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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1578-1589
c-Maf Induces Monocytic Differentiation and Apoptosis in Bipotent
Myeloid Progenitors
By
Shrikanth P. Hegde,
JingFeng Zhao,
Richard A. Ashmun, and
Linda H. Shapiro
From the Departments of Pathology and Laboratory Medicine and
Tumor Cell Biology, St Jude Children's Research Hospital,
Memphis, TN.
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ABSTRACT |
The transcriptional mechanisms that drive colony-forming unit
granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not
fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a
developmentally regulated pattern, peaking at the promyelocyte stage,
or in cell model systems, appearing soon after the induction of
monocytic differentiation. We wished to determine if this
developmentally related interaction is a consequence of myeloid
differentiation or an intrinsic differentiating stimulus. Because the
elevated Myb:Maf status seen in differentiating cells can be
recapitulated by overexpression of c-Maf in myeloid cell lines, we
inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid
progenitor cells. Elevated levels of c-Maf protein led to marked
increases in Myb:Maf complexes and the accumulation of
monocyte/macrophage cells, followed by eventual programmed cell death.
Analysis of targets that could mediate these phenotypic changes
indicated that c-Maf likely plays a key role in myeloid cell
development through dual mechanisms; inhibition of a select set of
c-Myb regulated targets, such as Bcl-2 and CD13/APN,
coupled with the activation of as yet undefined
differentiation-promoting genes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PRECISE REGULATION OF the c-Myb
transcription factor is essential for normal hematopoiesis.
Interference with T-cell-specific c-Myb expression in transgenic mice
causes growth arrest and reduced proliferative capacity of the T-cell
compartment.1 c-Myb null mice die late in gestation and
display greatly reduced numbers of hematopoietic progenitors,
presumably due to their reduced capacity to establish a normal
proliferative program.2 However, these progenitors can
still differentiate, suggesting that c-Myb sustains hematopoietic
progenitor cells in a proliferative mode, thereby regulating the shift
between proliferation and differentiation.2-4 Consequently,
inhibition of Myb activity during hematopoiesis could block
proliferation, setting the stage for differentiation on receipt of the
appropriate inductive signals.
Members of the Maf family of basic region/leucine zipper (bZIP)
transcription factors play an essential role in growth and development
by regulating tissue-specific gene expression.5-7 These
proteins activate or repress transcription depending on their
particular protein partner and the context of the target promoter. For
example, selective expression of c-Maf dictates the ratio of T-helper
cell subsets by positively regulating interleukin-4 (IL-4)
expression.5 Similarly, the differentially expressed and
highly homologous MafB protein physically binds to and inhibits the
activity of the Ets-1 protein in avian myeloid cells.7 Maf
family proteins are essential to both avian and mammalian development
and differentiation. The tissue restricted avian L-Maf regulates
multiple lens-specific genes, and ectopic expression of this protein is
sufficient to induce lens differentiation in ectodermal and cultured
cells.6 Finally, c-Maf null mice have severe multisystem
defects with high mortality rates in late gestation (L. Glimcher,
personal communication, July 1998, and Kim et
al8), illustrating a fundamental role for c-Maf in
mammalian development.
We have recently demonstrated9 that c-Myb and c-Maf
physically interact, resulting in the inhibition of Myb-dependent gene transcription in early myeloid cells through a mechanism that requires
the binding of c-Myb, but not Maf, to DNA. Although c-Maf mRNA and
protein levels remain constant during myeloid development, the
formation of inhibitory Myb:Maf-DNA complexes is developmentally regulated, with the highest levels found in promyelocytic cells and in
progenitors soon after their induction to differentiate. By contrast,
markedly lower levels of these complexes are present in either immature
myeloblasts or later developmental stages, and during terminal
differentiation. The regulation of complex formation appears to be
independent of Myb, as addition of Myb protein does not affect Myb:Maf
complex levels, or do levels of free Myb protein correlate with complex
levels.9 This developmental pattern of protein interaction
suggests that Maf modulation of c-Myb activity may be an important
regulatory pathway for the control of transcription and the initiation
of myeloid cell differentiation.
Enforced expression of c-Maf in early myeloid cells lacking Myb:Maf
complexes results in the formation of inhibitory complexes, thereby
recapitulating the regulatory interactions normally occurring in
maturing myeloid cells.9 Because downregulation of Myb may prepare cells for differentiation, we postulated that conditional expression of c-Maf would both inhibit obligatory Myb targets and
provide the necessary inductive signals to drive cells to differentiate. Here we report that induction of c-Maf in 2 bipotential human myeloid cell lines induces the appearance and accumulation of
cells characteristic of the monocytic phenotype. Importantly, levels of
Myb:Maf complexes in these induced clones increased in a pattern
similar to that seen on phorbol-induced monocytic differentiation, supporting the hypothesis that c-Maf plays a crucial
role in myeloid cell differentiation. Finally, prolonged c-Maf
induction in these cell lines results in a decrease in Bcl-2 protein levels and subsequent apoptotic cell death, consistent with inhibition of Myb activity and terminal monocytic differentiation.
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MATERIALS AND METHODS |
Cell lines.
