Gp91-phox Gene Expression Research Articles

Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all-trans retinoic acid-induced superoxide generating activity of U937 cells

Ellagitannins (esters composed of glucose and ellagic acid) are hydrolyzed to generate ellagic acid in gut followed by conversion of ellagic acid to urolithins such as urolithin A by intestinal bacteria. Since urolithins are absorbed by gut easier than ellagitannins and ellagic acid, and show various physiological activities (e.g. anti-cancer, anti-cardiovascular disease, anti-diabetes mellitus, anti-obesity and anti-Alzheimer disease activities), they are expected as excellent health-promoting phytochemicals. Here, using human monoblast U937 cells, we investigated the effect of ellagic acid and urolithin A on the superoxide anion (O2−)-generating system of phagocytes, which is consisted of five specific protein factors (membrane proteins: p22-phox and gp91-phox, cytosolic proteins: p40-phox, p47-phox and p67-phox). Twenty micromolar of urolithin A enhanced the all-trans retinoic acid (ATRA)-induced O2−-generating activity (to ~175%) while 20 μM ellagic acid inhibited the ATRA-induced O2−-generating activity (to ~70%). Semiquantitative RT-PCR showed that transcription level of gp91-phox was certainly decreased (to ~70%) in ATRA plus ellagic acid-treated cells, while that of gp91-phox was significantly increased (to ~160%) in ATRA plus urolithin A-treated cells. Chromatin immunoprecipitation assay suggested that urolithin A enhanced acetylations of Lys-9 residues of histone H3 within chromatin surrounding the promoter region of gp91-phox gene, but ellagic acid suppressed the acetylations. Immunoblotting also revealed that ATRA plus urolithin A-treatment up-regulated protein levels of p22-phox (to ~160%) and gp91-phox (to ~170%) although ATRA plus ellagic acid-treatment down-regulated protein levels of p22-phox (to ~70%) and gp91-phox (to ~60%). These results suggested that conversion of ellagic acid to urolithin A in gut may bring about reverse effects on the gp91-phox gene expression, resulting in opposite alterations in O2−-generating activity of intestinal macrophages.

Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells

The membrane bound cytochrome b558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O2−)-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O2−-generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O2−-generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O2−-generating activity via up-regulation of gp91-phox gene expression in U937 cells.

0194 : Angiotensin II type 2 receptor reduces metabolic and vascular effects of type 1 diabetes in the mouse

The renin-angiotensin system has a key role in cardiovascular homeostasis, mainly through activation of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. Although AT2R opposes the effects of AT1R, with vasodilator and antitrophic properties, its effect in diabetes remains debated. Thus we determined the role of AT2R in endothelium-mediated dilation in a mouse model of type 1 diabetes. Diabetes was induced in three-month-old mice by streptozotocin (150mg/kg, ip). After 45 days, mesenteric resistance arteries were isolated and mounted on a wire myograph to measure vascular reactivity. The absence of AT2R reduced glucose tolerance and body weight and increased STZ-induced hyperglycemia, hyperinsulinemia, creatiniemia, blood thromboxaneA2, PGF2alpha and isoprostane. AT2R, COX-2 and NADPHoxidase subunits (gp91phox and p22phox) gene expression were higher in arteries of diabetic mice than in control animals. COX-2 level and oxidative stress were also higher in mesenteric and renal arteries in AT2R-/- mice. Endothelium-dependent relaxation, reduced in diabetic mice arteries, was normalized after cycloxygenase-2 (COX-2) (CAY10404) blockade or ROS reduction with tempol plus catalase in both AT2R-/- and AT2R+/+ mice. AT2R-dependent dilation in isolated perfused mesenteric arteries was greater in diabetic mice than in control animals. Thus, in a model of type 1 diabetes AT2R reduces the severity of diabetes possibly through a reduction of the production of ROS and COX-2-derived vasoconstrictor agents.

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

BackgroundFructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. MethodsWistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. ResultsComparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91phox and p22phox) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. ConclusionsFructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. General significanceControl of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.

The Importance of Myeloperoxidase in Apocynin-Mediated NADPH Oxidase Inhibition

Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFNγ/TNFα and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFNγ < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFNγ/TNFα-differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFNγ/TNFα, and PMN and has a subtle increase in p67phox in HL60 IFNγ/TNFα. The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO.

