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Originally published online as doi:10.1189/jlb.1205759 on May 9, 2006

Published online before print May 9, 2006
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(Journal of Leukocyte Biology. 2006;80:196-203.)
© 2006 by Society for Leukocyte Biology

All-trans retinoic acid-induced expression of bactericidal/permeability-increasing protein (BPI) in human myeloid cells correlates to binding of C/EBPß and C/EBP{epsilon} to the BPI promoter

Andreas Lennartsson*, Karina Vidovic*, Malene Bjerregaard Pass{dagger}, Jack B. Cowland{dagger} and Urban Gullberg*,1

* Division of Hematology and Transfusion Medicine, Lund University, Sweden; and
{dagger} Granulocyte Research Laboratory, Rigshospitalet, Copenhagen, Denmark

1 Correspondence: Department of Hematology, BMC, C14, S-221 84 Lund, Sweden. E-mail: urban.gullberg{at}med.lu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bactericidal/permeability-increasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBPß and C/EBP{epsilon} to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBPß and C/EBP{epsilon} provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins.

Key Words: transcriptional regulation • granulocyte • azurophil granule protein • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, the innate immune system is the first line of host defense. The system is an evolutionarily, conserved form of microbial defense, as indicated by shared pathogen recognition, signaling pathways, and functional mechanisms between mammals and Drosophila (reviewed in refs. [1 , 2 ]). Bactericidal/permeability-increasing protein (BPI) is a component of the innate defense, and binding of BPI to Gram-negative bacteria causes an increase in outer membrane permeability and eventually, inner membrane damage followed by cell death [3 , 4 ]. An evolutionarily conservation of BPI is indicated by the presence of BPI orthologs in several species, ranging from mouse to rainbow trout [5 , 6 ]. BPI consists of an amino-terminal domain important for high-affinity binding to the lipid A moiety of lipopolysaccharide (LPS) of Gram-negative bacteria and a carboxy-terminal domain mediating opsonization [7 8 9 ]. Consistent with its high affinity for LPS, BPI is a paralogue to the acute-phase LPS-binding protein (LBP; reviewed in ref. [10 ]). LBP is a 60-kDa glycoprotein that binds LPS and delivers it to membrane-bound CD14/Toll-like receptor 4 (TLR-4) on inflammatory cells, thus evoking a strong, inflammatory response with increased secretion of proinflammatory cytokines, e.g., tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin 1 (IL-1; reviewed in ref. [11 ]). In addition to the involvement of LBP, a LPS-MD2 complex was identified recently as a potent trigger of TLR-4 activation [12 ]. When secreted excessively, TNF-{alpha} and IL-1 may give rise to septic shock with multiorgan failure (reviewed in ref. [13 ]). Thus, septic shock can be viewed as an uncontrolled and exaggerated inflammatory response, although recent results indicate that sepsis in later stages may be characterized by a severely compromised immune system (reviewed in ref. [14 ]). BPI counteracts the LPS-initiated inflammatory response by binding and neutralizing LPS [15 ]. Therefore, BPI is of potential use in treatment of sepsis caused by Gram-negative infections and other situations where LPS plays a role [16 , 17 ].

The neutrophil granulocyte is the major effector cell in the human innate defense against invading microbes. Neutrophils store potent, microbicidal proteins and peptides in different granules. Several antimicrobial proteins, such as myeloperoxidase (MPO), serine proteases, defensins, and BPI, dominate the content of the azurophil granules (reviewed in refs. [18 , 19 ]). BPI is a major constituent in neutrophils [20 , 21 ] but is also present in granules of human eosinophils [22 ] and on the surface of monocytes [23 ]. Moreover, certain epithelial cells can be induced to synthesize BPI [24 ], and BPI-mRNA is also present in the testis [5 ].