Human cell lines included the myeloid leukemia lines HL-60 (American
Type Culture Collection [ATCC], Rockville, MD, CRL
1593) and U937 (ATCC, CRL 1593). Cells were grown in RPMI-1640 medium supplemented with 2 mmol/L L-glutamine and 10% fetal calf serum. HL-60
and U937 cells were induced to differentiate by using
12-0-tetradecanoylphorbol diester (TPA, 5 × 10 6 mol/L) added to the culture medium; cells were
harvested at indicated time points after addition. Anti-IL-4 and
matched control antibodies (R & D Systems, Minneapolis,
MN) were used according to the manufacturer's instructions.
Expression vectors and reporter plasmids.
The CD13/APN minimal reporter construct  411luc contains genomic
sequences from bp 411 through bp +65 of the CD13/APN myeloid promoter in the pGL2basic backbone (Promega, Madison,
WI)10. The conditional expression plasmid
pMT-CB6-cMaf was made by cloning the full-length murine c-Maf
cDNA11 into HindIII/XbaI cut
pMT-CB69. Ligation of the Myb-dominant interfering
construct (MEnT1, the generous gift of Dr Kathleen
Weston, CRC Centre for Cell and Molecular Biology,
Chester Beatty Laboratories, London, UK) into XbaI cut pMT-CB6
vector resulted in the conditional Myb dominant negative construct
pMT-CB6-MEnT.
Transfection of recombinant plasmids and reporter gene assays.
To analyze inhibition of Myb transcriptional activity, c-Maf stably
transfected U937 or HL-60 cloned cell lines were induced with 150 µmol/L ZnSO4 for the indicated time intervals before electroporation with 5 µg of the CD13/APN 411luc promoter
construct10 and 2 µg of the control  -actin-secreted
alkaline phosphatase (SEAP) plasmid as described.10 The
transfection efficiency for each construct was normalized to the
control level of SEAP activity12; the reported values were
calculated as relative light units (RLU) per unit of SEAP activity.
Each point was determined at least 4 times.
Northern and Western blot analysis.
polyA+ RNA was purified from cell lines using the FastTrack
2.0 kit (Invitrogen, Carlsbod, CA). Total RNA was
extracted using Tri Reagent (Molecular Research Center, Inc,
Cincinnati, OH). A total of 10 µg of poly A+ RNA or 20 µg of total RNA from the indicated cell lines was separated on a 1%
agarose-formaldehyde gel, transferred to nylon membranes, and
sequentially probed with the BstEII/NcoI fragment containing the
5' region of murine c-Maf (which excludes the bZIP domain), the
3-kb BamHI fragment of the murine CSF-1R cDNA, and a
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA
probe (Clontech, Palo Alto, CA). For Western blot
analysis, proteins were separated on a 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and
transferred onto nitrocellulose. Membranes were blocked with 5% milk
in Tris-buffered saline (TBS) buffer; incubated
with the primary antibody (rabbit anti v-Maf and goat
anti-actin polyclonal antisera, Santa Cruz
Biotechnology, Santa Cruz, CA; monoclonal mouse
anti-human Bcl-2, Upstate Biotechnology, Lake Placid,
NY; or anti-Myc for detection of Myc-tagged MEnT, Calbiochem, San Diego, CA), followed by horseradish peroxidase
(HRP)-linked secondary antibody. Specific HRP-conjugated protein
complexes were detected according to the ECL protocol (Amersham,
Arlington Heights, IL).
DNA binding assays.
For DNA binding assays, whole-cell lysates from myeloid cells were
prepared by resuspending and washing pelleted cells in cold
phosphate-buffered saline (PBS) and resuspending the pellet in lysis
buffer (20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L dithiothreitol [DTT], 20% glycerol, 50 mmol/L KCl) containing a
protease inhibitor cocktail (Boehringer-Mannheim Complete Inhibitor
Cocktail Tablets; Boehringer-Mannheim, Indianapolis,
IN). After addition of Triton X-100 to 0.5% final concentration
(vol/vol), the lysate was incubated on ice for 60 minutes, and the
debris pelleted for 15 minutes at 14,000 rpm. Cleared lysate was
quantitated and stored at 80°C until use. Cell lysates (20 µg) were preincubated in binding buffer (10 mmol/L HEPES, pH 7.9, 50 mmol/L KCl, 5 mmol/L MgCl2, 10% glycerol, 1 mmol/L DTT,
and 1 µg poly d[I-C]) in 25-µL reactions with or without 0.1 µg
of the consensus double-stranded oligonucleotide competitors (Myb
consensus site: mimAmyb-CTAGGACATTAT AACGGTTTTTTAGT10; Maf
consensus site: MARE-TCGAGCTCGGAATTGCTGACT CAGCATTACTC13) for 15 minutes at 4°C before the addition of probe. For supershift experiments, 1 µL of an anti-v-Maf polyclonal antibody (Santa Cruz
Biotechnology, cross-reactive with c-Maf) was preincubated with lysate
for 10 minutes at 4°C. The 32P-end-labeled genomic
fragment probe (CD13/APN XbaI/DdeI fragment; containing
sequences bp 433 through 3059) was then added
and incubated for an additional 15 minutes at 4°C. Binding
reactions were electrophoresed through a 3.5% acrylamide gel
containing 10% glycerol in TAE buffer at 4°C. Gels were dried and
exposed to x-ray film (Kodak, Rochester, NY).
Apoptosis analysis.
