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HEMATOPOIESIS
From the Fels Institute for Cancer Research and
Molecular Biology and Department of Biochemistry, Temple University
School of Medicine, Philadelphia, PA.
To understand the molecular mechanism by which interleukin-6 (IL-6)
regulates myeloid differentiation primary response (MyD) genes at the
onset of M1 myeloid differentiation, we used JunB as a representative
MyD gene to isolate and characterize IL-6 responsive elements. An IL-6
responsive element was localized between To understand the molecular mechanism by which
interleukin-6 (IL-6) regulates myeloid differentiation primary response
(MyD) genes at the onset of M1 myeloid differentiation, we used JunB as
a representative MyD gene to isolate and characterize IL-6 responsive
elements. An IL-6 response element (IL-6RE), localized between The CCAAT box is a widespread regulatory sequence that has been
identified in many eukaryotic promoters and is usually located between
NF-Y is a ubiquitous factor that is evolutionarily conserved. It was
originally identified as the component that interacts with the Ea Y box
of the major histocompatibility complex class II
promoter.9 It consists of 3 subunits, NF-YA, NF-YB, and NF-YC, all of which are required for DNA interaction.20
NF-YB and NF-YC belong to the histone H2A-H2B subfamily; they have
conserved domains of 90 and 84 amino acids, containing histonelike fold motifs important for subunit interaction,21 allowing for
NF-YB-NF-YC dimerization, which is a prerequisite for NF-YA
interaction. NF-Y plays a crucial role in transcriptional activity at
several levels. It increases the DNA affinity of other transcription
factors that bind to neighboring enhancer elements, participates in the
correct positioning of other transcription factors, and interacts with the TBP complex and proteins that have intrinsic histone
acetyltransferase activity.22-25
In this study, we identified NF-Y as the major protein that binds to
the Cells and cytokines
Infection of BM cells and colony formation assays
Assays for differentiation-associated properties Morphologic differentiation was determined by counting at least 300 cells on May-Grünwald-Giemsa-stained cytospin smears and scoring the proportion of blast cells, mature granulocytes, and macrophages as previously described.26,27 Immature blast cells are characterized by scant cytoplasm and round or oval nuclei; cells at intermediate monocyte stages of differentiation are flattened, with a larger cytoplasm-to-nucleus ratio, irregularly shaped nuclei, and few interspersed or no vacuoles; granulocyte intermediates are characterized by dented but not lobulated nuclei; mature macrophagelike cells are flattened and spread out cells interspersed with numerous vacuoles in a greatly enlarged cytoplasm; mature granulocytelike cells are characterized by enlarged cytoplasm and lobulated nuclei. Analysis of the expression of the specific cell surface marker of macrophages was done by fluorescence-activated cell-sorting (FACS) analysis by using fluorescein isothiocyanate (FITC)-conjugated F4/80 (Caltag Laboratories, Burlingame, CA) as previously described.26 Cells (2 × 106) were harvested by centrifugation at 200 rpm and washed twice with 1 × phosphate-buffered saline (PBS). To eliminate background, cells were resuspended in 1% bovine serum albumin and allowed to incubate at room temperature for 30 minutes. Cells were further incubated with FITC-conjugated F4/80, washed 2 times, resuspended in 1 × PBS, and analyzed by using the Coulter Epic Elite system (Coulter, Miami, FL) for variation in signal.General recombinant DNA techniques and probes The use of the XhoP2GD construct was described in detail.1 The DNA probes for the murine MyD genes have been previously described.28-30 Probes were purified and labeled as described.1 The retroviral expression plasmid, MSCV EB-neo, used in this study was a generous gift from Dr Robert G. Hawley (University of Toronto, Toronto, Ontario, Canada). The DN NF-YA29 (NF-YA DN) was a gift from Dr Peter Edwards (UCLA, Los Angeles, CA).31 The EcoRI/BglII 1.0-kb NF-YA29 insert was subcloned into the EcoRI and BglII restrictions sites of MSCV EB-neo by directional ligation.Gross deletions The 102GD was obtained by digesting XhoP2GD with
HpaI and NcoI, and the resulting 1.3-kilobase
(kb) fragment was isolated. This fragment was further digested with
BfaI and XcmI, and the 355-bp fragment that was
obtained was cloned into the XbaI site of
pCAT-Basic.