The content of the different granule subtypes of the neutrophil is determined by the synthesis of proteins belonging to different granule subtypes separated in time during maturation [19 ]. Azurophil granule proteins are the first to be expressed during the promyelocyte (PM) stage, followed by specific granule proteins in myelocytes (MC). Accordingly, transcription of BPI is regulated positively by acute myeloid leukemia 1 (AML 1) and PU.1 [25 ], factors also involved in transcription of other azurophil granule proteins. Several independent observations indicate, however, that transcriptional regulation of BPI is distinct from that of other azurophil granule proteins; newborn children are specifically deficient in BPI and are still equipped with normal amounts of other azurophil granule proteins, such as MPO and defensins [26 , 27 ], arguing for a distinct regulation of BPI expression. Moreover, in cases of specific granule deficiency (SGD), a rare disease with absent gene expression of several proteins stored in specific granules, BPI is also affected [28 ]. This disease has been linked to homozygous elimination of the neutrophil transcription factor CCAAT/enhancer-binding protein (C/EBP){epsilon} [28 , 29 ], suggesting that C/EBP{epsilon} is involved in the regulation of BPI, while most other azurophil granule proteins are regulated by C/EBP{alpha} [30 , 31 ]. The human PM cell line NB4 does not express detectable amounts of BPI. Treatment of the cells with all-trans retinoic acid (ATRA) results in induction of neutrophil maturation, including up-regulation of C/EBPß and C/EBP{epsilon} [32 , 33 ]. One of the questions we asked was whether this induced differentiation includes up-regulation of BPI expression and whether it affects the binding of C/EBPs to the BPI promoter.

We now report that in vivo, BPI mRNA is expressed in MC and metamyelocytes (MM), cells also showing expression of mRNAs for specific granule proteins. ATRA strongly induces BPI expression in human myeloid NB4 cells by mechanisms dependent on de novo protein synthesis. Moreover, ATRA-induced expression correlates to binding of C/EBPß and C/EBP{epsilon} to the proximal promoter of BPI. We conclude that binding of C/EBPß and C/EBP{epsilon}, but not of C/EBP{alpha}, to the BPI promoter distinguishes BPI from other investigated azurophil granule proteins and provides an explanation for the expression pattern of BPI during neutropoiesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human PM NB4 cells [34 ] were maintained in RPMI 1640 with 10% fetal calf serum (PAA Laboratories GmbH, Pasching, Austria). In induction experiments, ATRA (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 10 µM for the incubation times indicated. For inhibition of protein synthesis, cycloheximide (Sigma-Aldrich) was added to a final concentration of 100 µg/ml.

Isolation of neutrophils and their precursors from bone marrow and peripheral blood
Isolation of neutrophil precursors and polymorphonuclear neutrophils (PMNs) was performed as described previously [35 ]. Briefly, bone marrow samples from healthy volunteers were separated by centrifugation on a two-layer PercollTM (Amersham Biosciences, Buckinghamshire, UK) gradient, which resulted in separation of bone marrow cells into three bands containing neutrophil precursors of different maturity: myeloblasts (MB) and PM, MC + MM, and band cells and segmented neutrophils (BC+SC), respectively. PMNs from peripheral blood were isolated by centrifugation on Lymphoprep (Nygaard, Oslo, Norway). Non-neutrophil cells were removed from the cell populations by depletion using the magnetic cell sorter system (Miltenyi Biotec, Bergisch Gladbach, Germany).

Northern blot
RNA was extracted using Trizol LS reagent (Invitrogen Corp., Carlsbad, CA). An amount of 15 µg total RNA was separated on a formaldehyde gel, followed by transfer to a GeneScreen plus membrane (NEN Life Science Products, Boston, MA). Equal loading, RNA quality, and transfer efficiency were checked by Radiant Red RNA stain (Bio-Rad Laboratories, Hercules, CA). A 730-base pair (bp) cDNA, corresponding to the amino-terminal half of BPI, was labeled with [32P]deoxy-cytidine 5'-triphosphate using the RediprimeTM II kit (Amersham Biosciences). High-stringency hybridization was performed at 68°C with the ExpressHyb solution (BD Biosciences-Clontech, Palo Alto, CA). The membrane was washed with 2x saline sodium citrate (SSC), 0.05% sodium dodecyl sulfate (SDS), 3 x 10 min at room temperature, and 0.1x SSC, 0.1% SDS, 2 x 20 min at 50°C. Membranes were analyzed using a Molecular Imaging FX analyzer (Bio-Rad Laboratories).

Northern blotting of RNA from PMNs and neutrophil precursor populations and subsequent hybridization was performed as described previously [36 ]. The blot was hybridized to a 1695-bp BPI cDNA probe, and ß-actin was used as equal loading control. cDNA probes for MPO and lactoferrin (LF) were as described [36 ]. The blot was developed and quantified using a PhosphorImager (FUJI X Bas 2000). The intensity of the signals from the probes was normalized to the intensity of the internal standard ß-actin, as described [36 ].