DNA from ZnSO4 induced cells was isolated at the indicated
intervals (see Fig 8C) using described methods.14 Briefly,
cells were harvested, washed with PBS and resuspended in lysis buffer (1% NP-40 in 20 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 7.5; 10 mL per 1 × 106 cells). After centrifugation, SDS was added to
1% and the supernatants were treated for 2 hours with RNase A (5 mg/mL
final concentration) at 56°C followed by digestion with proteinase
K (2.5 mg/mL final concentration) for 2 hours at 37°C. The DNA was
precipitated, separated by electrophoresis in 1% agarose, and
visualized by ethidium bromide staining. TdT-mediated dUTP nick end
labeling (TUNEL) assays were performed as
described15 on cells treated with zinc for 24 hours (MEnT)
or 48 hours (c-Maf).
Reverse transcription-polymerase chain reaction (RT-PCR).
RNA was extracted with TRI-Reagent (Molecular Research Center,
Inc) according to the method provided. Fifteen
micrograms of total RNA was used as template for first-strand
complementary DNA (cDNA) synthesis using Superscript II reverse
transcriptase (GIBCO/BRL, Gaithersburg, MD) with 100 U ribonuclease inhibitor, 1 µL random primer
(GIBCO/BRL), and nuclease-free water to 36 µL. The reaction was
heated to 70°C for 10 minutes and then quick chilled on ice.
Second-strand synthesis was performed in 5X first strand buffer (12 µL), 10 mmol/L each dNTP (3 µL), and 3 µL
SuperScript II, and the reaction incubated at 25°C for 10 minutes,
42°C for 50 minutes, and at 75°C for 15 minutes. PCR
amplification was performed with 1 µL cDNA, 10 µL 10X PCR buffer, 8 µL dNTP (2.5 mmol/L each), 1 µL 3' primer (100 mmol/L), 1 µL 5' primer (100 mmol/L), add dH2O up to 99.5 µL, and 0.5 µL AmpliTaq Gold. The cDNA was heat denatured at 94°C for 9 minutes, then amplified through 35 cycles of 94°C, 45 seconds,
58°C, 45 seconds, and 72°C, 45 seconds. For c-Maf RT-PCR,
5' primer: TGCACTTCGACGACCGCTTCTCGG, 3' primer:
AAGGTGGCTAGCTGGAATCGCG. MafG RT-PCR primers are as published16; IL-4 and b-actin primers were purchased from
Stratagene (La Jolla, CA). Positive control plasmid
template containing the IL-4 cDNA was purchased from ATCC.
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RESULTS |
c-Maf promotes monocytic/macrophage differentiation of HL-60 and U937
myeloid cells.
In myeloid cells, the highest levels of Myb:Maf protein complexes are
found in those representing early stages of differentiation or in cells
soon after the induction of monocytic differentiation. By contrast,
markedly lower levels are apparent in either immature myeloblasts or
later developmental stages, linking the interaction of these 2 proteins
with myeloid differentiation.9 To address whether this
interaction is a consequence of myeloid differentiation signals or a
differentiating stimulus in and of itself, we engineered HL-60 and U937
cells (containing equivalent, low levels of endogenous c-Maf mRNA,
Fig 1A) to inducibly express the
full-length murine c-Maf cDNA11 using the
metallothionine-based expression vector pMT-CB6.9 Although
protein expression from the individual clones varied, extremely high
induction of c-Maf was detected in several clonal lines (Fig 1B). The
low level of endogenous c-Maf protein in parental cells is not
detectable in whole-cell lysates, but is evident on immunoprecipitation
followed by Western blot analysis.9 Consistent with our
model, electrophoretic mobility shift assay (EMSA)
analysis of myeloid cell lysates indicated a dose-dependent increase in
the levels of Myb:Maf containing complexes binding to the
CD13/APN target promoter fragment on zinc induction of c-Maf-containing clonal lines (Figs 1C, lanes 4 and 5; 1D, lanes 10 to
16), in effect, mirroring the increase observed in cell lysates from
TPA-induced (monocytic lineage) parental cell lines (Fig 1D, lanes 17 and 18). These complexes consist of physically interacting Myb and Maf
proteins bound to the Myb consensus site in the target promoter
fragment as confirmed by competitor oligonucleotide and specific
antibody abrogation of the complexes (Fig 1C, lanes 6 to 8 and Hegde et
al9). Maf itself does not contact the CD13/APN promoter probe.9 Therefore, these c-Maf-expressing clones
recapitulate the Myb:Maf status of myeloid cells driven to
differentiate along the monocyte/macrophage pathway.
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| Fig 1.