Smaller gross deletions were obtained by using the polymerase chain
reaction (PCR). For all PCR amplifications, the same 3' primer,
5'-GATCCTCTAGAGGGCTCGCTGCGG-3', was used, which changed a
SmaI restriction site into XbaI. The Northern blot analysis RNA extraction, loading of the gel, and hybridization conditions were all described in detail elsewhere.1 -Actin and
glyceraldehyde-3-phosphate dehydrogenase 3 (GADPH3) probes were
commercially obtained from Clonetech (Palo Alto, CA). Relative
messenger RNA (mRNA) levels were determined with the aid of a Fuji BAS
2000 phosphoimage analyser.
Electrophoretic mobility shift assays All nuclear extracts and electrophoretic mobility shift assay (EMSA) reactions were performed as mentioned before.1 NF-Y antibodies (1 µg) were incubated with extracts for 2 hours on ice prior to the addition of labeled probe. NF-YA and NF-YB antibodies obtained from Rockland (Gilbertsville, PA) were used in Western analysis, and antibodies obtained from Santa Cruz (Santa Cruz, CA) were used in EMSA experiments. All remaining antibodies were obtained from Santa Cruz. Most antibodies (2-5 µg), unless otherwise indicated, were incubated with extracts for 30 minutes at room temperature prior to the addition of labeled probe.Synthetic oligonucleotides and minimal promoter CAT gene constructs The synthesized oligonucleotide (UPENN), names, and sequence were as follows (only coding strands are indicated): JunBWT, 5'-GATCCCCCGCGTCGGCCAATCGGAGTGCACTTCCGCAGCTGA-3'; JunBMut, 5'-GATCCCCCGCGTCGGCCTGCAGGTCGACCCTTCCGCAGCTGA-3'; JunB Mut (67/65),
5'-GATCCCCCGCGTCCTGCAATCGGAGTGCACTTCCGCAGCTGA-3'; JunB
Mut (63/62),
5'-GATCCCCCGCGTCGGCCTGTCGGAGTGCACTTCCGCAGCTGA-3'; JunB Mut
(61/60),
5'-GATCCCCCGCGTCGGCCAAAGGGAGTGCACTTCCGCAGCTGA-3'; JunB
Mut (54/52),
5'-GATCCCCCGCGTCGGCCAATCGGAGTACCCTTCCGCAGCTGA-3'; NF-Y,
5'-GATCCTTTTTCTGATTGGTTAAAAGA-3'; NF-Y Mut,
5'-GATCCTTTTTCTGCGGTTTTAAAAGA-3'; NF-1,
5'-TTTTGGATTGAAGCCAATATGATAA-3'; Egr-1,
5'-GATCCATCCCGGCGCGGGGGCGAGGGCGTA-3'; Sp-1,
5'-ATTCGATCGGGGCGGGGCGAGC-3'; and C/EBP consensus binding sequence,
5'-TGCAGATTGCGCAATCTGCA-3'. For protein interaction to the longer probe
the following oligonucleotides were used: GC-CAAT,
5'-GATCCTCTAGGAGGGGGCCGCGGGGGCCTGGCCTCCCGCGTCGGCCAATCGGAGTGCACTTCCGCAGCTGA-3'; GCmut-CAAT, 5'-GATCCTCTAGGAGGTTTCCGCGTTTTCCTGGCCTCCCGCGTCGGCCAATCGGAGTGCACTTCCGCAGCTGA-3'; GC-CAATmut, 5'-GATCCTCTAGGAGGGGGCCGCGGGGGCCTGGCCTCCCGCGTCGGCCTGCAGGTCGACCCTTCCGCAGCTGA-3'; and GCmut-CAATmut,
5'-GATCCTCTAGGAGGTTTCCGCGTTTTCCTGGCCTCCCGCGTCGGCCTGCAGGTCGACCCTTCCGCAGCTGA-3'. Mutations are indicated by underlined sequences. The Sp-1, NF-1, and
C/EBP oligonucleotides were obtained from Santa Cruz Biotechnology. Complementary oligonucleotides were made so that there was a
BamHI restriction site at the 5' end and a BglII
site at the 3' end, respectively. DNA probes (1 µg) were made by
annealing complementary oligonucleotides by heating to 75°C for 10 minutes and then slowly cooling to room temperature.