Biosynthetic labeling and immunoprecipitation
Biosynthetic radiolabeling and immunoprecipitation of newly synthesized proteins were performed with rabbit polyclonal BPI antibody [21 ], as described elsewhere [37 ]. Immunoprecipitated material was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using 10–20% Tris-glycine gels and fluorography.

Real-time reverse transcription-polymerase chain reaction (RT-PCR)
RT of 400 ng total RNA was performed using the Taqman kit (Applied Biosystems, Foster City, CA) and random hexamer primer. Real-time PCR was performed with Taqman probes in the 7000 Sequence Detection system (Applied Biosystems), according to the manufacturer’s instructions. Specific primers and probes for human BPI, {alpha}-defensin 1-3, cathepsin G, proteinase 3, and LF were purchased from Applied Biosystems (Assay-by-Demand). Real-time RT-PCR of ß2-microglobulin (Applied Biosystems) was used as an internal control. Data were collected and analyzed with the sequence detector, v.1.1, software (Applied Biosystems). Relative quantitative data were calculated based on the {Delta}{Delta}CT method, where normalization is performed as follows: {Delta}CT = CT (sample) – CT (ß2-microglobulin); {Delta}{Delta}CT = {Delta}CT (induced) – {Delta}CT (uninduced); and relative quantification = 2–{Delta}{Delta}CT (reviewed in ref. [38 ]).

Protein extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described previously [39 ], with the modification that 0.6% Nonidet P-40 was included in the lysis buffer and that protease inhibitors, Complete (Roche Diagnostics GmbH, Mannheim, Germany), were used in all buffers. Probe was prepared by labeling DNA oligonucleotide probe with [{gamma}-32P]adenosine 5'-triphosphate, as described [25 ]. The oligonucleotide 5'-GGTTCCTTCCTCCTTTCCACATTCTACTGACT-3', corresponding to –107/–76 bp, according to the numeration seen in Go Go Go Figure 4 and ref. [25 ], was used as probe. Nuclear extract was incubated with the labeled DNA probe for 20 min at room temperature in binding buffer (HEPES, pH 7.9, 25 mM KCl, 1.25 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM ZnCl2, and 50% glycerol), supplemented with 0.3 µg poly(dIdC)/15 µl reaction mixture (Amersham Biosciences). The antibodies [C/EBP{alpha} (14aa)X, sc-61X, C/EBPß (1c-19)X, sc-150X, C/EBP{delta} (m-17), sc-636, C/EBP{epsilon} (c-22) sc-158, all from Santa Cruz Biotechnology, CA] were added together with labeled probe. The samples were separated on 6% polyacrylamide Tris-boric acid-EDTA gel and analyzed in a Molecular Imaging FX analyzer (Bio-Rad Laboratories).


Figure 1
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  		Figure 1. In vivo BPI mRNA expression profile during neutrophil differentiation. Total RNA was extracted from bone marrow-derived granulocytic precursor populations enriched for MB + PM, MC + MM, BC + SC, and from PMNs. (Upper panel) RNA was subjected to Northern blotting and high-stringency hybridization with cDNA for BPI, LF, and MPO. A ß-actin probe was used as equal loading control. Results from two donors are shown. Positions of 28S and 18S rRNA are indicated. (Lower panel) Schematic representation of hybridization intensities. For each probe, the cell population showing maximal expression is given Value 1; the expression levels of the other cell populations are shown relative to this. All expression levels are normalized to the intensity of the internal standard ß-actin.

 

Figure 2
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  		Figure 2. Expression of BPI in bone marrow cells and cell lines. (A) Total RNA was extracted and subjected to Northern blotting and high-stringency hybridization with cDNA for BPI. Expression of BPI is shown in normal bone marrow progenitor cells (positive controls), as well as in NB4 cells treated with 10 µM ATRA for 24 h. Position of 18S rNA is indicated with an arrow to the left. (B) NB4 cells were incubated for 24 h in the presence or absence of ATRA (10 µM), after which, biosynthetic labeling and immunoprecipitation of BPI were performed. Positions of molecular weight markers are indicated with arrows to the left.