c-Maf in myeloid cell lines and stable cell clones. (A)
Endogenous expression of c-Maf mRNA expression levels in human myeloid
cell lines as determined by Northern blot analysis of
polyA+ RNA from the HL-60 promyelocytic or U937
monoblastic cell lines. A single blot was initially probed with the
c-Maf specific cDNA probe, stripped, and reprobed with c-Myb, and
finally, -actin as a control for RNA loading and integrity. Exposure
times for optimal detection of c-Maf mRNA were routinely significantly
longer than for other probes (1 week v 1 day). (B) Conditional
expression c-Maf in stably transfected clones. Individual HL-60 or U937
clonal cell lines containing either CB6 vector alone (left 2 lanes) or
c-Maf (right lanes) encoding plasmids were cultured for 24 hours with
150 µmol/L ZnSO4. Cell lysates were analyzed beside
control lysates from a previously established c-Maf inducible cell line
(+).9 The gels were transferred and probed for c-Maf
expression with an antiserum that recognized the c-Maf protein. (C)
Myb:Maf complexes increase on c-Maf induction. An end-labeled 129-bp
promoter fragment probe containing the functionally defined
CD13/APN myeloid promoter elements9 was incubated
with uninduced (-) or induced (+) whole-cell lysates from control
vector-containing (CB6, lanes 2 and 3), c-Maf-containing (c-Maf clone
6, lanes 4 to 8) stable HL-60 clones. Unlabeled competitor
oligonucleotides containing either the consensus Myb site from the
mim-1 promoter (mimAmyb, lane 6) or the consensus Maf binding
site (MARE, lane 8) were added to the assays in 100-fold molar excess
before addition of probe. Maf binding to its consensus oligonucleotide
interferes with its ability to complex with Myb.9
Antibodies recognizing the c-Maf protein (lane 7) were added to binding
reactions before probe addition. (D) The increase in Myb:Maf complexes
is dose-dependent and comparable to induction on monocytic
differentiation. The identical promoter fragment probe used in (C) was
incubated with uninduced (0) or induced (indicated concentrations)
whole-cell lysates from control vector-containing (CB6, lanes 10 and
11), c-Maf-containing (c-Maf, lanes 12 to 16) stable HL-60 clonal
lines or from untreated (lane 17), TPA-treated (lane 18) HL-60 parental
cells.
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Our initial observations of three different pMT-c-Maf-containing U937
clonal cell lines showed striking morphological changes after 48 hours
of zinc induction. These cells proliferated more slowly, were
noticeably larger, extensively vacuolated and multinucleated with a
decreased nuclear:cytoplasmic ratio (Fig 2, lower left panel), characteristic of macrophage morphology. To verify this phenotypic designation, cells were stained for  -napthyl-butyrate esterase (ANB, Fig 2, right panel), a standard indicator of monocytic specification.17 The development of deep red cytoplasmic
staining in 50% to 60% of the c-Maf-overexpressing cells and
TPA-treated control cells verified the presence of ANB esterase and
confirmed that c-Maf overexpression triggered monocytic
differentiation.
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| Fig 2.
Morphologic and lineage analysis of cloned U937 cell
lines conditionally expressing c-Maf. Cytospin preparations of parental
U937 cells either untreated (top row) or treated with TPA (second row);
or U937 clonal lines containing either vector control (U937 CB6, third
row) or c-Maf expression constructs (U937 c-Maf, bottom row) incubated
for 48 hours with 150 µmol/L ZnSO4. Left column:
Wright-Giemsa stain for morphological examination; right column:
 -napthyl butyrate esterase stain specific for monocytic lineage.
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To confirm these observations in other myeloid progenitors, we also
examined c-Maf-expressing clones established in HL-60 cells that
readily differentiate into monocytes when treated with TPA.18 c-Maf-overexpressing HL-60 cells also displayed
characteristics of differentiation along the monocytic lineage,
including ANB esterase positive cells with a decreased
nuclear:cytoplasmic ratio (Fig 3A). These
changes occurred more rapidly in the HL-60 cells than in the U937
lines: early morphologic changes and esterase activity were apparent
after 12 hours of zinc treatment (data not shown), which correlated
closely with c-Maf protein levels (Fig 3B). By 24 to 48 hours after
zinc induction, 70% to 90% of the cells exhibited morphology
characteristic of differentiated monocytic cells, comparable to that
seen on TPA treatment (89% to 95%). A smaller proportion of
multinucleated cells was apparent in c-Maf containing HL-60 as compared
with U937 cell cultures (4% v 15% containing 2 to 15 nuclei),
and this ratio increased slightly on longer zinc incubation (10% at 48 to 72 hours). ANB esterase staining of cytospin preparations indicated
that 45% to 50% of c-Maf-expressing cells displayed this
monocyte-specific enzyme activity, identical to ratios obtained in
parental cells or HL-60 c-Maf clone 6 cells induced with TPA ( 50%).
Similar morphologic and ANB data were obtained with HL-60 c-Maf clone 4, while identical treatment of vector containing control cells showed
no analogous changes (data not shown). Finally, the induction of c-Maf
in these clones altered their normal growth kinetics, as illustrated by
the reduced proliferative capacity of c-Maf containing clones, but not
vector control clones after exposure to zinc (Fig 3C).
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| Fig 3.
Analysis of cloned HL-60 cell lines conditionally
expressing c-Maf. (A) Lineage analysis of stable HL-60 lines expressing
c-Maf. Cell lines containing either vector control (CB6, top row) or
c-Maf (c-Maf, middle row) were untreated (none) or incubated with 150 µmol/L ZnSO4 (Zn+2), or parental HL-60
cells (bottom row) were untreated (none) or TPA treated (TPA). Cytospin
preparations were stained with Wright-Giemsa (left two panels) or
-napthyl butyrate esterase monocyte-specific stain (right two
panels).
(B) Conditional expression of c-Maf over time.
Individual HL-60 clonal cell lines containing either CB6 vector (HL-60
CB6 clone 3) or CB6-c-Maf-(HL-60 c-Maf clones 4 and 6) were induced
with ZnS04 for the indicated time periods and lysates were
probed for c-Maf expression with an antiserum recognizing the c-Maf
protein. (C) Induction of c-Maf alters normal growth kinetics. Viable
cell numbers were determined for vector control-containing (CB6) or
c-Maf-containing (c-Maf clones 4 and 6) stable cell lines by trypan
blue dye exclusion at the indicated intervals after zinc exposure.
Representative data from 2 individual experiments are presented.
