in two other cell types of white cell lineage, mast cells, and basophils. In addition, the mRNA for p40-phox is expressed in megakaryocytic cells, but not in erythroid cells.

NADPH oxidase promotes NF-κB activation and proliferation in human airway smooth muscle

2002 ◽  
Vol 282 (4) ◽  
pp. L782-L795 ◽  
Author(s):  
Sukhdev S. Brar ◽  
Thomas P. Kennedy ◽  
Anne B. Sturrock ◽  
Thomas P. Huecksteadt ◽  
Mark T. Quinn ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Nadph Oxidase ◽  
Airway Smooth Muscle ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nuclear Factor Κb ◽  
Nicotinamide Adenine ◽  
Phagocytic Cells ◽  
Diphenylene Iodonium ◽  
Reduced Nicotinamide Adenine Dinucleotide

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O[Formula: see text]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O[Formula: see text] as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22phox. Transfection with p22phoxantisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-κB (NF-κB), and overexpression of a superrepressor form of the NF-κB inhibitor IκBα significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22phoxregulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-κB.

scholarly journals MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Danxia Zhu ◽  
Cheng Fang ◽  
Wenting He ◽  
Chen Wu ◽  
Xiaodong Li ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Expression Vector ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

scholarly journals Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 804-811 ◽  
Author(s):  
TG Gabig ◽  
CD Crean ◽  
PL Mantel ◽  
R Rosli
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Cell Lines ◽  
Wild Type ◽  
Free System ◽  
Hl60 Cells ◽  
Cell Free System ◽  
Nadph Oxidase Activation ◽  
O2 Production

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1–166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

scholarly journals 156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox.

1994 ◽  
Vol 180 (6) ◽  
pp. 2329-2334 ◽  
Author(s):  
J H Leusen ◽  
B G Bolscher ◽  
P M Hilarius ◽  
R S Weening ◽  
W Kaulfersch ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Light Chain ◽  
Electron Flow ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Gene Encoding ◽  
Cytosolic Proteins ◽  
Cytochrome B558 ◽  
A Cell ◽  
Src Homology ◽  
P22 Phox

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH-dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47-phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.

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