 

Figure 3
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  		Figure 3. ATRA-induced expression of BPI mRNA in NB4 cells is dependent on de novo protein synthesis. NB4 cells were treated with 10 µM ATRA for up to 24 h. Cycloheximide (100 µg/ml) was added to parallel incubations to investigate dependence on protein synthesis. Total RNA was extracted, and BPI mRNA expression was determined with real-time-RT-PCR. The figure shows levels of BPI mRNA normalized to the level in uninduced control cells, mean values ± ranges (n=2).

 

Figure 4
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  		Figure 4. Sequence of the EMSA probe with C/EBP site in relation to partial sequence of the BPI promoter (adopted from ref. [25 ]). Numeration from the ATG translation start site in bold. Transcription start sites are indicated with arrows; C/EBP- and PU.1-binding sites are underlined [25 ].

 
Chromatin immunoprecipitation (ChIP)
ChIP was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, 1 million cells were cross-linked with 1% formaldehyde for 20 min, harvested, and sonicated 4 x 40 s at 100% power using a UP 50H sonicator (Hielscher Systems GmbH, Teltow, Germany). After 30 min preclearing with salmon sperm DNA/protein A slurry, 6 µg indicated antibody (see above) was added and incubated overnight at 4°C. The immunocomplexes were washed, after which the cross-linking was reversed by incubation at 65°C for 4 h in the presence of NaCl. After proteinase K treatment, the immunoprecipitated DNA was recovered with phenol/chloroform extraction and ethanol precipitation. The DNA was resuspended in 40 µl water, of which 15 µl was used as template in a 32-cycle PCR amplification, using forward primer 5'-CACACACATACACACACGCACG-3' and reverse primer 5'-AAGAGTCGGCTGGGGAAATG-3'.

Western blotting
Western blotting analysis was performed, as described previously [40 ]. Briefly, 5 x 106 cells were harvested, washed, and lysed. Extracted proteins were separated by SDS-PAGE and transferred to a Hybond-P membrane (Amersham Biosciences). Antibodies against the different C/EBPs (Santa Cruz Biotechnology) were used as primary antibodies and a peroxidase-conjugated swine anti-rabbit antiserum (Dako, Glostrup, Denmark) as secondary antibody. The electrochemiluminescence Western blotting detection system (Amersham Biosciences) was used for detection.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vivo BPI mRNA expression profile during neutrophil differentiation
Three different granulocytic cell populations were purified from normal human bone marrow and depleted for non-neutrophilic cells as described [35 , 36 ]. The neutrophil precursors were separated in populations highly enriched for MB + PM, MC + MM, and BC + SC. Mature PMNs were extracted from peripheral blood. The different cell populations were investigated for BPI, LF, and MPO mRNA expression by Northern blotting technique. As shown in Figure 1 , BPI displayed a distinct mRNA expression profile, with a sharp peak in the MC + MM population. This expression pattern is similar to that of the specific granule protein LF (Fig. 1) but quite distinct from the expression of MPO, which shows most pronounced expression in the MB + PM population (Fig. 1) . Thus, the expression profile of BPI is different from that of other azurophil granule proteins such as MPO, which are mainly expressed during the MB and PM stage of maturation [36 ]. Rather, BPI shares the expression profile of mRNAs encoding LF and other specific granule proteins [36 ]. No BPI mRNA expression could be detected in peripheral neutrophils.

ATRA induces BPI expression in NB4 cells
NB4 is a cell line derived from a patient with acute PM leukemia and harbors the t(15;17) chromosomal translocation [34 ]. NB4 cells undergo partial granulocytic maturation upon induction with ATRA. The maturation is only partial, as the cells undergo apparent phenotypic maturation but fail to express secondary granule protein genes [41 ]. In agreement with the in vivo expression profile of BPI, showing absence of BPI mRNA in PM (Fig. 1) , uninduced NB4 cells expressed no BPI. However, treatment of NB4 cells with 10 µM ATRA for 24 h resulted in an up-regulation of BPI at the level of mRNA and protein, as judged by Northern blotting and biosynthetic labeling, respectively (Fig. 2 ). For quantification of the increase in mRNA, real-time RT-PCR was performed. In addition, changes in mRNA of other azurophil and secondary granule proteins in response to ATRA were analyzed. It is interesting that an ~60-fold increase in BPI mRNA was specific for this transcript, inasmuch as mRNA expression for the azurophil granule proteins, cathepsin G and proteinase 3, was actually down-modulated after ATRA treatment (Table 1 ). Moreover, the expression of {alpha}-defensin 1-3 mRNA was almost unchanged, although a slight increase could be seen (Table 1) . No expression of the specific granule protein LF could be detected in accordance with previous data [41 ]. We conclude that treatment of NB4 cells with ATRA results in robust expression of BPI mRNA, and levels of other azurophil and specific granule protein mRNAs are decreased or unaffected.


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  		Table 1. Specific ATRA-Induced Expression of BPI in NB4 Cells

 
ATRA-induced expression of BPI mRNA is dependent on de novo protein synthesis
Real-time RT-PCR analysis revealed that BPI mRNA induction was detectable after 12 h (data not shown and Fig. 3 ) and that an ~60-fold increase was attained after 24 h of ATRA treatment (Table 1) . To investigate whether BPI mRNA induction was dependent on de novo protein synthesis, we inhibited protein synthesis with 100 µg/ml cycloheximide. As shown in Figure 3 , cycloheximide completely abrogated ATRA-induced expression of BPI mRNA, indicating indirect effects of ATRA, possibly involving de novo synthesis of transcription factors.

ATRA-induced expression of BPI mRNA correlates to C/EBPß and C/EBP{epsilon} binding to the BPI promoter
The finding that ATRA-induced expression of BPI mRNA is dependent on de novo protein synthesis suggests that ATRA induces synthesis of a transcription factor that binds to the promoter of BPI. Different C/EBP family members have been shown to be ATRA-responsive in myeloid cells [32 , 33 ], and previous characterization of the BPI promoter has revealed C/EBP-binding sites in the proximal promoter [25 ] (Fig. 4 ). Moreover, mutations of the C/EBP sites have been shown to reduce the promoter activity in HL-60 cells [25 ]. Therefore, we hypothesized that ATRA induces C/EBP binding to the proximal BPI promoter. To correlate activation of BPI expression by ATRA to C/EBP binding, we performed ChIP analysis and EMSA. Prior to ATRA induction of NB4 cells, no binding of C/EBP to the BPI promoter could be detected by ChIP (Fig. 5 ). However, after 24 h of ATRA treatment, binding of C/EBP was clearly detected. The binding was specific for C/EBPß and C/EBP{epsilon}, insofar as binding of C/EBP{alpha} and C/EBP{delta} was not detected (Fig. 5) . Consequently, ChIP demonstrates that ATRA treatment induces C/EBPß and C/EBP{epsilon} binding to the proximal promoter of BPI.


Figure 5
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  		Figure 5. ChIP analysis to calculate the degree of binding of C/EBPß and C/EBP{epsilon} to the BPI promoter. NB4 cells incubated for 24 h with or without 10 µM ATRA were used in ChIP analyses. Specific antibodies for the various C/EBP family members were used in the immunoprecipitation, and antibodies against cyclin D1 were used as a negative control. The last lane is a negative control containing no template. Typical result is shown.

 
To gain further support for binding of C/EBPß and C/EBP{epsilon} to the BPI promoter in response to ATRA, EMSA was performed. Nuclear extracts of ATRA-induced or uninduced NB4 cells were mixed with an oligonucleotide corresponding to –107 to –76 bp in the promoter, containing a putative C/EBP site (Fig. 4) . We have, in previous analyses, shown that this upstream C/EBP site is the most important for BPI expression [25 ]. Therefore, an EMSA probe containing this C/EBP site was used in our assay (Fig. 4) . Indeed, a shift appeared after induction with ATRA, which could not be obtained with uninduced nuclear extract and consequently, was specific for ATRA-induced cells (Fig. 6 ). Although addition of antibodies against different C/EBP family members did not result in any obvious supershift, the specific shift disappeared after addition of antibodies against C/EBPß, C/EBP{delta}, and C/EBP{epsilon} but not after addition of antibodies against C/EBP{alpha}. The elimination of the shift is likely to have been the result of antibody interaction with the DNA-binding protein, disturbing its DNA-binding capacity, indicating binding of C/EBPß, C/EBP{delta}, and C/EBP{epsilon} to the oligonucleotide. As the induced shift remained unaffected after incubation with antibody against C/EBP{alpha}, the data suggest that C/EBP{alpha} is not bound to this part of the BPI promoter after induction with ATRA. We conclude from the ChIP analysis and EMSA that ATRA-induced expression of BPI in NB4 cells correlates to the appearance of C/EBPß and C/EBP{epsilon} bound to the proximal promoter. Although EMSA also indicated binding of C/EBP{delta}, this observation was not supported by ChIP analysis.