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The induction of monocytic differentiation by c-Maf was further
confirmed in individual HL-60 clones cultured in zinc (protein levels
shown in Fig 1B) by flow cytometry and Northern blot analysis (Fig 4). Elevated cell-surface levels of
the myeloid maturation marker CD11b to levels equivalent to those seen
on TPA induction and the monocyte-restricted CD14 protein to levels
higher than TPA induced cells (Fig 4A), and the appearance of low, but
significant, levels of CSF-1 receptor mRNA (Fig 4B) confirms the
monocytic phenotype of the c-Maf-containing cells. Thus, the enforced
expression of c-Maf in multiple independent clones in two different
cell lines induces phenotypic changes consistent with monocytic
differentiation.
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| Fig 4.
c-Maf induces the expression of maturation markers in
HL-60 cell lines. (A) Fluorescence-activated cell sorting (FACS)
analysis of the expression of the CD14 and CD11b myeloid maturation
markers on untreated HL-60 cells, HL-60 cells treated with TPA for 24 hours, and 4 individual c-Maf expressing HL-60 clones treated with zinc
for 24 hours. Negative control antibody binding is indicated by dashed
lines. (B) Expression of c-Maf mRNA (top panel), CSF-1 receptor
(CSF-1R, center panel), or control G3PDH (lower panel), as determined
by Northern blot analysis of total RNA from uninduced (, lane 1) or
zinc-induced (+) control vector-containing (CB6, lane 2),
c-Maf-containing (lanes 1, 3, and 4) stable HL-60 clones; or
TPA-treated HL-60 parental cells (lane 5). A single blot was initially
probed with the c-Maf-specific cDNA probe, stripped, and reprobed with
CSF-1R and G3PDH; G3PDH served as a control for RNA loading and
integrity.
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IL-4 does not play a role in inducing monocytic differentiation.
What are the possible mechanisms of monocytic differentiation induced
by c-Maf? Relatively few hematopoietic target genes have been
identified that are directly regulated by c-Maf through its activity as
a DNA-binding transcription factor. Recent data have indicated that
c-Maf directly activates the expression of IL-4 in T-helper cells and
can induce the expression of the endogenous IL-4 gene in IL-4-negative
B-cell lines.5 In several models of experimental
inflammation, exogenous IL-4 can initiate the formation of
multinucleated giant cells (MGC) through macrophage fusion.19-23 MGC contain multiple nuclei within extensively
spread cytoplasm,24 similar to the phenotype of a
percentage of c-Maf-induced monocytes, raising the possibility that
overexpression of c-Maf in myeloid cell lines could also induce ectopic
IL-4 production. To address this issue, we performed RT-PCR analysis
(Fig 5A). Although c-Maf mRNA is highly
induced in our myeloid clones (lanes 8 and 9), its upregulation does
not initiate IL-4 mRNA synthesis (lanes 3 and 4). In addition, we added
neutralizing antibodies to IL-4 to cultures of zinc-induced c-Maf
clonal cell lines; these antibodies had no discernable effect on the
previously observed morphologic changes or ANB esterase positivity (Fig
5B). Similarly, addition of inhibitory concentrations of cyclosporin A
(a potent and specific inhibitor of the calcineurin-dependent
transcription factors required for IL-4 transcription25,26)
had no effect on the c-Maf-induced phenotypic changes (data not
shown). These results indicate that in our system, c-Maf induction does
not initiate endogenous IL-4 expression and, therefore, IL-4 does not
contribute to the observed monocytic differentiation.
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| Fig 5.
IL-4 expression is not induced in c-Maf-expressing cell
lines. (A) IL-4 mRNA is not induced on c-Maf protein expression. RT-PCR
analysis of cDNA templates from induced CB6 vector control (lanes 1, 3, 6, and 8) or c-Maf-containing clone 6 (c-Maf, lanes 2, 4, 7, and 9)
using primer pairs detecting -actin control (lanes 1 and 2), IL-4
(lanes 3 to 5), MafG (lanes 6 and 7), or c-Maf (lanes 8 and 9). M,
marker lanes; +, IL-4 positive control DNA template. (B) IL-4
neutralizing antibodies do not affect c-Maf-induced monocytic
differentiation. Neutralizing antibodies directed against IL-4 or
isotype-matched control antibodies were added to cultures of HL-60
c-Maf clone 6 cells at the time of zinc induction. Top row:
Wright-Giemsa stain; bottom row: monocyte-specific ANB esterase stain
of cytospin preparations.
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Inhibition of Myb activity is not sufficient to drive
differentiation.
During myeloid differentiation, c-Maf forms inhibitory complexes with
c-Myb, which function independently of Maf's binding to
DNA.9 Because the c-Maf protein controls gene expression through DNA-dependent, as well as DNA-independent, mechanisms, Maf-induced differentiation could be triggered by either or both of
these mechanisms.5-7,9,11,27 To confirm that c-Maf
induction altered Myb transactivation in our stable HL-60 clones, we
transiently transfected these cells with Myb-dependent CD13/APN
promoter-driven reporter constructs.9,10 c-Maf expression
inhibited luciferase activity of the reporter gene in these cells in a
dose-dependent fashion with 90% inhibition at the highest dose tested
(Fig 6). Therefore, Myb-dependent
transcription is functionally impaired in these cells and in the U937
clonal lines as well (data not shown).
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| Fig 6.