GCN5 Regulates the Superoxide-Generating System in Leukocytes Via Controlling gp91-phox Gene Expression

The superoxide anion (O(2)(-))-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other cells, including B lymphocytes. The O(2)(-)-generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O(2)(-)-generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 cell line, we show that GCN5 deficiency causes loss of the O(2)(-)-generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ∼4%) in GCN5-deficient cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 cells. When human monoblastic U937 cells were cultured in the presence of IFN-γ, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O(2)(-). Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-γ revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated, but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O(2)(-)-generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O(2)(-)-generating system in leukocytes.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study, we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMK I-like kinase (CKLiK) and CaMKKalpha, in regulating neutrophil differentiation and functional activation. Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation vs inhibition of CaMKs on neutrophil maturation. Expression of CaMKKalpha was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes, but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91(phox) gene expression. The CaMK inhibitor KN-93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKalpha inhibitor, STO-609. Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation.

Phase I/II Gene Therapy Study for Chronic Granulomatous Disease: Results, Lessons and Perspectives.

Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.

Up‐regulation of NADPH oxidase components and increased production of interferon‐gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P=0.036). Monocytes from SCD patients showed higher levels of p47(phox) phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P=0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P>0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Effects of BAY 41-2272, an activator of nitric oxide-independent site of soluble guanylate cyclase, on human NADPH oxidase system from THP-1 cells

We investigated the effects of the 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1 H-pyrazolo[3,4- b] pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) on the NADPH oxidase activity, gp91 phox gene expression, cyclic guanosine-3′,5′-monophosphate (cGMP) and cyclic adenosine-3′,5′-monophosphate (cAMP) levels in the human myelomonocytic THP-1 cell line. THP-1 cells treated with BAY 41-2272 (0.3–10 μM) for 48 h significantly increased the superoxide anion (O 2 •−) release. This increase was not affected when cells were pre-treated with the specific cGMP-phosphodiesterase inhibitor zaprinast, the soluble guanylate cyclase inhibitor 1 H-[1,2,4] oxidiazolo[4,3-α] quinoxalin-1-one (ODQ), the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl) adenine (SQ 22,536) or the nitric oxide synthase inhibitor N ω-nitro-l-arginine methyl ester (l-NAME). In addition, BAY 41-2272 (3 and 10 μM; 48 h) was able to increase gp91 phox gene expression on THP-1 cells. The pre-treatment with zaprinast, 3-isobutyl- l-methyl-xanthine (IBMX; 0.5 mM), ODQ, SQ 22,536 or l-NAME caused no additional effect on the expression of gp91 phox evoked by BAY 41-2272. Treatment of THP-1 cells with BAY 41-2272 caused a significant increase in cGMP and cAMP levels. Our findings show that BAY 41-2272 caused a significant increase on the O 2 •− release and gp91 phox gene expression by THP-1 cells, and an elevation of intracellular cGMP and cAMP levels. However, we could not detect a clear correlation between both O 2 •− release and gp91 phox gene expression with activation of cGMP and cAMP signaling pathways.

Sa.90. BAY 41-2272, an Activator of Nitric Oxide-Independent Site of Soluble Guanylate Cyclase, Induces gp91phox Gene Expression and Activates the Human NADPH Oxidase System in Monocytic THP-1 Cells

Sa.90. BAY 41-2272, an Activator of Nitric Oxide-Independent Site of Soluble Guanylate Cyclase, Induces gp91phox Gene Expression and Activates the Human NADPH Oxidase System in Monocytic THP-1 Cells

The Effect of IFN-γ and TNF-α on the NADPH Oxidase System of Human Colostrum Macrophages, Blood Monocytes, and THP-1 Cells