Methyl interference assay For methyl interference experiments either coding or noncoding strands of the JunBWT probe were 5' end labeled by using 5' DNA Terminus Labeling System with [~32P]ATP (NEN). The complementary strands were annealed at 75°C for 10 minutes and allowed to slowly cool to room temperature, ensuring that the probe was labeled only at
one 5' end. Probes were gel purified and isolated as mentioned before.
Limited methylation on labeled probe (107 cpm) was carried
out with dimethylsulfate (Sigma) in 200 µL dimethylsulfate buffer
(Sigma) for 2.5 minutes at 30°C. The methylated probe (50 000 cpm)
was used in gel shift binding reactions with 20 µg nuclear extract and 5 pg poly (dl-dC) and electrophoresed on a 5%
nondenaturing polyacrylamide gel. Following autoradiography, bound and
free probes were excised and eluted with extraction buffer (10 mM Tris [pH 8.0], 10 mM EDTA, and 200 mM NaCl) overnight at room temperature, passed through Dextran-sulfate (Sigma) capillary columns, washed with
washing buffer (10 mM Tris [pH 8.0], 10 mM EDTA, and 300 mM NaCl),
and eluted with elution buffer (10 mM Tris [pH 8.0], 10 mM EDTA, and
600 mM NaCl). The recovered DNA was cleaved with 1 M piperidine (Sigma)
at 90°C for 30 minutes, lyophilized and resuspended in 10 µL formamide sample loading buffer (80% formamide, 10 mM
NaOH, 1 mM EDTA, 0.1% xylene cyanole, and 0.1% bromophenol blue). An
equal number of counts were loaded on a 12% polyacrylamide DNA
sequencing gel.
Western blot analysis Whole extracts were prepared from 3 × 107 cells, which were washed twice with 5 mL cold PBS and lysed in RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris [pH 8.0], 2 µg/mL Aprotinin, 2 µg/mL Benzamidine, 2 µg/mL Pepstatin A, 2 µg/mL Leupeptin, and 100 µg/mL 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride [AEBSF]). Cells were allowed to sit on ice for 30 minutes, and extracts were collected as supernatants after centrifugation. Protein concentrations were determined according to the Bradford method, using a Bio-Rad protein assay kit.Unless otherwise indicated, 50 µg of each protein extract was electrophoresed on 7.5% to 12.5% SDS-polyacrylamide gels (SDS-PAGE), and transferred to polyvinylidene diflouride (Millipore, Bedford, MA) membranes using 3-(cyclohexylamino)-l-propanesulfonic acid (CAPS) (Sigma) transfer buffer for 30 minutes at 65 V. For Western blot analysis, membranes were blocked with Blotto A solution (5% nonfat dry milk in 1 × PBS and 0.05% Tween-20) for 1 hour at room temperature, probed with primary antibodies overnight at 4°C, and finally incubated with secondary antibody antirabbit or antigoat immunoglobulin horseradish peroxidase-linked whole antibody (1:5000 dilution; Amersham Life Science, Cleveland, OH) for 2 hours at room temperature. The antibodies were detected by using enhanced chemiluminescence reagents (Amersham). Rabbit polyclonal NF-YA and NF-YB antibodies were obtained from Rockland (Gilbertsville, PA) and used at 1:1000 and 1:500 dilution, respectively. Goat polyclonal NF-YC antibody, obtained from Santa Cruz, was used at a 1:1000 dilution.
CCAAT box is the critical component of the 65/52 IL-6RE in its promoter region.1 This site contains both a CCAAT box and an IR
repeat region (Figure 1A). Previously, we
have shown that a specific protein complex is bound to the 65/52
IL-6RE.1 To more precisely define the binding site of the
65/52 IL-6RE to the nuclear protein complex, mutant
oligonucleotides were used in gel retardation experiments, each having
2 or 3 bases of the wild-type 65/52 IL-6RE (JunBWT) sequence
mutated (Figure 1B).