Myb-dependent transcription is functionally impaired in
clonal lines of HL-60 cells expressing c-Maf. HL-60 c-Maf clone 6 ( )
or CB6 vector control clone 3 ( ) were incubated at the indicated
ZnSO4 concentrations for 12 hours to induce c-Maf protein
expression, and then transiently transfected with 5 µg of the
411luc reporter construct containing sequences sufficient for
wild-type level, tissue-appropriate expression from the
CD13/APN myeloid promoter. Luciferase activities were assayed
at 6 hours and normalized for differences in transfection efficiency
with SEAP activity produced by the control -actin-SEAP plasmid.
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Myb mRNA and protein levels normally decrease during hematopoietic cell
differentiation.28-30 Downregulation is obligatory for
differentiation to proceed, as enforced expression of Myb blocks
differentiation of hematopoietic cell lines.31-34 Given the
functional relationship between Myb levels and hematopoietic cell
differentiation, it was possible that inhibition of Myb activity alone
was sufficient to produce the commitment and phenotypic changes
observed in the c-Maf-expressing cell lines. To test this possibility,
we established stable lines that conditionally express a dominant
interfering Myb containing the Drosophila Engrailed repressor
domain fused to the Myb DNA binding domain. Ectopic expression of this
chimeric protein has been shown to actively inhibit transcription from
multiple promoters containing Myb consensus sites.1,10,35
Its conditional expression in multiple stably transfected HL-60 clones
failed to induce the cell-surface markers (Fig 7) or cytosolic ANB esterase activity
alterations (data not shown) seen during parallel induction of
c-Maf-expressing clones over a range of time points. Therefore,
inhibition of Myb activity alone is not sufficient to initiate
c-Maf-like monocytic differentiation in these cells, suggesting that
c-Maf subserves other functions during monocytic commitment.
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| Fig 7.
MEnT does not induce the expression of monocyte specific
markers in HL-60 cell lines. Flow cytometric analysis of the expression
of the CD14 and CD11b maturation markers on untreated, zinc treated (18 hours), or TPA treated (18 hours) HL-60 MEnT-containing cells. Negative
control antibody binding is indicated by dashed lines. Data shown are a
representative tracing of clonal line, HL-60 MEnT clone 1 (Western blot
showing protein expression Fig 9A).
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Maf-induced differentiation terminates in apoptosis.
Active inhibition of Myb in avian myeloid and mammalian T cells results
in the downregulation of the antiapoptotic effector, Bcl-2, a direct
transcriptional target of Myb, followed by induction of
apoptosis.35,36 We observed that induction of either c-Maf or the dominant interfering Myb construct (MEnT) in HL-60 clonal lines
generates substantial cell death when compared with zinc-treated control cells (Fig 3C and data not shown). Flow cytometric analysis of
the DNA content of propidium iodide stained cells showed the appearance
of a significant percentage of c-Maf, and a higher ratio of positive
control MEnT expressing lines, present in a sub-G1 peak by
24 hours and as shown at 72 hours (Fig 8A),
indicative of apoptotic cell death. Untreated cells (Fig 8A) or vector
control cells treated with zinc (data not shown) did not result in
similar DNA content profiles. We confirmed that c-Maf containing cells were dying by apoptosis by TUNEL assay (Fig 8B) and DNA fragmentation analysis (Fig 8C). This latter assay also indicated that c-Maf-induced apoptosis is delayed relative to that induced by the positive control
MEnT fusion protein (24 to 48 hours v 6 hours), probably reflecting differences in the range of Myb targets inhibited by Maf
versus MEnT.
View larger version (31K):
[in this window]
[in a new window]
| Fig 8.
c-Maf-expressing cells die by apoptosis. (A) c-Maf- and
MEnT-containing cells show a sub-G1 peak indicative of
apoptotic cell death. Positive control MEnT-containing (MEnT, top two
panels) or c-Maf-containing (c-Maf, lower panels) that had been
untreated or treated with ZnSO4 for 72 hours, stained with
propidium iodide, and analyzed for DNA content by flow cytometry.
Representative data is presented. (B) c-Maf- and MEnT-expressing cells
show increased TUNEL staining. HL-60 clones overexpressing MEnT, CB6
vector, or c-Maf were treated with zinc for 48 hours (c-Maf, CB6) or 24 hours (MEnT), stained, and assessed by flow cytometry. Representative
data are presented. (C) c-Maf- and MEnT-containing cells show DNA
"laddering" characteristic of apoptosis. DNA was extracted from
negative control CB6 (lanes 1 and 2), c-Maf-containing (c-Maf, lanes 3 to 6), or positive control MEnT-containing cells (lanes 7 to 9) that
had been treated with ZnSO4 for the indicated time periods
and separated by agarose gel electrophoresis.
|
|
Because it was possible that apoptosis was triggered by Maf
downregulation of Bcl-2 through inhibition of Myb activity, we determined Bcl-2 protein levels in the induced cells by Western blot
analysis (Fig 9). As expected, conditional
expression of the MEnT Myb interfering protein in 2 HL-60 clonal cell
lines resulted in decreased Bcl-2 expression (Fig 9A). Induction of c-Maf in HL-60 cells also reduced Bcl-2 protein levels between 12 and
24 hours of induction (Fig 9B), directly corresponding with the
upregulation of Maf protein and the formation of inhibitory Myb:Maf
complexes (Fig 1C). By contrast, addition of zinc to vector containing
control cell lines showed no change in Bcl-2 levels (Fig 9B). Overall,
these data suggest that Maf inhibits Myb transregulatory activity,
which in turn, downregulates Bcl-2, resulting in apoptotic cell death.