The aim of this work was to analyze the effect of Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in human colostrum macrophages (CM), peripheral blood monocytes (PBM), and myelomonocytic THP-1 cells. We also investigated the effect of IFN-gamma on the release of TNF-alpha by these cells. Our results show that under basal culture conditions, CM release more superoxide than PBM and THP-1 cells (p < 0.05). The addition of IFN-gamma, alone or in combination with TNF-alpha, increased spontaneous superoxide release by PBM and THP-1 cells (p < 0.05) and increased phorbol myristate acetate (PMA)-stimulated superoxide release by CM, PBM, and THP-1 cells (p < 0.05). The NADPH oxidase activity of THP-1 cells consistently remained lower than that of CM or PBM, despite a dramatic response to IFN-gamma and TNF-alpha. Under basal conditions, gp91-phox gene expression was significantly higher in CM and PBM compared with THP-1 cells (p < 0.05). The addition of IFN-gamma alone or in combination with TNF-alpha caused a dramatic increase in gp91-phox gene expression in THP-1 cells (p < 0.05) but not in CM or PBM. Under basal conditions or in the presence of IFN-gamma, CM released more TNF-alpha than PBM or THP-1 cells (p < 0.05). In addition, PBM released more TNF-gamma than THP-1 cells (p < 0.05). IFN-gamma did not significantly augment the release of TNF-alpha by these cells (p > 0.05). Thus, IFN-gamma and TNF-alpha induced equivalent gp91-phox gene expression in THP-1 cells compared with CM or PBM but did not bring about equivalent NADPH oxidase activity. TNF-alpha release was higher in more mature cells. This partial divergence of gp91- phox gene expression, NADPH oxidase activity, and TNF-alpha release is probably a consequence of different events of myeloid cell biology and relates at least in part to cell differentiation state.

The effect of IFN-gamma and TNF-alpha on the eosinophilic differentiation and NADPH oxidase activation of human HL-60 clone 15 cells.

The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.

NADPH oxidase and enhanced superoxide generation in intrauterine undernourished rats: involvement of the renin-angiotensin system.

We previously reported that intrauterine undernutrition increased the oxidative stress by decreasing superoxide dismutase activity. In the present study, we tested whether NADPH oxidase, xanthine oxidase, cyclooxygenase or nitric oxide synthase are responsible for the increased O(2)(-) generation observed in rats submitted to intrauterine undernutrition. In addition, we investigated the effect of angiotensin II (ANG II) on O(2)(-) production via activation of NADPH oxidase. Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 16 weeks of age, the rats were used for the study of intravital fluorescence microscopy; microvascular reactivity, local ANG II concentration and AT(1), p22(phox) and gp91(phox) gene expression. In this study only the male offspring was used. Treatment of mesenteric arterioles with the xanthine oxidase inhibitor oxypurinol, the nitric oxide synthase inhibitor L-NAME or the cyclooxygenase inhibitor diclofenac did not significantly change superoxide production. Thus, these vascular sources of superoxide were not responsible for the increased superoxide concentration. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased superoxide generation and improved vascular function. On the other hand, intrauterine undernutrition did not alter the gene expression for p22(phox) and gp91(phox). The fact that the local ANG II concentration was increased and the attenuation of oxidative stress by blocking AT(1) receptor with losartan, led us to suggest that ANG II induces O(2)(-) generation in intrauterine undernourished rats. Our study shows that NADPH oxidase inhibition attenuated superoxide anion generation and ameliorated vascular function in rats submitted to intrauterine undernutrition. Although it is not clear which mechanisms are responsible for the increase in NADPH oxidase activity, a role for ANG II-mediated superoxide production via activation of NADPH oxidase is suggested.

Eosinophil-specific Regulation of gp91 phox Gene Expression by Transcription Factors GATA-1 and GATA-2

The glycoprotein gp91(phox) is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91(phox) gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91(phox) promoter in various types of gp91(phox)-expressing cells. A 10-base pair (bp) region from bp -105 to -96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp -101 to -96 (-98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the -98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the -105/+12 construct in a -98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91(phox) gene in human eosinophils.

Differential Gene Expression in the Activation and Maturation of Human Monocytes

Differential-display or RNA fingerprint was applied to identify genes differentially expressed in monocyte maturation induced by an immunomodulating peptide on human peripheral blood mononuclear cells. Two unknown sequences (06c22 and 06c71) and p21 protein (cyclin dependent kinase inhibitor) were repressed, and three genes activated: Cathepsin D, DRP2 (dihydropirimidinase related protein 2), and gp91phox (91-kDa subunit of citochrome b558). Phenotype of evolving monocytes was analyzed by flow cytometry and mRNA level of identified genes determined by reverse transcription-PCR. The expression pattern of identified genes seemed to correlate with different monocyte subsets, monocyte-derived cells, and expected functional changes. After peptide addition, immature monocytes were initially activated, increasing the expression of CD25, CD69, and HLA-DR markers. This was accompanied by repression of p21 and the two unknown sequences, along with the simultaneous activation of Cathepsin D and DRP2. Later, the differentiation marker CD16 rose, and gp91phox gene expression activated. Further maturation led certain monocytes to express marker CD23 and gp91phox expression to reach a maximum, while Cathepsin D and DRP2 dropped to preactivation levels. Results reflect part of the evolution of immature monocytes toward macrophages and monocyte-derived dendritic cell precursors.

GATA-3 represses gp91 phox gene expression in eosinophil-committed HL-60-C15 cells

To study the regulatory mechanism of gp91 phox gene expression in eosinophils, we transiently transfected eosinophil-committed HL-60-C15 cells with gp91 phox promoter constructs, and identified a negative element from bp −267 to −246 of the gp91 phox gene, the deletion of which caused an 83% increase in promoter activity. Electrophoresis mobility shift assays demonstrated GATA-3 binds to the GATA consensus site from bp −256 to −250. An 81% increment in promoter activity was obtained when a mutation was introduced in the GATA-3 binding site of the bp −267 to +12 construct, which is comparable to that of the bp −245 to +12 construct. We therefore conclude that GATA-3 specifically binding to the GATA site negatively regulates the expression of the gene in HL-60-C15 cells.

PU.1 as an essential activator for the expression of gp91(phox) gene in human peripheral neutrophils, monocytes, and B lymphocytes.

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91(phox)) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91(phox) gene revealed a single-base mutation (C --> T) at position -53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position -53 of the gp91(phox) promoter, and the mutation at position -53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position -50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position -56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions -53 and -50 significantly reduced the gp91(phox) promoter activity; however, the mutation at position -56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91(phox) promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C --> T) at position -52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91(phox) gene in human neutrophils, monocytes, and B lymphocytes.

Repressor Activity of CCAAT Displacement Protein in HL-60 Myeloid Leukemia Cells

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.

Evidence for the Involvement of Distinct Signal Transduction Pathways in the Regulation of Constitutive and Interferon γ-Dependent Gene Expression of NADPH Oxidase Components (gp91-phox, p47-phox, and p22-phox) and High-Affinity Receptor for IgG (FcγR-I) in Human Polymorphonuclear Leukocytes

AbstractWe recently showed that mRNA levels coding the high-affinity Fcγ receptor for IgG (FcγR-I, CD64) and two of the components of the phagocytic superoxide anion-generating system—the heavy-chain subunit of cytochrome b558 (gp91-phox) and the 47-Kd cytosolic factor (p47-phox)—are modulated by interferon gamma (IFN-γ). In this study, we examined whether dexamethasone (DEX) affects gp91-phox and p47-phox mRNA expression of human polymorphonuclear leukocytes (PMN), treated or not with IFN-γ. We also investigated whether staurosporine, a general inhibitor of protein kinases, influences gp91-phox, p47-phox, and FcγR-I gene expression in PMN treated with or without IFN-γ. We found that (1) gp91-phox mRNA steady-state levels, expressed in control or IFN-γ-treated PMN, were significantly inhibited, in a dose-dependent fashion, by both DEX and staurosporine; (2) p47-phox mRNA steady-state levels, expressed in control or IFN-γ-treated PMN, were not influenced by DEX, but were markedly depressed by staurosporine; (3) no changes of spectrophotometric cytochrome b558 were found in PMN treated for up to 20 hours with the inhibitors, regardless of the presence of IFN-γ; (4) both DEX and staurosporine dose-dependently inhibited IFN-γ-induced FcγR-I mRNA and protein expression; and (5) stability of gp91-phox and FcγR-I messages in IFN-γ-treated PMN was not altered by the presence of DEX. Our results demonstrate that gp91-phox, p47-phox, and FcγR-I gene expression of PMN is governed by specific and independent biochemical pathways. Moreover, IFN-γ activates different signal transduction pathways to modulate mRNA expression of gp91-phox, p47-phox, and FcγR-I. © 1992 by The American Society of Hematology. 0006-4971/92/7903-0021$3.00/0