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PHAGOCYTES
From the Department of Medicine, University of Texas
Health Science Center and the South Texas Veterans Health Care System,
Audie L. Murphy Division, San Antonio; and Fondation pour Recherches
Médicales, University of Geneva, Switzerland.
The p40phox protein, a regulatory component
of the phagocyte NADPH oxidase, is preferentially expressed in cells of
myeloid lineage. We investigated transcriptional regulation of the
p40phox gene in HL-60 myeloid cells.
Deletion analysis of approximately 6 kb of the 5'-flanking sequence of
the gene demonstrated that the proximal 106 base pair of the promoter
exhibited maximum reporter activity. This region contains 3 potential
binding sites for PU.1, a myeloid-restricted member of the
ets family of transcription factors. Mutation or deletion
of each PU.1 site decreased promoter activity, and the level of
activity mediated by each site correlated with its binding avidity for
PU.1, as determined by gel shift competition assays. Mutation of all 3 sites abolished promoter activity in myeloid cells. PU.1-dependent
expression was also observed in the Raji B-cell line, whereas the
moderate level of promoter reporter activity in the nonmyeloid HeLa
cell line was independent of PU.1. Chromatin immunoprecipitation assay
demonstrated occupation of the PU.1 sites by PU.1 in vivo in HL-60
cells. Cotransfection of the pGL3-p40-106 reporter construct with a
dominant-negative PU.1 mutant dramatically reduced promoter activity,
whereas the overexpression of PU.1 increased promoter activity.
Promoter activity and transcript levels of
p40phox increased in HL-60 cells during
dimethyl sulfoxide-induced differentiation toward the granulocyte
phenotype, and this was associated with increased cellular levels of
PU.1 protein. Our findings demonstrate that PU.1 binding at multiple
sites is required for p40phox gene
transcription in myeloid cells and that granulocytic differentiation is
associated with the coordinated up-regulation of PU.1 and
p40phox expression.
(Blood. 2002;99:4578-4587) NADPH oxidase is the major inducible source of
superoxide and superoxide-derived reactive oxygen species in phagocytes
and is a key enzyme in host defense against microbial
infection.1-3 Genetic deficiency of this enzyme results in
the inherited disorder chronic granulomatous disease (CGD), which is
characterized by impaired phagocyte microbicidal activity and a
clinical syndrome of recurrent life-threatening
infections.1-3 The reactive oxygen species generated by
this enzyme are also toxic to host cells and may account for much of
the tissue injury that occurs during inflammation.4-6
Therefore, the expression and activity of the phagocyte NADPH oxidase
components must be tightly regulated.
The fully assembled active NADPH oxidase complex is composed of
membrane-bound and cytosolic components.7,8 The
membrane-associated core component of the oxidase is a heterodimeric
b-type cytochrome comprised of light (p22phox)
and heavy (gp91phox) chain polypeptides.
Cytosolic components include p67phox,
p47phox,
p40phox, and the small GTPase protein,
Rac2. X-linked CGD, the most prevalent form of the disorder, is a
result of mutations in the gp91phox gene,
whereas autosomal recessive CGD is associated with mutations of the
p47phox, p67phox, or
p22phox genes.1-3
Numerous studies have demonstrated essential roles for
gp91phox, p22phox,
p67phox, p47phox, and
Rac2 in phagocyte NADPH oxidase function.9-20
Reconstitution of the oxidase by transfection into cell lines deficient
in the oxidase proteins or recombination of the components in vitro
shows that efficient superoxide generation by the NADPH oxidase complex is absolutely dependent on these components. The role of
p40phox, on the other hand, is less
clearly defined because it does not appear to be absolutely necessary
for the core enzymatic function, namely the univalent reduction of
oxygen. Nevertheless, several lines of evidence suggest that
p40phox is directly involved in the
normal physiologic function of the oxidase. Thus,
p40phox forms a trimolecular complex
with p47phox and p67phox
in the cytosol of resting phagocytes and on activation translocates to
the membrane with these components.21-23 Moreover,
p40phox can bind
p67phox and p47phox, and
the strong binding of p40phox to
p67phox can be disrupted by the activated
guanosine triphosphate-bound form of Rac, a key intermediate in
oxidase activation.23-31 The SH3 domain of
p40phox competes for the C-terminal,
proline-rich domain in p47phox, which also
interacts with the C-terminal SH3 domain of
p67phox and thereby down-regulates NADPH oxidase
activity.24,25,29,30 In contrast, other studies suggest
that p40phox actually participates in
the activation of NADPH oxidase by increasing the affinity of
p47phox for the flavocytochrome.32
More recently, it has been reported that the 57-kd actin-binding
protein coronin associates with the p40phox-p67phox-p47phox
cytosolic complex through the C-terminal domain of
p40phox and that it colocalizes with
the phox proteins in the periphery of the phagocytic vacuole
during phagocytosis and activation of the oxidase.33
Finally, recent studies have shown that
p40phox is hyperphosphorylated during
NADPH oxidase activation and dephosphorylated during enzyme
inactivation.34,35 In summary, these studies suggest that
p40phox acts as a regulator of oxidase
activity and as an adapter that links oxidase to the cytoskeletal
elements that participate in cell activation.
Tissue distribution studies have demonstrated that, similar to the
gp91phox, p47phox, and
p67phox components of NADPH oxidase,
p40phox is preferentially expressed in
cells of myeloid lineage.36,37 Transcription of many
myeloid-specific genes is regulated by PU.1, a member of the
ets family of transcription factors.38
Gene-targeting studies have demonstrated that PU.1 is essential for the
normal embryonic development of hematopoietic cells of myeloid, B-cell, and T-cell lineages.39,40 We have previously characterized the promoter of the p47phox gene and
demonstrated that PU.1 is essential for p47phox
transcription in myeloid cells.41 PU.1 appears to regulate transcription by binding to a single cis element located in
the proximal region of the p47phox gene. In this
report, we describe our studies on the transcriptional regulation of
the p40phox gene in the HL-60 myeloid
cell line. Our data show that p40phox
transcription is regulated by 3 PU.1 binding sites located within the
proximal region of the p40phox promoter
and that the PU.1 transcription factor binds to each site, in vitro and
in vivo. The central role of PU.1 in the regulation of
p40phox expression supports previous
observations that this phox protein is mainly restricted to
cells of myeloid lineage and is of particular importance in phagocyte function.
Materials
Human p40phox genomic cloning
and sequencing
Luciferase vector construction Reporter vectors were constructed in the pGL3-Basic luciferase vector. Promoter regions were amplified using human genomic DNA as template. The reverse primer was similar to the nested reverse primer used above, but a site for cleavage by XhoI was added (5'-CACTCGAGAGGCTGAGTTCACCTCTC-3'). The forward primers corresponded to the upstream sequences of desired promoter regions, with a KpnI cleavage site added at the 5' end. PCR products were digested with KpnI and XhoI and were cloned into the pGL3-Basic reporter vector. Inserts of the constructs generated all extend downstream to +104 relative to the transcription start site of the p40phox gene.36 Mutations of the PU.1 binding sites were generated by site-directed mutagenesis (QuikChange kit; Stratagene, La Jolla, CA) using the primers illustrated in Figure 2B. Constructs were confirmed by restriction mapping and sequencing.Cell culture and transient transfections As previously described,41 the human myeloid cell lines HL-60, THP-1, U937, and PLB985 and the human B-cell line Raji were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM HEPES, penicillin, and streptomycin. Transfection was carried out by electroporation as we have described.41 Briefly, approximately 107 cells were resuspended in 0.5 mL complete medium containing 20 µg luciferase reporter constructs and 0.2 µg of a Renilla luciferase vector (pRL-null; Promega) as a transfection efficiency control. In Figure 1, transfection was also carried out with equimolar amounts (7 pmol) of each construct, plus an unrelated plasmid, pUC19, to provide an equal amount of total DNA. For cotransfection assays, 10 µg each reporter construct and PU.1 expression plasmid (a generous gift from Dr M. Klemsz, Indiana University, Indianapolis) were used. Electroporation was carried out at 960 µF and 250 V for HL-60 cells or 320 V for Raji cells. At 48 hours the cells were washed 3 times in phosphate-buffered saline (pH 7.4), lysed in 100 µL 1× reporter lysis buffer (Promega), and centrifuged at 400g for 5 minutes at ambient temperature, and 20-µL aliquots of the supernatants were tested for reporter gene activity using the dual luciferase assay system (Promega) and a Turner Designs TD-20/20 luminometer. The human cervical carcinoma epithelial cell line HeLa was grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and transfected by Lipofectamine Plus reagent (Gibco) according to the manufacturer's protocol.
In vitro synthesis Mouse PU.1 cDNA42 was subcloned into pBluescript SK and was translated in vitro using T3 RNA polymerase and the TnT-coupled reticulocyte lysate system (Promega). Synthesized [35S]-methionine-labeled PU.1 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. A predominant band measuring approximately 38 kd was observed, consistent with the molecular mass previously reported.43 The control un-programmed sample (ie, no cDNA) produced no corresponding band.Nuclear extracts Cells were disrupted by nitrogen cavitation using a technique originally described for neutrophils,44 and nuclear extracts were prepared as we have previously reported in detail.41 Extracts were collected and stored in aliquots at 70°C. The protein concentration was determined using the
Bradford reagent (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assay Complementary DNA oligonucleotides were annealed by heating in 1× NET at 95°C for 5 minutes and cooling at ambient temperature. Probes were then labeled with [ -32P]ATP and T4
polynucleotide kinase. For gel shift assays, nuclear extract (6 µg)
was incubated for 20 minutes at ambient temperature with
5 × 104 cpm of the labeled DNA probe in 20 µL
binding buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 5% glycerol, 1 µg/µL bovine serum
albumin, and 2 µg poly-d(I-C). For supershift assays, 2 µL specific
antibody was added, and the reaction was continued for 15 minutes.
Samples were loaded on 5% nondenaturing polyacrylamide gels, and
electrophoresis was carried out at 200 V in 25 mM Tris (pH 8.5) with
190 mM glycerol and 1 mM EDTA. Competition assays were carried out in
the same manner, except that the above reaction mixture was
preincubated with competitor DNA for 10 minutes at 4°C before the
labeled probe was added. Signals were quantitated with a PhosphorImager
and ImageQuant software (Molecular Dynamics). Relative binding avidity of various DNA probes for PU.1 were determined by comparisons of band
intensities observed in the presence of varied amounts of probes or a
series of dilutions of competing oligonucleotides.
Chromatin immunoprecipitation analysis Chromatin immunoprecipitation (ChIP) analysis was carried out as described by Boyd et al.45 Briefly, formaldehyde was added directly to the cell culture to a final concentration of 1% (wt/vol), and the cells were incubated at 23°C for 10 minutes. Glycine was added to stop the reaction, and the cells were collected, washed, and allowed to swell on ice for 10 minutes in 5 mM PIPES (pH 8.0) containing 85 mM KCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, and 100 ng/mL leupeptin and aprotinin. Pelleted nuclei were collected and incubated on ice for 10 minutes in 50 mM Tris HCl (pH 8.1), 1% SDS, 10 mM EDTA, and the protease inhibitors, sonicated on ice to break the chromatin DNA, and precleared with Staph A cells (protein A-positive Staphylococcus aureus cells; Roche Molecular Biochemicals, Indianapolis, IN). Precleared chromatin from 2 × 107 cells was incubated with 1.5 µg affinity-purified rabbit polyclonal antibody to PU.1 (anti-PU.1 sc-352X; Santa Cruz Biotechnology) or without antibody, and it was rotated at 4°C for 12 to 16 hours. Immunoprecipitation was carried out with Staph A cells. The supernatant from the reaction lacking primary antibody was saved as total input of chromatin and was processed in the same way as the eluted immunoprecipitates, starting with the cross-link reversal step. Cross-linking was reversed by incubation at 65°C for 5 hours in the presence of 300 mM NaCl and 0.01% RNase A. Samples were then precipitated, resusupended, and treated with proteinase K, followed by extraction with phenol-chloroform-isoamyl alcohol and precipitation with sodium acetate and ethanol plus tRNA and glycogen as carriers. Pellets were collected by microcentrifugation, resuspended in 30 µL H2O, and analyzed by PCR. Total input samples were resuspended in 100 µL H2O and then diluted 1:100 before PCR. PCR reactions were carried out in a total volume of 25 µL containing 2-3 µL immunoprecipitate or diluted total input as the template and using recombinant Taq DNA polymerase (Life Technologies). After 35 cycles of amplification, PCR products were separated on 1.5% agarose containing ethidium bromide. PCR primers (p40-PU.1 forward, 5'-GCGCCAAGGACTGACATC-3' and reverse, 5'-GAGCAGGTGGTGCGTCTC-3') were designed to amplify a 299-bp region of the p40phox promoter that contains the 3 PU.1 sites.Northern blot analysis Total cellular RNA was isolated using the Ultraspec reagent (Biotecx Laboratories, Houston, TX) and dissolved in nuclease-free water. Samples of RNA (10 µg) were separated by electrophoresis in 1% agarose under denaturing conditions (formaldehyde), transferred to Nytran Plus membrane (Schleicher & Schuell, Keene, NH), UV cross-linked, and probed with p40phox cDNA labeled using the High Prime DNA labeling kit, (Roche Molecular Biochemicals) and [ -32P]-dCTP (DuPont-NEN). Following
autoradiography at 70°C, the membrane was stripped and probed with
32P-labeled cDNA for human acidic ribosomal phosphoprotein
36B4 as a loading control.
The 5'-flanking region of the human p40phox gene exhibits promoter activity in myeloid cells Previous studies have shown that human p40phox mRNA is expressed almost exclusively in hematopoietic cells that exhibit NADPH oxidase activity.36,37 To identify the sites of promoter activity in the p40phox gene, regions of the 5'-flanking sequence were prepared by PCR, cloned into the luciferase reporter vector pGL3-Basic, and assayed by transient transfection and expression in the promyelocytic HL-60 cell line. The highest level of activity (approximately 37-fold increase over pGL3-Basic vector control) was observed with the construct pGL3-p40-106 (Figure 1), which contained the first 106 nucleotides upstream of the p40phox transcription start site.36 Constructs extending further upstream exhibited progressively lower luciferase activity, suggesting that the minimal p40phox promoter lies within the first 106 nucleotides of the 5'-flanking sequence of the p40phox gene and that negative regulatory elements may be present in upstream regions. Other myeloid cell lines tested (THP-1, U937, and PLB985) showed similar results (data not shown).Three PU.1 consensus sites in the proximal p40phox promoter bind PU.1 Inspection of the p40phox promoter for potential regulatory elements identified several core-binding sequences (GAGGAA) for the PU.1 transcription factor. Applying criteria for optimal flanking nucleotides that we identified previously,46 3 of these PU.1 sites (PU.1a,95 to 100;
PU.1b, 65 to 60; and PU.1c, +80 to +85; Figure
2A) were considered to have a high
probability of binding PU.1. To determine whether these sites bound
PU.1, electrophoretic mobility shift assay (EMSA) was carried out using
32P-labeled double-stranded oligonucleotides corresponding
to these regions (Figure 2B) and nuclear extracts of HL-60 cells.
Several DNA-protein complexes were formed between the PU.1 probes and HL-60 nuclear extracts (Figure 2C). Effective competition with excess
unlabeled wild-type, but not mutated, DNA probes demonstrated that
these bands were specific. The addition of specific antibody to PU.1
resulted in a dramatic decrease in band intensity and in the appearance
of new supershifted bands, providing clear evidence that the complexes
contained PU.1 protein. Comparison with previous studies suggested that
the major bands of slower mobility contained intact PU.1 protein,
whereas the faster-migrating bands were probably formed by PU.1
degradation products.46
Intact PU.1 binding sites are required for promoter activity in HL-60 myeloid cells and Raji B cells, but not in nonmyeloid HeLa cells To investigate the role of these 3 p40phox PU.1 binding sites on promoter activity, a series of mutation and deletion promoter-luciferase constructs were prepared in which the 3 PU.1 sites were deleted or mutated individually or in combination. These constructs were tested as before by transient expression in HL-60 cells. Mutation of PU.1a, PU.1b, or PU.1c in the1599 bp
p40phox promoter construct reduced
activity by 82%, 63%, and 18%, respectively (Figure
3A). Similar results were observed with
the deletion series (Figure 3B). The p40phox
promoter activity in the 1599 bp construct was nearly abolished in the HL-60 cells when all 3 binding sites were mutated, indicating the essential requirement for PU.1 in this cell type (Figure 3B). Because PU.1 and p40phox are also
expressed in B lymphocytes, we investigated the roles of the PU.1 sites
on p40phox promoter activity in this
cell type using the Raji human B-cell line. The PU.1 sites in the
p40phox promoter bound to PU.1 protein
in Raji nuclear extracts (Figure 4A).
Furthermore, the promoter was active in these cells, though at levels
lower than those observed in the HL-60 cells (Figure 4B). More
important, however, the promoter function was dependent on the presence
of intact PU.1a and PU.1b sites (Figure 4B; PU.1c not tested). To
investigate further the requirement for the PU.1 sites in
p40phox promoter activity, the mutated
constructs were also analyzed in the nonmyeloid HeLa cell line, which
does not express PU.1. Although the activity of the wild type 1599 bp
p40phox promoter construct was high in
HeLa (Figure 4C), this activity was not affected by mutation of the
PU.1 sites. The relatively high basal activity seen in HeLa cells with
these constructs was surprising and might have been mediated by
transcription factors present in these cells that bind the 1599
p40phox promoter construct at sites
distinct from the PU.1 elements. These sites might normally be
repressed in nonmyeloid tissues in vivo through elements in the gene
not included in the 1599 p40phox
construct. Such factors would be absent, functionally inactive, or
PU.1-dependent in HL-60 and Raji cells because in both cases, mutation
of the PU.1 sites was sufficient to eliminate most promoter activity.
Promoter activity of the PU.1 sites correlates with PU.1 binding avidity Our previous studies indicated a correlation between the transactivation activity of PU.1 sites from a number of promoters and their affinity for the PU.1 protein.46 Thus, we hypothesized that the different levels of transactivation activity of the PU.1 sites in the p40phox promoter might be attributed in part to their relative PU.1 binding avidity. To investigate this possibility, the binding affinities of the 3 sites for PU.1 present in HL-60 nuclear extracts or in vitro synthesized PU.1 were estimated by 2 methods. First, increasing amounts of each labeled p40phox PU.1 oligonucleotide probe were incubated with a fixed amount of PU.1 protein (Figure 5A). Second, the ability of unlabeled p40phox PU.1 oligonucleotide probes to compete for binding with the well-characterized p47phox PU.1 site41,46 was determined (Figure 5B). Semiquantitative analysis of these binding studies suggested that the binding avidity of the PU.1a site is 2- to 4-fold greater than that of the PU.1b site (compare lane 2 with lanes 8 and 9 in Figure 5A and lane 2 with lane 6 in Figure 5B). The binding avidity of PU.1b is, in turn, 3- to 8-fold greater than that of the PU.1c site (compare lane 7 with lane 15 in Figure 5A and lane 5 with lane 9 in Figure 5B). Combining these EMSA results with the data obtained by deletion and mutation transfection studies indicated a direct relationship between the avidity with which these sites bind PU.1 and their capacity to dictate reporter gene transcription (PU.1a is greater than PU.1b is greater than PU.1c). However, the decrease in promoter activity on deletion of the PU.1c site was 20% when the other 2 PU.1 binding sites were intact, but it was 36% when the PU.1a site was deleted and 45% when the PU.1a and PU.1b sites were deleted (data not shown), suggesting that compensation may take place when one or another of the PU.1 sites is inactivated.
PU.1 binds to the p40phox promoter in vivo To determine whether PU.1 protein binds the p40phox promoter at the identified PU.1 sites in vivo, we performed ChIP analysis on HL-60 cells using a specific polyclonal antibody to PU.1. Following immunoprecipitation and reversal of cross-linking, the DNA was purified and used as a template for PCR amplification using primers that encompass the region of the p40phox promoter containing the 3 PU.1 binding sites. As a negative control, we carried out immunoprecipitation with antibody to c-Jun, consensus binding sites for which are absent from this region of the p40phox promoter. The p40phox primers produced an amplicon of predicted size from the total input DNA and the anti-PU.1-precipitated DNA, but not from anti-c-Jun-precipitated DNA (Figure 6). Furthermore, PCR primers for an unrelated gene, CCR5, generated products only from the total input DNA, confirming the specificity of the assay. The CCR5 primers were designed to amplify a region of the CCR5 gene that does not contain PU.1 or c-Jun binding sites. These data indicate that endogenous PU.1 protein in HL-60 cells binds the p40phox promoter in the region containing the PU.1 sites and thus may promote transcription of the gene in vivo.
Overexpression of PU.1 increases p40phox promoter activity, whereas a dominant-negative mutant of PU.1 decreases activity To provide further evidence that PU.1 is critical for p40phox promoter activity, the p40phox luciferase construct pGL3-p40-106 or pGL-3-Basic control were transfected into HL-60 cells with or without cotransfection of either the PU.1 expression plasmid pJ6-mPU.1 or the dominant-negative mutant plasmid pJ6-NN, in which the transactivation domain is deleted. Overexpression of PU.1 increased the activity of the p40phox promoter by approximately 2.5-fold (Figure 7), whereas expression of the dominant-negative mutant PU.1 decreased activity by approximately 50%, presumably because of competition with endogenous PU.1. That the transactivation was dependent on the binding of PU.1 to the putative PU.1 sites in the proximal portion of the p40phox promoter was confirmed in control experiments using the pGL3-p40-106mt construct, in which all 3 PU.1 sites were mutated. No effect of wild-type or mutant PU.1 was observed with this mutant reporter construct (Figure 7).
Induction of granulocyte differentiation in HL-60 cells increases PU.1 protein, p40phox mRNA expression, and p40phox promoter activity HL-60 cells can be induced to differentiate toward a granulocytic phenotype by treatment with dimethyl sulfoxide (DMSO).47 Components of the phagocyte NADPH oxidase system are induced by DMSO, and enzymatic activity can be detected within 1 to 2 days of treatment48-50 Although p40phox is not an essential component of the functional NADPH oxidase, it is expressed predominantly in phagocytic cells, complexed with the essential oxidase components p47phox and p67phox, and, in all probability, has a role in enzyme activation and regulation.29,32 To investigate the influence of granulocytic differentiation on p40phox gene expression, we first examined the induction of p40phox mRNA during DMSO treatment. Total RNA was isolated from DMSO-treated HL-60 cells at daily intervals, and Northern blot analysis was carried out using a cDNA probe specific for human p40phox. Levels of p40phox mRNA increased approximately 8-fold within 1 day of treatment (Figure 8A). Subsequently, transcript levels decreased but were maintained at approximately 4-fold over controls for 2 to 3 days. To determine whether increased gene transcription contributed to this observed increase in p40phox gene expression, HL-60 cells were first transfected with the pGL-3-p40-106 reporter gene construct, then treated with DMSO for 38 hours, and luciferase reporter activity was measured. DMSO increased reporter gene activity by 3- to 4-fold over the untreated control (Figure 8B). However, when we transfected the construct in which all 3 PU.1 sites were mutated, only a small increase in luciferase activity was seen following DMSO treatment. This suggests that the increase in transcriptional activity induced by DMSO in the wild-type promoter was mediated through increased PU.1 activity. These data were supported by EMSA and immunoblot analysis because nuclear extracts from HL-60 cells taken 38 hours after DMSO treatment showed increased PU.1 binding activity (Figure 9A) and increased PU.1 protein using specific anti-PU.1 antibodies (Figure 9B).
PU.1, the most divergent member of the ets family of transcription factors, is important in the early development of multiple hematopoietic progenitors. Targeted disruption of the PU.1 locus results in multilineage defects that affect B and T lymphocytes, monocytes, osteoclasts, alveolar macrophages, and neutrophils.39,40 The PU.1 transcription factor is expressed specifically in hematopoietic tissues, particularly in cells of granulocytic, monocytic, and B lymphoid lineages.42,51-53 These patterns of expression are shared with the components of the phagocyte NADPH oxidase.1-3 Moreover, previous studies have shown that the components of the phagocytic respiratory burst oxidase are transcriptionally regulated by PU.1.41,54-57 For example, expression of the large subunit of the membrane-bound cytochrome b558, gp91phox is controlled by PU.1, along with other transcription factors.54-57 Of particular note, the PU.1-binding site is critical for function of the promoter of the gp91phox (CYBB) gene, and point mutations in this site lead to the CGD clinical phenotype.55,56 In the case of the cytosolic oxidase component p47phox, we have shown that its myeloid-specific expression is regulated primarily through a single essential cis element that binds PU.1 with high avidity.41 In addition, we and others have reported that p67phox transcription is regulated by a complex array of transcription factors that includes PU.1.57-59 Adding to the body of evidence for a key role of PU.1 in regulation of NADPH oxidase expression are our current data that the cytosolic oxidase component p40phox is also regulated by PU.1. We have shown that a unique combination of 3 PU.1-binding elements, 2 in the proximal promoter and one in the 5' UTR of the gene, comprise the major regulatory components of p40phox gene transcription in the HL-60 myeloid and Raji B-cell lines. Moreover, we have demonstrated that the 3 PU.1-binding sites also mediate increased expression of the p40phox gene observed during DMSO-induced granulocytic differentiation of HL-60 cells. Recent studies indicate that regulation by multiple PU.1 promoter elements may not be unusual for myeloid-restricted genes. Two functional PU.1 sites have been identified in the promoter of the myeloid-specific CD18 gene60 and, interestingly, in the promoter of the PU.1 gene itself.61 The PU.1-binding sites in the PU.1 promoter thus appear to act as a pathway for autoregulation, a potentially important concept for lineage development of myeloid cells. A recent report has indicated that a low level of expression of PU.1 in hematopoietic progenitors is associated with the development of B cells, whereas a high level of expression blocks B-cell development and promotes macrophage differentiation.62 Nevertheless, the specific functional implications of the presence of multiple functional PU.1 sites in a promoter are yet to be determined. It is possible that in some genes they could act in a synergistic or cooperative manner, allowing a relatively strong transcriptional response in the presence of low activity of the PU.1 transcription factor, a situation that might be found in the early stages of myeloid development. This may well be the case with the p40phox gene. Transcripts for p40phox can be readily identified in undifferentiated myeloid cell lines by Northern blot analysis,36 whereas the mRNA for p47phox, p67phox, and gp91phox is generally more difficult to detect.36,50,63,64 We showed previously that the avidity of binding of PU.1 by a particular cis element correlates with its capacity to dictate reporter gene transcription.46 The results of our analyses of the 3 PU.1 sites in the p40phox promoter support this earlier observation. The affinity of binding of PU.1 to the p40phox PU.1 oligonucleotide probes in the gel mobility shift assay and the transacting activity mediated by each site in the transfection studies were of the same rank order (PU.1a is greater than PU.1b is greater than PU.1c). The relatively high activity of the PU.1a site is probably caused by the presence of optimal (AT-rich) sequences flanking the 5' end of the core GAGGAA sequence.46 Interestingly, these sequences are not found in the weakly acting PU.1c site. The proximity of the 3 PU.1-binding elements to each other and their different activities suggest that together they produce a synergistic activation of p40phox gene transcription. However, our deletion-mutation studies indicated that the relative contribution to promoter activity of each of the 3 PU.1 sites was not reduced when the other sites were functionally lost. Therefore, at least in resting HL-60 cells, it appears more likely that each PU.1 site acts independently of the others. Transfection of the There is increasing evidence that PU.1 can act in association with accessory factors to transactivate genes. Early studies showed an essential functional requirement for the factor NF-EM5 (PIP) to act in conjunction with PU.1 to transactivate the immunoglobulin kappa gene.66 The recruitment of NF-EM5 to the PU.1-DNA complex was dependent on the activation of PU.1 by phosphorylation of serine 148, possibly by casein kinase II. In the immunoglobulin kappa gene, nonphosphorylated PU.1 was able to bind its cognate site but could not stimulate promoter activity. It is possible that the binding of PU.1 to the p40phox gene recruits such accessory factors into a ternary complex that in turn dictates promoter activity. Our data do not directly support such a model because the pattern of complexes formed on EMSA with the p40phox PU.1 probes was similar for HL-60 nuclear extracts and in vitro-synthesized PU.1 protein. However, the recruitment of accessory factors may require additional nucleotide sequences that extend beyond those used for the probes. The induction of granulocytic differentiation in HL-60 cells with DMSO led to an increase in the net cellular levels of p40phox mRNA. DMSO treatment also produced an increase in the total cellular levels of PU.1, particularly in the slower-migrating form of PU.1, which most likely corresponds to a hyper-phosphorylated form of the protein.67,68 These data are consistent with earlier studies using enriched human CD34+ progenitor cells in which the induction of myeloid differentiation with granulocyte macrophage-colony-stimulating factor resulted in an increase in PU.1 mRNA and protein.69,70 The increase in PU.1 protein in the HL-60 cells was accompanied by an increase in the levels of PU.1 binding to DNA and PU.1-mediated p40phox promoter activity. Taken together with our functional mutation studies, these data suggest that the increased p40phox gene transcription observed during maturation and differentiation is mediated primarily by the 3 identified PU.1 sites. This does not preclude the contribution of additional accessory or coactivator factors, perhaps recruited through the activation and phosphorylation of PU.1. However, these putative factors would have to function through the PU.1 elements because mutation of these sites reduced the promoter activity in the HL-60 cells to near-background levels.
We thank Dr Michael J. Klemsz for providing PU.1 expression plasmids and Dr Long Wang for assistance with graphics and statistical analyses.
Submitted July 27, 2001; accepted February 22, 2002.
Supported by grant AI20866 from the National Institutes of Health (R.A.C.) and by a grant from the Research Resources Program of the Howard Hughes Medical Institute (S.-L.L.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert A. Clark, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Dr, San Antonio, TX 78229-3900; e-mail: clarkra{at}uthscsa.edu.
1.
Babior BM.
NADPH oxidase: an update.
Blood.
1999;93:1464-1476 2. Clark RA. Activation of the neutrophil respiratory burst oxidase. J Infect Dis. 1999;179:S309-S317[CrossRef][Medline] [Order article via Infotrieve]. 3. Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM. Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine. 2000;79:170-200[CrossRef][Medline] [Order article via Infotrieve].
4.
Klebanoff SJ.
Oxygen metabolism and the toxic properties of phagocytes.
Ann Intern Med.
1980;93:480-489 5. Clark RA. Extracellular effects of the myeloperoxidase-hydrogen peroxide-halide system. In: Weissmann G, ed. Advances in Inflammation Research. New York, NY: Raven Press; 1983:107-146. 6. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med. 1989;320:365-376[Medline] [Order article via Infotrieve]. 7. Leusen JHW, Verhoeven AJ, Roos D. Interactions between the components of the human NADPH oxidase: intrigues in the phox family. J Lab Clin Med. 1996;128:461-476[CrossRef][Medline] [Order article via Infotrieve]. 8. DeLeo FR, Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol. 1996;60:677-691[Abstract].
9.
Volpp BD, Nauseef WM, Clark RA.
Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease.
Science.
1988;242:1295-1297
10.
Nunoi H, Rotrosen D, Gallin JI, Malech HL.
Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors.
Science.
1988;242:1298-1301
11.
Volpp BD, Nauseef WM, Donelson JE, Moser DR, Clark RA.
Cloning of the cDNA and functional expression of the 47 kilodalton cytosolic component of the human neutrophil respiratory burst oxidase.
Proc Natl Acad Sci USA.
1989;86:7195-7199 12. Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991;353:668-670[CrossRef][Medline] [Order article via Infotrieve].
13.
Rotrosen D, Yeung CL, Leto TL, Malech HL, Kwong CH.
Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase.
Science.
1992;256:1459-1462 14. Segal AW, West I, Wientjes F, et al. Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J. 1992;284:781-788[Medline] [Order article via Infotrieve].
15.
Knaus UG, Heyworth PG, Kinsella BT, Curnutte JT, Bokoch GM.
Purification and characterization of Rac 2: a cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.
J Biol Chem.
1992;267:23575-23582 16. Volpp BD, Lin Y. In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease. J Clin Invest. 1993;91:201-207[Medline] [Order article via Infotrieve].
17.
Sekhsaria S, Gallin JI, Linton GF, Mallory RM, Mulligan RC, Malech HL.
Peripheral blood progenitors as a target for genetic correction of p47 phox-deficient chronic granulomatous disease.
Proc Natl Acad Sci U S A.
1993;90:7446-7450
18.
Li F, Linton GF, Sekhsaria S, et al.
CD34+ peripheral blood progenitors as a target for genetic correction of the two flavocytochrome b558 defective forms of chronic granulomatous disease.
Blood.
1994;84:53-58
19.
Kume A, Dinauer MC.
Retrovirus-mediated reconstitution of respiratory burst activity in X-linked chronic granulomatous disease cells.
Blood.
1994;84:3311-3316
20.
Porter CD, Parkar MH, Verhoeven AJ, Levinsky RJ, Collins MKL, Kinnon C.
p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox.
Blood.
1994;84:2767-2775 21. Someya A, Nagaoka I, Yamashita T. Purification of the 260 kDa cytosolic complex involved in the superoxide production of guinea pig neutrophils. FEBS Lett. 1993;330:215-218[CrossRef][Medline] [Order article via Infotrieve]. 22. Wientjes FB, Hsuan JJ, Totty NF, Segal AW. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993;296:557-561[Medline] [Order article via Infotrieve]. 23. Tsunawaki S, Mizunari H, Nagata M, Tatsuzawa O, Kuratsuji T. A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox. Biochem Biophys Res Commun. 1994;199:1378-1387[CrossRef][Medline] [Order article via Infotrieve].
24.
Fuchs A, Dagher M-C, Vignais PV.
Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system.
J Biol Chem.
1995;270:5695-5697 25. Ito T, Nakamura R, Sumimoto H, Takeshige K, Sakaki Y. An SH3 domain-mediated interaction between the phagocyte NADPH oxidase factors p40phox and p47phox. FEBS Lett. 1996;385:229-232[CrossRef][Medline] [Order article via Infotrieve]. 26. Fuchs A, Dagher MC, Fauré J, Vignais PV. Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase: pinpointing the sites of interaction between p47phox, p67phox and p40phox using the two-hybrid system. Biochim Biophys Acta Mol Cell Res. 1996;1312:39-47[Medline] [Order article via Infotrieve]. 27. Wientjes FB, Panayotou G, Reeves E, Segal AW. Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox. Biochem J. 1996;317:919-924[Medline] [Order article via Infotrieve].
28.
Tsunawaki S, Kagara S, Yoshikawa K, Yoshida LS, Kuratsuji T, Namiki H.
Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.
J Exp Med.
1996;184:893-902
29.
Sathyamoorthy M, De Mendez I, Adams AG, Leto TL.
p40 phox down-regulates NADPH oxidase activity through interactions with its SH3 domain.
J Biol Chem.
1997;272:9141-9146 30. Wilson L, Butcher C, Finan P, Kellie S. SH3 domain-mediated interactions involving the phox components of the NADPH oxidase. Inflamm Res. 1997;46:265-271[CrossRef][Medline] [Order article via Infotrieve]. 31. Rinckel LA, Faris SL, Hitt ND, Kleinberg ME. Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation. Biochem Biophys Res Commun. 1999;263:118-122[CrossRef][Medline] [Order article via Infotrieve]. 32. Cross AR. p40phox participates in the activation of NADPH oxidase by increasing the affinity of p47phox for flavocytochrome b558. Biochem J. 2000;349:113-117[CrossRef][Medline] [Order article via Infotrieve]. 33. Grogan A, Reeves E, Keep N, et al. Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J Cell Sci. 1997;110:3071-3081[Abstract]. 34. Fuchs A, Bouin AP, Rabilloud T, Vignais PV. The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells. Eur J Biochem. 1997;249:531-539[Medline] [Order article via Infotrieve].
35.
Bouin AP, Grandvaux N, Vignais PV, Fuchs A.
p40phox is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase: implication of a protein kinase C type kinase in the phosphorylation process.
J Biol Chem.
1998;273:30097-30103
36.
Zhan S, Vazquez N, Wientjes FB, et al.
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;88:2714-2721 37. Mizuki K, Kadomatsu K, Hata K, et al. Functional modules and expression of mouse p40phox and p67phox, SH3-domain-containing proteins, involved in the phagocyte NADPH oxidase complex. Eur J Biochem. 1998;251:573-582[Medline] [Order article via Infotrieve]. 38. Simon MC, Olson M, Scott E, Hack A, Su G, Singh H. Terminal myeloid gene expression and differentiation requires the transcription factor PU.1. Curr Top Microbiol Immunol. 1996;211:113-119[Medline] [Order article via Infotrieve].
39.
Scott EW, Simon MC, Anastasi J, Singh H.
Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.
Science.
1994;265:1573-1577 40. McKercher SR, Torbett BE, Anderson KL, et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996;15:5647-5658[Medline] [Order article via Infotrieve].
41.
Li SL, Valente AJ, Zhao SJ, Clark RA.
PU.1 is essential for p47phox promoter activity in myeloid cells.