View larger version (47K):
[in this window]
[in a new window]
| Fig 9.
c-Maf-expressing cells downregulate Bcl-2 protein
levels. (A) Cells expressing MEnT have decreased Bcl-2 protein levels.
Western blot analysis of MEnT and Bcl-2 protein expression in uninduced
or induced HL-60 clones (clones 1 and 2) containing the
ZnSO4 inducible MEnT Myb dominant-interfering construct.
(B) Bcl-2 protein levels decrease coordinately with c-Maf expression.
Western blot analysis of c-Maf, Bcl-2, and actin control protein levels
in HL-60 c-Maf-containing clone 6 incubated with ZnSO4 for
the indicated time periods. Vector control cells were treated
identically and assayed for Bcl-2 and actin protein levels (CB6
control).
|
|
| |
DISCUSSION |
Here we show that elevated, conditional expression of c-Maf in bipotent
myeloid cell lines is sufficient to promote differentiation to
monocytes. Elevation of c-Maf causes an increase in the levels of
Myb:Maf complexes, recapitulating the increase seen when these lines
are induced to monocytic differentiation. Enforced expression of the
c-Maf protein correlated precisely with the induction of morphological
changes, the appearance of cell-surface markers, and the induction of
cytosolic enzyme activities characteristic of monocytic
differentiation. Maf's induction of differentiation was independent of
the expression of IL-4, a direct transcriptional target of c-Maf, but
was associated with the inhibition of c-Myb activity, including the Myb
targets CD13/APN and Bcl-2. This inhibition of Bcl-2
correlated with the induction of apoptosis following the
differentiation of c-Maf-expressing cells and was consistent with
Bcl-2 downregulation seen during normal terminal myeloid differentiation.37-39 However, inhibition of c-Myb alone
does not appear to account for all of the effects of c-Maf, as
conditional expression of dominant interfering Myb proteins in HL-60
cells did not induce c-Maf-like differentiation. Hence, c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of selected c-Myb regulated targets coupled with the activation of as yet undefined differentiation-promoting genes.
The morphology of the c-Maf-induced cells differs to some extent from
that of cells resulting from standard effectors of monocytic or
granulocytic differentiation, precluding characterization based on
morphology alone. While the monocyte specific indicator, ANB esterase
activity, is readily detected in 50% of the cells in cultures induced
to express c-Maf, characterization of granulocytic differentiation in
the remaining cells is less clear. However, expression of the CAAT
enhancer binding protein (CEBP) transcription factor is rapidly downregulated to undetectable levels in our c-Maf
containing clones following induction of c-Maf
expression,40 a pattern compatible with that seen in mature
peripheral blood monocytes and bipotent cell lines induced toward
monocytic differentiation.41 By contrast, high levels of
CEBP expression are seen in mature neutrophils and bipotent cell
lines induced to differentiate toward granulocytes,41
suggesting that the effects of c-Maf are selective for promoting
monocytic differentiation of myeloid cells.
An additional striking morphological change observed on induction of
c-Maf expression is the appearance of large, multinucleated, intensely
ANB esterase positive cells. These cells strongly resemble MGC, which
are characterized by randomly arranged nuclei within extensively spread
cytoplasm.24 MGC are a primary histologic feature of
chronic inflammatory processes such as tubercular
granulomas19,23 and result from macrophage fusion rather
than endomitosis. MGC are formed through a 2-step process; progenitor
cells must first mature to macrophages before membrane fusion can
occur.20,21 Therefore, the presence of cells that resemble
MGC in c-Maf-induced clonal lines is consistent with our assertion
that c-Maf drives cells toward monocytic differentiation.
Surprisingly, although c-Maf can initiate IL-4 expression in IL-4
negative B cells,5 and IL-4 promotes MGC
formation,20-22 attempts to implicate IL-4 in the formation
of the large cells in our cultures have been unsuccessful. Endogenous
IL-4 mRNA was not upregulated in c-Maf expressing myeloid cells and
blocking of IL-4 activity with neutralizing antibodies had no effect on c-Maf-induced phenotypic alterations or differentiation (Fig 5). It is
thus possible that other effectors shown to promote MGC formation, such
as IL-13 or interferon- (IFN-),42 are responsible for
formation of MGC-like cells in our clones.
Downregulation of Myb expression is strictly required for the
progression of hematopoietic cell differentiation,31-34 but
the question of whether Myb plays a role in inducing cells to
differentiate is still unresolved. In our system, inhibition of Myb
alone by the MEnT construct does not induce monocytic differentiation. Although the arrest of Myb-driven transcription by the MEnT dominant interfering protein results in alterations in cell morphology, we see
no evidence of differentiation as determined by production of
alpha-napthyl-butyrate esterase or induction of monocytic cell-surface markers over a wide range of time points. By contrast, these indicators of monocytic differentiation are readily detectable in parallel cultures of c-Maf inducible lines. Therefore, our observations support
a hypothesis enjoining a broader role for c-Maf in inducing monocytic
differentiation, as inhibiting Myb activity alone does not appear to
recapitulate c-Maf-induced differentiation when assayed at early (6 to
18 hours) or later (72 hours) time points.40 Furthermore,
the fact that c-Maf-expressing cells do not immediately undergo
programmed cell death, even though Myb is inhibited, reinforces this
observation and may suggest that other antiapoptotic proteins either
continue to function or are activated by Maf expression, thereby
delaying apoptosis until after differentiation. Conversely, because the
MEnT construct used in this study inhibits transcription from any
promoter containing Myb binding sites and because Maf does not inhibit
all Myb-regulated genes,9 the MEnT encoded protein
undoubtedly inhibits a larger array of genes than does Myb:Maf alone.