Figure 6
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  		Figure 6. Direct binding of C/EBP to the BPI promoter shown by EMSA. A probe corresponding to –107/–76 bp, including the putative C/EBP-binding site (Fig. 4) , was incubated with nuclear extract from NB4 cells treated for 24 h with or without 10 µM ATRA. Arrows indicate the shifts, as well as free probe. Specific antibodies for C/EBP members were added to the reaction mixtures, as indicated.

 
C/EBPß and C/EBP{epsilon} expression is up-regulated by ATRA stimulation of NB4 cells
Results described above indicate that ATRA-induced expression of BPI involves binding of C/EBPß and C/EBP{epsilon} to the promoter of BPI (Figs. 5 and 6) . Given that the effect of ATRA on BPI expression is dependent on de novo protein synthesis (Fig. 3) , we asked whether ATRA increases the protein levels of C/EBPß and C/EBP{epsilon} in NB4 cells. As shown in Figure 7 , this is indeed the case; C/EBPß was not expressed in uninduced cells, but ATRA treatment for 24 h strongly induced C/EBPß protein (Fig. 7) . In the case of C/EBP{epsilon}, uninduced NB4 cells also expressed significant levels of protein, but ATRA treatment further increased C/EBP{epsilon} protein, especially the p27 isoform and more moderately, the p30 isoform (Fig. 7) . By contrast, the protein levels of C/EBP{alpha} and C/EBP{delta} were unaffected or decreased in response to ATRA (Fig. 7) . We conclude that ATRA increases the protein levels of C/EBPß and C/EBP{epsilon}.


Figure 7
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  		Figure 7. Western blot of C/EBP protein in ATRA-induced NB4 cells, which were incubated for 24 h with or without 10 µM ATRA and were lysed and subjected to SDS-PAGE and immunoblot analysis using antibodies against different C/EBP family members. Immunoblotting with antibody against actin was used as control of equal loading. Positions of C/EBP proteins and C/EBP{epsilon} protein isoforms are indicated with arrows.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that BPI is expressed in the MC and MM cell population, and most azurophil granule proteins are expressed during the PM stage of maturation [18 , 19 ]. Thus, BPI displays an expression pattern more similar to specific granule proteins than other azurophil granule proteins. BPI shares this characteristic with defensin, for which the levels of mRNA and protein during myeloid maturation reach their highest level after the peak of expression of most other azurophil proteins [36 ]. Moreover, BPI and defensins are the only azurophil granule proteins included in SGD [28 ], indicating transcriptional control mechanisms in common with those of genes encoding specific granule proteins.

The present data demonstrate that ATRA strongly induces transcription of BPI in human PM NB4 cells. The positive response of BPI mRNA to ATRA was not shared by proteinase 3 and cathepsin G, the expression of which was rather down-regulated. The effect of ATRA on expression of BPI involves de novo protein synthesis, as inhibition of protein synthesis completely abrogated an ATRA-induced increase of BPI mRNA. The fairly slow induction of BPI mRNA in response to ATRA is compatible with a requirement of de novo protein synthesis, which is also in agreement with previous failure to reveal retinoic acid response elements (RAREs) in the proximal promoter of BPI [25 ]. Given that induction of BPI expression by ATRA in NB4 cells correlated to increased levels of C/EBP{epsilon} and C/EBPß protein and also to direct binding of C/EBP{epsilon} and C/EBPß to the BPI promoter, as demonstrated by ChIP and EMSA, our data strongly propose that C/EBP{epsilon} and C/EBPß are mediators of the effects of ATRA on the BPI promoter. C/EBP{epsilon} was, in contrary to C/EBPß, also expressed in uninduced NB4 cells. However, no C/EBP{epsilon} binding to the BPI promoter could be detected in uninduced NB4 cells. Whether, this is a result of required heterodimer formation with C/EBPß or post-transcriptional modifications of C/EBP{epsilon}, such as phosphorylation [42 ], remains to be elucidated.

C/EBP{epsilon} is involved in transcriptional regulation of genes encoding specific granule proteins such as LF and collagenase [43 , 44 ], and C/EBP{epsilon} is essential for late granulocytic differentiation [45 ]. Our present results are consistent with previous reports that C/EBP{epsilon} is up-regulated in NB4 after treatment with ATRA via RAREs in the C/EBP{epsilon} promoter [32 , 46 ] and that C/EBPß expression and DNA binding are induced rapidly by ATRA via a PM leukemia-retinoic acid receptor {alpha}-dependent pathway in NB4 cells [33 ].

Evidence for the critical role of C/EBP{epsilon} in the regulation of BPI is provided by the previous observation that BPI expression is absent in SGD patients with homozygous mutation of C/EBP{epsilon} [28 , 29 ]. Besides BPI, defensins are the only azurophil granule components that are absent in SGD patients [47 ]. It is interesting that C/EBP{epsilon} binds to the promoter of neutrophil defensins after ATRA stimulation of NB4 cells [48 ]. It is therefore likely that defensins and BPI share common regulatory mechanisms, including C/EBP{epsilon}. In NB4 cells, however, ATRA caused only a modest increase in the amount of {alpha}-defensin mRNA, and the increase in BPI mRNA was almost 60-fold, suggesting that robust expression of {alpha}-defensins needs additional transcriptional activators or release of transcriptional repression.

Our results demonstrate that besides C/EBP{epsilon}, C/EBPß also contributes to the C/EBP DNA-binding complex. C/EBPß is important for monocytic maturation but is also involved in neutrophil [33 , 49 ] and eosinophil [50 ] differentiation. Moreover, expression of C/EBPß and C/EBP{epsilon} is up-regulated during neutrophil maturation in vivo [35 ]. Given that BPI is present primarily in neutrophils [20 , 21 ] but also in eosinophils [22 ] and on the surface of monocytes [23 ], C/EBPß may be a common regulator of BPI in all three cell types.

Results of the EMSA suggest that C/EBP{delta} also participates in ATRA-induced BPI regulation. C/EBP{delta} and C/EBP{epsilon} have been shown to activate transcription of genes encoding specific granule proteins [42 , 48 ]. Binding of C/EBP{delta} to the BPI promoter was not, however, confirmed by the ChIP analysis. Therefore, additional experiments are required to clearly demonstrate a role for C/EBP{delta} in BPI expression.

In our previous characterization of the proximal promoter of BPI, we identified a potential C/EBP-binding site (–100), partly overlapping a PU.1 site, which is functionally important for constitutive expression of BPI in HL-60 cells [25 ]. However, in contrast to HL-60 cells, PU.1 does not bind to this site in uninduced NB4 cells [25 ]. Taken together, data indicate that PU.1 and C/EBP can bind to sites close to each other and that both factors affect promoter activity in a positive way. In HL-60 cells, a transiently transfected BPI promoter driving the luciferase reporter gene clearly lost transcriptional activity after mutation of the C/EBP/PU.1 site [25 ]. Recent investigations about the transcriptional regulation of BPI in epithelial cells give further support for a prominent role of C/EBP [51 ]. Unfortunately, it was not possible to evaluate the effect of ATRA on promoter-reporter constructs in NB4 cells, as ATRA rapidly down-modulated all transiently transfected luciferase reporter cells, including positive and internal controls, with strong viral promoters. The reason for this is unclear, but it made it impossible to use ATRA in reporter experiments.

In conclusion, the present data demonstrate that BPI mRNA is expressed in vivo in a MC and MM cell population. Moreover, ATRA-induced BPI expression involves induction of C/EBPß and C/EBP{epsilon}, and binding of these transcription factors to the promoter correlates to BPI expression. Transcriptional activation dependent on C/EBPß and C/EBP{epsilon} may be a feature of the transcriptional control of BPI, which is related to the transcriptional control of specific granule proteins and distinct from that of most other azurophil granule proteins.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Foundation for Strategic Research, the Swedish Research Council (Project #11546), the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Georg Danielsson Foundation, the Gunnar Nilsson Cancer Foundation, the Österlund Foundation, and the Lundberg Foundation and from Funds of Lund University Hospital.

Received December 28, 2005; accepted March 28, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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