J Biol Chem.
1997;272:17802-17809 42. Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990;61:113-124[CrossRef][Medline] [Order article via Infotrieve].
43.
Himmelmann A, Thevenin C, Harrison K, Kehrl JH.
Analysis of the Bruton's tyrosine kinase gene promoter reveals critical PU.1 and SP1 sites.
Blood.
1996;87:1036-1044
44.
Borregaard N, Heiple JM, Simons ER, Clark RA.
Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.
J Cell Biol.
1983;97:52-61
45.
Boyd KE, Wells J, Gutman J, Bartley SM, Farnham PJ.
c-Myc target gene specificity is determined by a post-DNA binding mechanism.
Proc Natl Acad Sci U S A.
1998;95:13887-13892
46.
Li SL, Schlegel W, Valente AJ, Clark RA.
Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters.
J Biol Chem.
1999;274:32453-32460
47.
Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC.
Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethyl sulfoxide.
J Exp Med.
1979;149:969-974
48.
Tsiftsoglou AS, Wong W, Hyman R, Minden M, Robinson SH.
Analysis of commitment of human leukemia HL-60 cells to terminal granulocytic maturation.
Cancer Res.
1985;45:2334-2339 49. Levy R, Rotrosen D, Nagauker O, Leto TL, Malech HL. Induction of the respiratory burst in HL-60 cells: correlation of function and protein expression. J Immunol. 1990;145:2595-2601[Abstract].
50.
Hua J, Hasebe T, Someya A, Nakamura S, Sugimoto K, Nagaoka I.
Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage.
J Leukoc Biol.
2000;68:216-224 51. Goebl MG. The PU.1 transcription factor is the product of the putative oncogene Spi-1. Cell. 1990;61:1165-1166[CrossRef][Medline] [Order article via Infotrieve].
52.
Galson DL, Hensold JO, Bishop TR, et al.
Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis.
Mol Cell Biol.
1993;13:2929-2941 53. Hromas R, Orazi A, Neiman RS, et al. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood. 1993;82:2998-3004.
54.
Eklund EA, Jalava A, Kakar R.
PU.1, interferon regulatory factor 1, and interferon consensus sequence-binding protein cooperate to increase gp91phox expression.
J Biol Chem.
1998;273:13957-13965
55.
Suzuki S, Kumatori A, Haagen IA, et al.
PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes.
Proc Natl Acad Sci U S A.
1998;95:6085-6090
56.
Voo KS, Skalnik DG.
Elf-1 and PU.1 induce expression of gp91phox via a promoter element mutated in a subset of chronic granulomatous disease patients.
Blood.
1999;93:3512-3520
57.
Eklund EA, Kakar R.
Recruitment of CREB-binding protein by PU.1, IFN-regulatory factor-1, and the IFN consensus sequence-binding protein is necessary for IFN-gamma-induced p67phox and gp91phox expression.
J Immunol.
1999;163:6095-6105
58.
Li SL, Valente AJ, Wang L, Gamez MJ, Clark RA.
Transcriptional regulation of the p67phox gene: role of AP-1 in concert with myeloid-specific transcription factors.
J Biol Chem.
2001;276:39368-39378
59.
Gauss KA, Bunger PL, Quinn MT.
AP-1 is essential for p67phox promoter activity.
J Leukoc Biol.
2002;71:163-172
60.
Rosmarin AG, Caprio D, Levy R, Simkevich C.
CD18 ( 61. Chen H, Ray-Gallet D, Zhang P, et al. PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene. 1995;11:1549-1560[Medline] [Order article via Infotrieve].
62.
DeKoter RP, Singh H.
Regulation of B lymphocyte and macrophage development by graded expression of PU.1.
Science.
2000;288:1439-1441 63. Obermeier H, Sellmayer A, Danesch U, Aepfelbacher M. Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells. Biochim Biophys Acta Mol Cell Res. 1995;1269:25-31[Medline] [Order article via Infotrieve].
64.
Yu LX, Zhen L, Dinauer MC.
Biosynthesis of the phagocyte NADPH oxidase cytochrome b558: role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits.
J Biol Chem.
1997;272:27288-27294
65.
Moulton KS, Semple K, Wu H, Glass CK.
Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif.
Mol Cell Biol.
1994;14:4408-4418
66.
Eisenbeis CF, Singh H, Storb U.
Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator.
Genes Dev.
1995;9:1377-1387 67. Mao C, Ray-Gallet D, Tavitian A, Moreau-Gachelin F. Differential phosphorylations of Spi-B and Spi-1 transcription factors. Oncogene. 1996;12:863-873[Medline] [Order article via Infotrieve]. 68. Lodie TA, Savedra RJ, Golenbock DT, Van Beveren CP, Maki RA, Fenton MJ. Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J Immunol. 1997;158:1848-1856[Abstract].
69.
Cheng T, Shen H, Giokas D, Gere J, Tenen DG, Scadden DT.
Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells.
Proc Natl Acad Sci U S A.
1996;93:13158-13163
70.
Voso MT, Burn TC, Wulf G, Lim B, Leone G, Tenen DG.
Inhibition of hematopoiesis by competitive binding of transcription factor PU.1.
Proc Natl Acad Sci U S A.
1994;91:7932-7936
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  V. Thomas, S. Samanta, and E. Fikrig Anaplasma phagocytophilum Increases Cathepsin L Activity, Thereby Globally Influencing Neutrophil Function Infect. Immun., November 1, 2008; 76(11): 4905 - 4912. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