It is possible that physiologic Myb downregulation does induce
differentiation, but the global repression of Myb transcription by MEnT
forces cells to undergo an accelerated apoptosis, bypassing or
surpassing the normal differentiation phase induced by the less abrupt
physiologic Myb downregulation. Cell lines expressing functionally
defined mutant Maf constructs will help to clarify this question.
In the hematopoietic system, terminal differentiation of
myeloid cells is tightly associated with programmed cell
death.37,38,43 Levels of Bcl-2 mRNA and its encoded protein
coordinately decrease during normal myelomonocytic maturation and
terminal monocytic differentiation of HL-60 cells,37-39
providing a mechanistic link between normal differentiation and
programmed cell death. The gene encoding the antiapoptotic
protein Bcl-2 is a direct transcriptional target of Myb, and inhibition
of Myb activity leads to apoptosis in hematopoietic
cells.35,36 In agreement, we observed apoptotic cell death
in c-Maf expressing HL-60 clones that paralleled c-Maf expression,
Myb:Maf inhibitory complex formation, cell differentiation, and a
decline in Bcl-2 protein levels. Whether this Bcl-2 decrease is the
result of Maf's inhibition of Myb transcriptional activity or is a
consequence of the physiologic Bcl-2 decline seen during myeloid cell
differentiation remains to be determined.
If Myb mRNA levels normally decline during development, would it be
necessary to invoke a second developmental strategy to inhibit
Myb-regulated genes? It is possible that to prepare cells for
differentiation, it is necessary to selectively refine Myb's positive
effects on a subset of responder genes before its physiologic downregulation. Myb:Maf complex levels do not strictly correlate with
Myb mRNA and protein levels in myeloid cell lines: despite low levels
of Myb:Maf in complex, U937 cells contain Myb mRNA levels equivalent to
those in HL-60 cells, where concentrations of Myb:Maf in complex with
DNA are significantly higher (Fig 1A, and Hegde et al9).
This suggests that Myb levels alone do not dictate Myb:Maf complex
formation, so that high Myb levels could transactivate some target
genes even as Myb:Maf complexes are blocking the expression of others.
Interestingly, in promoter assays using fibroblasts that do not express
c-Myb and thus lack Myb:Maf complexes, c-Myb positively autoregulates
its own transcription through binding to Myb consensus sites located in
its 5'-flanking region.44 However, Myb transcription
is negatively regulated through these same Myb sites when assays are
performed in Myb-expressing T cells.45 While we have not
studied Myb:Maf complex formation in T cells, it is intriguing to
speculate that the negative regulation observed in these cells results
from endogenous Myb:Maf inhibition of Myb transcription as an initial
step in the differentiation-linked decline of Myb mRNA. Studies to
address these questions during myeloid cell development are important
to our further understanding of Maf's role in myelopoiesis.
| |
ACKNOWLEDGMENT |
We thank Drs Rick Bram, David Shapiro, John Cleveland, and Paul Brindle
for helpful comments, Dr Kathy Weston for her generous gift of
plasmids, John Zacher for photomicrography, Liz Mann for technical
help, and John Gilbert for editorial assistance.
| |
FOOTNOTES |
Submitted January 13, 1999; accepted May 3, 1999.
Supported by Grant No. CA-70909 from the National Institutes of Health
(to L.H.S.), by Grant No. CA-21765 from the National Cancer Institute
Cancer Center Support (CORE), and by the American Lebanese Syrian
Associated Charities (ALSAC), St Jude Children's Research Hospital.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Linda H. Shapiro, PhD,
Department of Pathology and Laboratory Medicine, St Jude Children's
Research Hospital, Memphis, TN 38105; e-mail:
linda.shapiro{at}stjude.org.
| |
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O. Sordet, C. Rebe, S. Plenchette, Y. Zermati, O. Hermine, W. Vainchenker, C. Garrido, E. Solary, and L. Dubrez-Daloz
Specific involvement of caspases in the differentiation of monocytes into macrophages
Blood,
December 15, 2002;
100(13):
4446 - 4453.
[Abstract]
[Full Text]
[PDF]
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M. J. Loza and B. Perussia
Peripheral Immature CD2-/low T Cell Development from Type 2 to Type 1 Cytokine Production
J. Immunol.,
September 15, 2002;
169(6):
3061 - 3068.
[Abstract]
[Full Text]
[PDF]
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M. Sakai, M. S. Serria, H. Ikeda, K. Yoshida, J. Imaki, and S. Nishi
Regulation of c-maf gene expression by Pax6 in cultured cells
Nucleic Acids Res.,
March 1, 2001;
29(5):
1228 - 1237.
[Abstract]
[Full Text]
[PDF]
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K. Kataoka, K. Yoshitomo-Nakagawa, S. Shioda, and M. Nishizawa
A Set of Hox Proteins Interact with the Maf Oncoprotein to Inhibit Its DNA Binding, Transactivation, and Transforming Activities
J. Biol. Chem.,
January 5, 2001;
276(1):
819 - 826.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION