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Originally published online as doi:10.1189/jlb.0203061 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:111-117.)
© 2003 by Society for Leukocyte Biology

PU.1 regulates glutathione peroxidase expression in neutrophils

Stacy L. Throm and Michael J. Klemsz

Department of Microbiology and Immunology, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis

Correspondence: Michael J. Klemsz, Ph.D., Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Dr., MS5010, Indianapolis, IN 46202. E-mail: mklemsz{at}iupui.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on knockout models, the transcription factor PU.1 has been shown to be important for the maturation of neutrophils. As the list of genes PU.1 directly regulates in neutrophils is still quite limited, defining PU.1 target genes for this lineage will provide valuable insight into how this factor regulates neutrophil development and terminal function. Using the combined techniques of representational difference analysis and a cDNA library screen, we identified four genes that were differentially expressed in the PU.1-expressing 503PU myeloid cell line but not the PU.1 null parent cell line 503. Two of these genes, glutathione peroxidase (GPx) and serine leukoprotease inhibitor, are involved in protecting neutrophils from the products they make to destroy pathogens and were analyzed further to determine if PU.1 directly regulates their expression. These studies showed that PU.1 directly regulated the expression of only the GPx gene through binding sites in the promoter and a 3' regulatory region. Thus, PU.1 not only regulates the expression of molecules involved in the production of reactive oxygen species but also a gene that protects the neutrophils from these same destructive enzymes.

Key Words: transcription • blood cell • gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor PU.1 is a founding member of the Ets family of proteins and is identical to the spi-1 oncogene, identified as the site of proviral integration of the spleen focus-forming virus in Friend virus-induced murine erythroleukemia (MEL) [1 2 3 ]. PU.1 is normally expressed in CD34+ hematopoeitic progenitor cells [4 ] and in myeloid and lymphoid lineages during blood cell development [5 , 6 ]. Studies on PU.1 knockout mice have shown that it is required for the development of macrophages, B cells, and neutrophils, as well as dendritic cells [7 8 9 ]. PU.1 has been shown to regulate the expression of a growing list of genes in each of these lineages. Within the neutrophil, PU.1 regulates several genes important for their development and function, including CD11b, CD45, and the receptors for granulocyte macrophage-colony stimulating factor (GM-CSF) and G-CSF [10 11 12 13 ]. In addition, PU.1 has been shown to regulate the expression of several components of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex.

Neutrophils are critical effector cells in the host response to microbes because of their ability to phagocytose and kill these pathogens. One way they accomplish this is by the production of reactive oxygen species (ROS) through the NADPH oxidase complex. The active complex contains two membrane and five cytosolic components. Analyses of these genes have shown that PU.1 is involved in regulating the expression of most of these molecules, including the membrane component gp91phox and the cytosolic components p67phox, p47phox, and p40phox [14 15 16 17 18 ]. Thus, it is not surprising that neutrophil-like cells isolated from PU.1 knockout mice are less efficient at phagocytosis and killing than wild-type (WT) controls [19 ]. In response to phorbol 12-myristate 13-acetate (PMA) stimulation, no superoxide (O2-) was produced in the PU.1-/- neutrophil-like cells compared with a tenfold increase in O2- production in control cells [19 ]. The lack of O2- production in the neutrophil-like cells from the PU.1-/- mice was attributed to the loss of gp91phox expression in these cells. Re-expression of PU.1 using a retrovirus in the 503 myeloid cell line, which was derived from the knockout mouse, restored expression of gp91phox and the ability of these cells to produce reactive O2- in response to PMA [20 ]. These studies suggest that PU.1 is necessary for not only neutrophil development but also the expression of terminal genes used by these cells to fight invading pathogens.

To fully understand the role of PU.1 in regulating blood cell development and in particular, neutrophil development, we initiated studies to define additional genes directly regulated by this transcription factor. To accomplish this, we used the 503 myeloid cell line, which was derived from the neonatal liver of PU.1 knockout mice [20 ]. These cells are interleukin (IL)-3-dependent and blocked at the promyelocyte stage, based on the expression of components of primary granules but not those of secondary granules. Re-expression of PU.1 in this line generated the 503PU line. Expression of genes known to be regulated by PU.1, including CD11b, gp91phox, and CD45, was seen in the 503PU line [11 , 20 ]. As secondary granule components are also expressed, the 503PU line appears to have progressed to the myelocyte stage of neutrophil development. Comparison of gene expression profiles in these matched lines by differential display (DD) and representational difference analysis (RDA) resulted in the identification of many genes that were potentially regulated by PU.1. With the exception of one gene, all were shown to be false positives by Northern blot analysis. In an attempt to overcome this problem, we combined the technique of RDA with a bacterial library screen. Using this technique, four out of 11 possible targets were shown to be up-regulated in the 503PU line by Northern blot analysis. Two of the genes we identified with the combination technique, glutathione peroxidase (GPx) and serine leukoprotease inhibitor (SLPI), are involved in protecting neutrophils from the products they make to destroy pathogens. Analysis of these genes showed that PU.1 directly regulated the expression of the GPx gene. Thus, PU.1 has a dual role in neutrophils, regulating the expression of genes involved in the production of ROS as well as a gene that protects neutrophils from these destructive enzymes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
The monkey kidney cell line COS-7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% Fetalclone I (Hyclone, Logan, UT) fetal bovine serum (FBS) at 37°C in 7.5% CO2. The murine macrophage cell line P388D1 was cultured in DMEM containing 10% Fetalclone I at 37°C in 7.5% CO2. The IL-3-dependent murine myeloid cell lines 503 and 503PU were cultured in Iscove’s modified Eagle’s medium containing 20% FBS (Hyclone), 100 U/ml penicillin, 100 U/ml streptomycin, 1% ß-mercaptoethanol, and 1% L-glutamine. This was supplemented with 100 U recombinant murine IL-3 (R&D Systems, Minneapolis, MN) per ml at 37°C in 7.5% CO2.

Transfection analyses and reporter gene expression
Cells were seeded at a density of 4 x 105 cells/well in a six-well plate and were allowed to grow for 18 h. Cells were transfected using Geneporter I (Gene Therapy Systems, San Diego, CA). Briefly, 2 µg DNA (1 µg reporter and 1 µg expression)/well mixed with 0.5 ml serum-free DMEM combined with10 µl Geneporter/well mixed with 0.5 ml serum-free DMEM was incubated for 45 min at room temperature. The cells were washed once with serum-free media, and the 1 ml DNA/Geneporter mix was added directly to each well. Cells were incubated at 37°C in 7.5% CO2. After 4 h, 1 ml DMEM containing 2x serum (10% serum for COS-7 and 20% serum for P388) was added to each well. Cells were harvested after an additional 24 h of incubation. Firefly luciferase activity was measured using a Lumat LB 9501 luminometer (Berthold Systems, Pittsburg, PA). Luciferase assay reagent [105 µl; 20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)2- 5 H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, 470 µM luciferin, 530 µM adenosine 5'-triphosphate] was injected into a tube containing 20 µl lysate, and luciferase activity was measured for 20 s with no delays. Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, Madison, WI) per the manufacturer’s instructions. Briefly, cells were lysed as described above using the lysis buffer provided. Light units of total lysates were determined by using 100 µl of the luciferase assay reagent (firefly) provided and measuring for 10 s. The renilla luciferase assay reagent (100 µl) was added to the same tube, vortexed, and measured for an additional 10 s.

Plasmid constructions
The WT human GPx promoter and 3' region were cloned using polymerase chain reaction (PCR) from genomic DNA derived from the human monocytic cell line THP-1, based on published sequences [21 ]. The mutant human GPx promoter was made using overlap PCR with oligonucleotides containing a 3-bp mutation in the PU.1 binding site as described [22 ]. The fragments were ligated into Bluescript KS+ for sequencing and into the pXP2 luciferase reporter vector [23] to generate hGPXpxp2 and MhGPXpxp2 used in the transfection assays. The Nae reporter plasmid (naepxp2) is a 296-bp fragment of the PU.1 promoter cloned in-frame with the luciferase gene in the reporter vector pXP2. WT and mutant oligonucleotides generated from the sequence within the 3' regulatory region of GPx containing the potential PU.1 binding site were cloned into a HindIII restriction site 5' of the PU.1 promoter in this plasmid, generating GPx3'naepxp2.

Gel-shift assays
PU.1 cDNA cloned into Bluescript KS+ was used as template for in vitro transcription and translation of full-length PU.1 protein. Protein was made using the transcription-translation (TNT) T7/T3 coupled reticulocyte lysate system (Promega) according to the manufacturer’s instructions. Briefly, the 50-µl reaction contained 1 µg DNA, 25 µl TNT rabbit reticulocyte lysate, 0.5 µl each of the amino acid (AA) mixtures, AA-methionine and AA-leucine, 1 µl recombinant RNasin, 1 µl T3 polymerase, and 19 µl diethylpyrocarbonate water. The reaction was incubated at 30°C for 90 min, and the protein was stored at -80°C until use. Double-stranded GPx promoter and GPx 3' oligonucleotides were labeled with T4 kinase. For some experiments, the oligonucleotides were cloned into the HindIII site of Bluescript KS+ and sequenced to identify single binding-site plasmids. After restriction digestion, the single sites were end-labeled in a reaction containing 1x NEBuffer 3, 0.5 mM each dATP, thymidine 5'-triphosphate, and dGTP, 50 µCi 32P dCTP (3000 Ci/mM), and 5 U Klenow. A second restriction enzyme digestion (XhoI or PstI) was performed to excise the labeled DNA fragment from the vector. For competition analyses, assay conditions were as described above, except unlabeled oligonucleotide was added just before addition of labeled probe in the binding reaction. For supershifting of DNA–protein complexes, 1 µg anti-PU.1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction after the 15-min incubation and was allowed to incubate an additional 15 min at room temperature.

Screening for differentially expressed genes
Standard RDA and DD procedures identify a much higher number of false positives than actual differentially expressed clones. To overcome this limitation, we combined a cDNA subtraction RDA experiment with a subsequent library screen. Poly A+ RNA isolated from the 503 and 503PU cell lines was used in the Clontech PCR-Select cDNA subtraction kit, according to the manufacturer’s recommendation, to generate a pool of cDNA enriched from the PU.1-expressing cell line (BD Biosciences Clontech, Palo Alto, CA). This pool of cDNA was cloned into the topoisomerase T/A cloning vector (Invitrogen, Carlsbad, CA), and the resultant library was transformed and plated on Luria-Bertani (LB) agar media-containing ampicillin. For an initial screen, 240 colonies were duplicate-plated onto nitrocellulose membranes, placed on two 15-cm LB agar plates. The following day, the colonies were lysed, and the membranes were cross-linked with UV irradiation to fix the DNA. The membranes were placed in a sealed bag with prehybridization solution. We generated two different probes from subtracted populations of cDNA, 503 cDNA subtracted with 503PU cDNA or 503PU cDNA subtracted with 503 cDNA. By incorporating 32P dCTP into the final PCR step, these pools of cDNA were radiolabeled. After hybridization, the membranes were exposed to X-ray film, and the patterns between the membranes were compared to identify potential clones. Eleven colonies were selected for further analysis. After isolation of plasmid DNA from each colony, the insert cDNA was isolated and used as a probe for Northern blots of 503 versus 503PU poly A+ RNA. Four cDNAs were confirmed to be differentially expressed by Northern analysis. Each was sequenced, and this information was used for a BLAST search against the National Center for Biotechnology Information (NCBI; Bethesda, MD) database.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of fragment differentially expressed between the 503 and 503PU myeloid cell lines
Using Northern blot analysis, all fragments (with one exception) isolated using RDA or DD were not actually differentially expressed in the 503 and 503PU system. To reduce the number of false positives, we sought to develop a better method to identify genes whose expression is limited to the 503PU cell line and could be potentially directly regulated by the transcription factor PU.1. To accomplish this, we combined a cDNA subtraction procedure based on RDA with a cDNA plasmid library screen. An initial pilot screen of 240 colonies from the library yielded 11 potential positive clones. Northern blot analysis performed using poly A+ RNA generated from 503 and 503PU cells confirmed that four of the fragments (PU-3, PU-75, PU-82, PU-132) displayed increased expression in the 503PU cells as compared with the 503 cells. The sequence for each fragment was used to perform a BLAST search against the database at NCBI. All four fragments matched to known genes. Two of the cDNAs, fragments PU-75, identified as the actin-binding protein profilin, and fragment PU-132, identified as eukaryotic elongation factor 1{gamma}, were not studied further in this report. Fragment PU-82 was identical to SLPI, which had been the only gene we had identified as differentially expressed using RDA or DD alone. The fourth fragment, PU-3, was identical to GPx. Northern blot analysis showed that these two genes are highly expressed in the 503PU line, as compared with the 503 line (Fig. 1 ). As SLPI and GPx protect neutrophils from toxic molecules they make to destroy pathogens, we sought to determine whether PU.1 directly regulated the expression of either gene.



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  		Figure 1. Differential expression of genes. Northern blot analysis was performed on poly A+ RNA samples from 503 and 503PU cells. The blots were probed with fragments of two genes identified, SLPI and GPx. A fragment for the PU.1 gene and the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was used as control.

 
Identification of potential PU.1 binding sites for the GPx gene
Sequence analysis showed no consensus binding sites for PU.1 across the entire SLPI locus. As Ets family members can sometimes bind to nonconsensus sites, we cloned fragments of the SLPI locus into a luciferase reporter plasmid and used them in cotransfection assays along with a PU.1 expression vector. Band-shift analysis was also performed using these same fragments. Based on negative results from both sets of experiments (data not shown), we concluded that PU.1 does not directly regulate the expression of SLPI in the 503PU cell line.

We next turned our attention to the GPx locus. Upon examination of this locus and based on published reports, we analyzed two potential regulatory regions for possible PU.1 binding sites [23 , 24 ]. Two different cis-elements were identified as strong possibilities. The first site was in the promoter region upstream of the gene, and the second was in the 3' region containing the 3'-untranslated region (UTR; Fig. 2 ). These two potential PU.1 binding sites were also conserved between murine and human genomic sequences for the GPx gene.



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  		Figure 2. Potential PU.1 binding sites in the GPx locus. Analysis of the human GPx locus showed two potential PU.1 binding sites. The sequences for the two potential sites used to create oligonucleotides used in band-shift analysis (see Fig. 3 ) are shown in their relative position on the GPx locus. The core sequence is underlined. Mutant oligonucleotides used in subsequent experiments have changed the core sequence from GGAA to TCTA.

 
Specific binding of PU.1 to the putative PU.1 binding sites within the GPx regulatory regions
To determine if PU.1 were able to directly bind the potential PU.1 binding site in the GPx promoter and 3' region, bandshift analyses were performed. Double-stranded oligonucleotides of the promoter PU.1 site (hGPxp bandshift) were synthesized and end-labeled for use in binding reactions. In vitro-translated PU.1 (PU) and PU.1 from nuclear extracts made from the murine macrophage cell line RAW264.7 bound to this site (Fig. 3A ). The use of an anti-PU.1 antibody showed the band was PU.1, as the PU.1 band was missing and shifted most likely into a nonspecific band further up the gel (Fig. 3A) . Competition analyses were performed using unlabeled oligonucleotide DNA. WT CD45 PU-1 and hGPxp binding sites were able to compete for binding of the in vitro-translated PU.1 protein, and the mutated oligonucleotides for each of these sites were not able to compete for binding (Fig. 3B) . Additionally, when anti-PU.1 antibody was added to the binding reaction, the PU.1/DNA complex was supershifted (Fig. 3B) . These data suggested that PU.1 could directly bind the PU.1 site in the GPx promoter.



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  		Figure 3. Bandshift analysis of the PU.1 binding sites in the GPx locus. Bandshifts were performed to determine if PU.1 could bind to the GPx promoter or the 3' regulatory region. PU.1 shows the location of the PU.1 protein bound to each probe. (A) Oligonucleotides representing the GPx promoter site were used in a bandshift experiment with in vitro-translated PU.1 (PU) or nuclear extracts from the murine macrophage cell line RAW264.7 (Raw). Supershift analysis (ab) was performed with an antibody specific to PU.1. ns, Nonspecific bands. (B) Oligonucleotides representing the GPx promoter site were used in a bandshift with in vitro-translated PU.1 protein (PU). R, Rabbit reticulocyte alone. Molar excess (100-fold) of WT (wt) oligonucleotides for the PU.1 sites in the CD45 promoter or the GPx promoter competed for binding. Mutant (mut) oligonucleotides for each site, which has three bases of the core GGAA sequence mutated, did not compete for binding. Supershift analysis (ab) was performed with an antibody specific to PU.1, and the supershifted band is marked as SS. (C) Oligonucleotides representing the 3' site were used in a bandshift with in vitro-translated PU.1 protein (PU) or nuclear extracts derived from the murine macrophage cell line RAW264.7 (Raw). Supershift analysis (ab) is as discussed above. SS, Supershifted band. (D) Competition analysis using oligonucleotides from the 3' site. Molar excess (100-fold) of WT (wt) 3' or mutant (mut) 3' oligonucleotides was used in a bandshift experiment with in vitro-generated PU.1 protein.

 
Bandshift analyses were also performed with the 3' UTR regulatory region of the GPx gene. End-labeled, double-stranded oligonucleotide DNA containing the PU.1 binding site from the GPx 3' region was end-labeled and used in binding reactions. In vitro-translated PU.1 and nuclear extracts from the murine macrophage cell line RAW264.7 bound to this site from the GPx 3' region (Fig. 3C) . An anti-PU.1 antibody supershifted this complex, showing that it contained PU.1. Competition analysis showed that unlabeled, WT GPx 3' oligonucleotides could successfully block binding of in vitro-translated PU.1 protein from binding to this site, but mutant oligonucleotides could not compete for binding (Fig. 3D) . These data suggested that PU.1 bound to sites in two different regulatory regions of the GPx locus.

PU.1-dependent transactivation of the GPx promoter and 3' regions
To determine if the binding of PU.1 to the two sites in the GPx locus had functional relevance, cotransfection analyses were performed. WT and PU.1 binding site-mutated GPx promoter regions were cloned into the promoterless luciferase reporter plasmid pXP2. Cotransfection of these plasmids with the expression plasmid PU.1/pCB6 or the empty pCB6 vector into COS-7 cells showed that PU.1 increased reporter activity through the GPx promoter (Fig. 4A ). To complement this result, the WT GPx and mutated GPx promoter plasmids were transfected into the murine macrophage cell line P388D1. Results showed that mutation of the PU.1 binding site in the GPx promoter resulted in a 2.5-fold decrease in luciferase expression (Fig. 4B) . These data suggested that PU.1 could regulate expression of the GPx gene through its promoter.



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  		Figure 4. Transfection analysis of the GPx promoter and 3' regulatory regions. The GPx promoter and 3' regulatory regions were cloned into luciferase reporter plasmids. (A) The GPx promoter luciferase plasmid hGPXpxp2 was transfected into COS-7 cells with the PU.1 expression plasmid PUpCB6 or the empty pCB6 plasmid. The results from 10 transfections showed a 1.9 ± 0.3-fold increase in luciferase activity when PU.1 was present. (B) Transfection of the WT (wt) or mutant (mut) hGPXpxp2 plasmids into the murine macrophage line P388D1. The presence of the WT PU.1 site showed a 2.4-fold increase in luciferase light units, as compared with a mutant PU.1 site. Light units are shown to illustrate the high levels of activity of the GPx promoter in this cell line that expresses PU.1. (C) Transfection of the naepxp2 or the GPx3'naepxp2 plasmid with or without the PU.1pCB6 expression plasmid into COS-7 cells. The results from seven transfections showed that PU.1 transactivated the GPx3'naepxp2 reporter plasmid 3.2 ± 0.2-fold. In comparison, PU.1 only transactivated the naepxp2 reporter plamsid 1.8 ± 0.1-fold. (D) The naepxp2 and GPx3'naepxp2 plasmids were transfected into the murine macrophage line P388D1. The 3' regulatory region from the GPx gene increased the level of luciferase activity greater than twofold above the levels seen for the naepxp2 plasmid.

 
To determine whether PU.1 also could regulate GPx expression through the 3' regulatory region, a 250-bp fragment containing the PU.1 binding site was cloned upstream of a heterologous PU.1 promoter fragment in the pXP2 plasmid (naepxp2). This was done because of high light units generated using the endogenous GPx promoter. Full-length WT naepxp2 and GPx3'naepxp2 plasmids were cotransfected into COS-7 cells with and without the PU.1 expression plasmid PU.1/pCB6. As shown, PU.1 was able to increase reporter gene activity ~1.7-fold for the naepxp2 plasmid. This is a result of a PU.1 binding site located in the PU.1 promoter. The addition of the GPX 3' region increased the level of luciferase activity approximately twofold above the level seen for the naepXP2 plasmid alone (Fig. 4C) . In support of this result, both reporter plasmids were transfected into the murine macrophage line P388D1. The data showed that the addition of the 3' region to naepxp2 increased the level of light units approximately twofold in these cells (Fig. 4D) . These data suggest that the PU.1 can regulate GPx expression through the 3' site and that this site could cooperate with the promoter site. This would result in PU.1 being able to regulate the expression of the GPx gene to higher levels by using both sites.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have used the 503 myeloid cell line and its PU.1-expressing counterpart 503PU to identify genes that are directly regulated by PU.1. After determining that the high false-positive rates of conventional RDA and DD approaches prevented us from finding any genes that meet these criteria, we combined the techniques of RDA and a library screen. Using Northern blot analysis, we showed that four (out of 11 possible) fragments were actually differentially expressed in this system. Two of these genes, SLPI and GPx, are involved in protecting neutrophils from the destructive enzymes they produce to kill pathogens. Band-shift and transfection studies showed that PU.1 directly regulated the expression of the GPx gene through the promoter and a 3' regulatory region. This is the first study using a differential screening technique to not only identify targets of PU.1 but also to show that PU.1 directly regulates the expression of a target gene. These studies suggest that PU.1 is important for regulating the expression of destructive enzymes and the molecules that protect the neutrophil from these same enzymes.

Our studies on the 503 model are the first to specifically look for PU.1 gene targets in neutrophils. In addition, we have not only used Northern blot analysis to confirm differential expression but also showed whether the identified genes were directly regulated by PU.1. Three other studies have identified PU.1 gene targets using DD or cDNA subtraction techniques in other systems. The first study identified genes differentially expressed in fetal liver between PU.1 null mice and WT controls [24 ]. Liver-specific genes were suppressed, and the samples were enriched for early myeloid-specific genes found in the WT PU.1-expressing control. Genes identified in this study included lysozyme M, lactoferrin, gp91phox, gelatinase 3, procathepsin E, and several genes whose identity was unknown. Only procathepsin E represented a gene target not previously suggested as being regulated directly or indirectly by PU.1. This study, however, did not perform Northern blot analysis to confirm the up-regulation of this gene in the PU.1+/+ population nor show whether PU.1 directly regulated any genes that they identified. A second group used DD to identify genes up-regulated when PU.1 was overexpressed in MEL [25 ]. This group identified and confirmed by Northern blot analysis that osteopontin, eosinophil cationic protein, and B144 were potential PU.1 target genes. No further analyses such as promoter studies were performed to show that these genes were directly regulated by PU.1. A more recent, third study used a GM-CSF-dependent, early monocyte line derived from PU.1-/- fetal liver and a subline in which PU.1 has been re-expressed [26 ]. This group used suppressive, subtractive hybridization to identify three genes whose expression was higher in the PU.1 line. The differential expression of these three known genes (MRP14, Dap12, and CD53) was confirmed by Northern blot analysis, but no studies were performed to determine if PU.1 directly regulated the expression of any of these genes.

GPx is a selenium-containing protein whose expression is controlled at the levels of transcription and translation. Studies have shown that removal of selenium from the diet of rats resulted in decreased liver-specific GPx enzymatic activity and GPx mRNA levels [27 ]. Other in vitro studies on a human hepatoma cell line and the human myeloid cell line HL-60 have suggested that the depletion of selenium affected the levels of GPx protein but not the transcription of this gene [28 , 29 ]. This may be a result of the inability to incorporate the nonstandard AA selenocysteine into GPx at a UGA codon, which would terminate translation. Additional studies have shown that the presence of this UGA codon in GPx in selenium-free conditions may also result in an accelerated decay of the mRNA for GPx as a result of premature termination of translation [30 ]. Thus, although GPx is expressed in virtually all cell types in the body as the major cellular defense against H2O2 and other toxic oxidant species, the regulation of its expression is complicated.

Within the blood system, GPx has been found at low levels in most cell types, and the highest levels were seen in erythrocytes, monocytes, and neutrophils [31 ]. One study examined the levels of GPx expression during the differentiation of the HL-60 and PLB-985 cell lines into monocytes and granulocytes [32 ]. Their results showed an increase in GPx mRNA expression by Northern blot and an increase in gene transcription by nuclear, run-on assays, as neither cell line differentiated. This correlated with the ability of these cells to generate ROS. This suggests that neutrophils produce GPx as a way to protect themselves from toxic oxygen radicals to help them eliminate as many pathogens as possible before they die from these same destructive products.

A study of a series of human GPx promoter reporter gene plasmids showed that the GPx promoter displayed activity in several breast cancer cell lines following transfection [33 ]. Deletions of regions of the promoter had similar effects on reporter gene activity, regardless of the levels of GPx expressed in the cell. This suggests that the promoter may not be tissue-specific. Another study looked at the regulation of this gene in a MEL [34 ]. These studies showed that several tissue-specific DNase I hypersensitive sites were present in the 3' end of the GPx locus but only in the erythroid cell line and other high GPx-expressing cells. The 3' region showed tissue specificity following ligation to the GPx promoter in transfection studies. In addition, this report suggested that potential ETS and GATA factor-binding sites within the 3' region were important for erythroid-specific expression of GPx. In a third study, it was shown in human osteogenic cell lines that p53 could up-regulate GPx expression [35 ]. Cotransfection analysis using a p53 expression vector and a 262-bp region of the GPx promoter showed a p53-dependent up-regulation of reporter gene expression. Our studies now show that in neutrophils, PU.1 directly regulates GPx expression through sites in the promoter and 3' regulatory regions.

Neutrophils must be able to produce toxic molecules able to kill invading organisms and at the same time, must produce molecules to protect the neutrophil itself from the toxic molecules. Using the 503 myeloid cell line, we have now shown through a differential screening that PU.1 regulates the expression of genes involved in both activities. This regulation is through a direct mechanism for the protective GPx gene, as it is for the gp91phox component of the oxidase complex. It is through this type of complete analysis that combining confirmation of differential expression of a gene by Northern blot with studies on the cis-acting elements of the genes, we can continue to define true targets for tissue-specific transcription factors such as PU.1.


   ACKNOWLEDGEMENTS
 
United States Public Health Service Grant CA71384 supported this work.


   FOOTNOTES
 
Current address of Stacy L. Throm: Department of Experimental Hematology, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105.

Received February 7, 2003; revised March 11, 2003; accepted March 12, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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