JunBMut ( Further evidence that the CCAAT box, and not the IR site, is important
for binding of the protein complex to the It was also of interest to examine whether the sequences that are
important for protein binding in vitro are also important for the
IL-6-mediated induction of JunB in vivo. To do this, the mutant
oligonucleotides were subcloned upstream from the JunB minimal promoter
in the JunB minimal promoter expression vector, which is unresponsive
to IL-6.1 These constructs were transiently transfected
into M1 cells and assayed for IL-6 responsiveness (Figure 1D). JunBMut
( NF-Y is the protein complex bound to the CCAAT box of the 65/52 IL-6RE,
competition experiments were performed by using JunBWT-labeled probe
and cold competitor oligonucleotides containing cognate sequences
specific for binding to C/EBP family proteins, NF-1/CTF, and NF-Y
(Figure 2A). Excess unlabeled C/EBP and
NF-1/CTF oligonucleotides did not compete with the probe for complex
formation (Figure 2A, lanes 2 and 3). However, the NF-Y consensus
sequence successfully competed with the labeled probe for protein
binding, whereas a NF-Y mutant oligonucleotide did not (Figure 2A,
lanes 4 and 5). This result indicates that NF-Y binds to the 65/52
IL-6RE.
Direct evidence that NF-Y is bound to the CCAAT box comes from antibody
competition experiments, in which antibodies specific against known
CCAAT box binding proteins were used. NF-Y is a heterotrimeric factor
composed of 3 subunits, NF-YA (40-43 kd), NF-YB (32 kd), and NF-YC (30 kd). As shown in Figure 2B, antibodies specific against each of these
subunits caused a super shift of the complex (lanes 16-18),
whereas antibodies against C/EBP family proteins and NF-1 had no effect
(lanes 2-14). This finding clearly indicates that NF-Y is bound to the
CCAAT box within the Because an intact CCAAT box is necessary for both protein binding to
the NF-Y proteins are constitutively expressed in M1 cells Previously, we have shown that there is no IL-6-inducible binding to the65/52 IL-6RE.1 Because it has been
demonstrated that NF-Y is a component of the 65/52 IL-6RE
protein-DNA complex, Western analysis was performed to examine NF-Y
expression during M1 myeloid differentiation (Figure
3A). NF-YA mRNA is differentially spliced
to produce 2 isoforms, a 40-kDa and 43-kDa protein. Only the 40-kDa
isoform was detected, both prior to and following IL-6-induced M1
differentiation. In addition, both NF-YB and NF-YC expression, like
NF-YA, is not regulated by IL-6. Therefore, NF-YA, NF-YB, and NF-YC
proteins are constitutively expressed in M1 cells. Possibilities for
transcriptional activation of the 65/52 IL-6RE/NF-Y complex include
posttranslational activation of NF-Y by IL-6 and/or interaction with
and modification of other proteins.
Effect of the NF-YA DN mutant protein on IL-6-mediated induction of JunB Because NF-Y can bind to the CCAAT box in the65/52 IL-6RE and
the integrity of the CCAAT box is necessary for IL-6 induction of JunB,
it is important to know the functional significance of NF-Y in the
IL-6-mediated induction of JunB. Toward this end, we used a NF-YA DN
mutant, which allows heterotrimirization with NF-YB and NF-YC but
prevents binding to DNA.31 By using a MSCV retroviral
expression vector, M1 cell lines were established that express NF-YA
DN. Several neo-resistant clones were isolated and analyzed for NF-YA
DN expression and binding to the 65/52 IL-6RE. As shown in Figure
3B, the M1NF-YA DN clones expressed exogenous NF-YA DN transcripts,
whereas the parental M1 cells or M1 cells infected with the MSCV EB-neo
empty vector did not. The NF-YA DN transgene encodes for the 43-kDa
splice variant of NF-YA, and, when expressed in M1 cells, both the
larger exogenous NF-YA DN protein band and the endogenous 40-kDa NF-YA
protein band were detected (Figure 3C). The difference in intensity of
the protein bands was due to unequal loading of the gel.
To confirm that the NF-YA DN protein could inhibit NF-Y binding, EMSA experiments were performed with labeled JunBWT probe and extract from M1 cells expressing the NF-YA DN transgene (Figure 3D). It can be seen that NF-Y binding was suppressed 10- to 20-fold in the M1NF-YA DN cell lines, indicating that NF-YA DN could inhibit NF-Y transcriptional activity. To examine the effect of NF-YA DN protein on the IL-6 inducibility of JunB, the XhoP2GD CAT construct, which is IL-6 responsive, was transiently transfected into the different established cell lines and assayed for IL-6 induction of CAT expression (Figure 3E). By using this approach, IL-6 inducibility was inhibited by about 50% in the M1NF-YA DN cell lines, compared with the parental M1 or M1Neo control cells. Given the complexity of the transcriptional machinery, it is not surprising that there was not a linear relationship in the effect of NF-YA DN on suppression in NF-Y DNA binding and suppression of JunB induction that is driven by a limited promoter region of an exogenously transfected expression vector. Thus, to further establish the role NF-Y plays in JunB inducibility by IL6, endogenous JunB expression was examined in 3 independently isolated M1 NF-YA DN cell lines following IL-6 stimulation. As shown in Figure 3F, JunB mRNA was significantly suppressed in M1 NF-YA DN cells compared with M1Neo control cells on IL-6 stimulation. Furthermore, determining the ratio of JunB suppression to suppression of NF-Y DNA binding (Figure 3D) has indicated that suppression of JunB inducibility by NF-YA DN correlates well with suppression of NF-Y DNA binding (Figure 3F). These data are consistent with NF-Y-mediating induction of JunB gene expression by IL-6, indicating that NF-Y participates in the regulation of JunB induction by IL-6 in M1 myeloid cells. NF-Y plays a role in the induction of MyD genes by IL-6 So far we provided evidence that the immediate early activation of JunB, a representative MyD gene, is regulated by a novel IL-6 response element, which binds the NF-Y transcription factor. If NF-Y plays a role in regulating the coordinate activation of MyD genes at the onset of myeloid differentiation, NF-Y binding elements should be present in the promoter regions of these genes. By using the GAP aligning program of the GCG package (Madison, WI), NF-Y binding motifs, with the essential CCAAT motif intact, were found in the promoter regions of most MyD genes (Figure 4A).
To ascertain if NF-Y does play a role in the coordinate transcriptional activation of MyD genes by IL-6, MyD gene expression was examined in the M1 NF-YA DN cells following IL-6 stimulation. As shown in Figure 4B, all MyD genes were induced at lower levels in M1 NY-YA DN cells compared with the M1Neo control cells on IL-6 stimulation. Furthermore, it can be seen that suppression of MyD gene induction correlated with suppression of NF-Y DNA binding. These data are consistent with NF-Y-mediating induction of MyD gene expression by IL-6. Effect of NF-YA DN on M1 myeloid differentiation Because the MyD gene response was inhibited following IL-6 treatment of M1 NF-YA DN cells, we wanted to examine what effect inhibiting NF-Y function would have on terminal myeloid differentiation of M1 cells. NF-YA DN cells were observed to be less sensitive to IL-6 treatment at low concentrations compared with control cells, which was expected because of the reduced MyD response in these cells (data not shown). Analysis of cell morphology showed that at low concentration of IL-6 (10 ng/mL) M1NF-YA DN cells had a lower percentage of mature macrophages compared with the parental M1 and M1Neo controls (Figure 5, 10%-15% compared with 27%-28%). This results in a higher percentage of cells with blastlike and intermediate-stage morphology for M1NF-YA DN cells compared with M1 and M1Neo cells. To further corroborate the results of the cytologic examination, M1Neo and M1 M1NF-YA DN cells untreated and following treatment with IL-6 (10 ng/mL) were analyzed for the expression of the macrophage-specific cell surface differentiation marker F4/80. As indicated in Figure 5, a lower percentage of M1NF-YA DN cells treated with IL-6-expressed F4/80 compared with the parental M1 and M1Neo controls. Collectively, these data indicate that NF-Y plays a role in the progression of M1 myeloid differentiation following IL-6 stimulation.
NF-Y expression during myeloid differentiation of normal cells from BM Because all the previously described data were obtained using the M1 myeloid leukemia cell line, it was necessary to ascertain if the same holds true in normal BM cells induced for macrophage differentiation. BM cells were treated with MCSF and IL-6, which results in terminal macrophage differentiation (data not shown). RNA was extracted from these cells and analyzed for JunB expression (Figure 6A). JunB induction was detected by 12 hours following MCSF/IL-6 treatment and continued to be expressed up to 3 days. This induction is different from IL-6-induced M1 differentiation in which JunB expression reaches peak levels after only 1 hour.28,29 EMSA analysis, using labeled JunBWT probe with extracts from BM, either untreated or treated with MSCF/IL-6, showed induction of the protein-DNA complex (Figure 6B). Similar results were obtained when the BM cells were treated with MCSF alone (data not shown). To verify that NF-Y is a component of the protein-DNA complex, antibody competition experiments were performed, using extracts obtained from BM treated with MCSF/IL-6 for 3 days. As shown in Figure 6C, antibodies specific against all 3 NF-Y subunits disrupted the complex, indicating that NF-Y is indeed part of the induced protein-DNA complex. To determine if NF-Y protein expression is regulated, protein expression was analyzed using Western blots. NF-YA was induced following MCSF/IL-6 treatment, whereas NF-YB and NF-YC expression remained unchanged (Figure 6D). The regulation of NF-YA expression may account for the observed MCSF/IL-6-induced NF-Y binding to65/53 IL-6RE during BM differentiation; however, it is possible
that posttranslational modifications are also required for the
induced binding.
Effect of NF-YA DN expression on myeloid differentiation of BM cells Because NF-Y is a positive modulator of M1 myeloid cell differentiation and is induced during myeloid differentiation of BM, it was asked if NF-Y plays a role in normal hematopoietic cell development. To do this, we used high-efficiency retroviral transduction to infect normal BM cells with a retroviral expression vector expressing the NF-YA DN transgene. Retroviral particles were generated by transfecting the pMSCV retroviral vectors (MSCV-neo and MSCV-NF-YA29) into the high-efficiency Bosc23 packaging cell line. Normal BM cells were then infected with the resulting retroviral particles, seeded in methylcellulose supplemented with G418, and treated with either IL-3 or MCSF plus IL-6. After 8 days, the neo-resistant colonies were counted. As shown in Table 1, no difference in colony numbers was observed for Neo-infected and NF-YA DN-infected BM cells when the cells were treated with IL-3 alone, which promotes extensive proliferation as well as differentiation. However, when the cells were induced for macrophage differentiation with MCSF plus IL-6, more colonies were observed in the NF-YA DN-infected BM than the Neo-infected controls (Table 1). Inhibiting NF-Y function reduces terminal differentiation; the inhibition of differentiation would account for the higher colony number observed for MCSF plus IL-6-treated BM expressing the NF-YA DN transgene (Table 1). To further corroborate this notion, analysis of cell morphology was carried out in which the neo-resistant colonies were removed by pipet from the methylcellulose and were used to prepare cytospin smears. As shown in Figure 7A, there was no difference in cell morphology for Neo-infected BM and NF-YA DN-infected BM, when the cells were treated with IL-3 alone. However, when treated with MCSF plus IL-6, NF-YA DN-infected BM had fewer mature cells, with a concomitant increase in intermediate differentiated cells compared with Neo-infected BM (Figure 7B). Similar results were obtained when the cells were treated with MCSF alone (data not shown). Analysis for expression of the macrophage specific cell surface marker F4/80 correlated with the data obtained by the morphologic analysis (Figure 7A,B) Taken together, this data indicate that, like in M1 cells, NF-Y plays a role in modulating normal myeloid differentiation.
At the onset of M1 differentiation, a set of MyD primary response
genes is induced. JunB was chosen as a paradigm to dissect the
molecular mechanisms of the IL-6-mediated induction of MyD response
genes. We have identified a novel IL-6 responsive element on the JunB
promoter, termed By using antibodies against known CCAAT box binding factors in
protein-DNA binding experiments, it was shown that NF-Y binds specifically to the Direct evidence for NF-Y involvement in JunB induction comes from experiments using the NF-YA DN transgene, which inhibits NF-Y interaction with DNA. It has been shown that inhibition of NF-Y leads to a decrease in JunB induction. Furthermore, the observation that NF-Y binding motifs are present in the promoter regions of other MyD genes and that their induction is also regulated by NF-Y suggests that NF-Y plays a role in the regulation of coordinate induction of MyD genes by IL-6. NF-Y has been shown to be involved in regulating the expression of other myeloid genes, including myeloperoxidase in response to granulocyte colony-stimulating factor,38 and the ferritin heavy chain39 and gpc91-phox40 on spontaneous differentiation of peripheral blood monocytes to macrophages in vitro. Blocking the IL-6-mediated MyD response by expression of the NF-YA DN transgene leads to a decrease in mature macrophages, verifying the role of NF-Y in terminal myeloid differentiation of M1 cells. Unlike in M1 cells, inducible NF-Y binding was detected in BM cells on myeloid differentiation. When BM cells are stimulated with MCSF plus IL-6, the cells first pro |
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Abstract
Introduction
