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Mediates Induction of the CD11c 
2 Integrin Gene Promoter1
Renal Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin family of
adhesion molecules that, together with CD18, forms a heterodimeric
receptor on the surface of myeloid, NK, dendritic, and certain
leukemic, lymphoma, and activated lymphoid cells. Monocytic
differentiation is associated with an induction of both CD11c and CD18
gene expression. The resulting CD11c/CD18 receptor mediates firm
adhesion to the vascular endothelium, transendothelial migration,
chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked
in vitro by treatment of the promonocytic cell line U937 with PMA.
Recently, we reported that in U937 cells, expression of the
CD11c gene is controlled by an unidentified
transcription factor that binds ssDNA. This finding suggested that DNA
secondary structure plays an important role in controlling the
CD11c gene and prompted us to search for additional
ssDNA-binding activities with which this gene interacts. In this study,
we report that in U937 cells, expression of the CD11c
gene is mediated by the ssDNA-binding protein Pur
. During
PMA-induced differentiation, the ability of Pur to activate the
CD11c promoter in U937 cells increases, as does that of
Sp1. Together, these increases in the functional activity of both
Pur and Sp1 combine to induce CD11c
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin
family of cell surface glycoproteins mediate a broad range of the
adhesive functions of lymphoid and myeloid cells. They are composed of
distinct subunits encoded by the CD11a,
CD11b, CD11c, and CD11d genes
noncovalently associated with a common subunit encoded by the
CD18 gene (1, 2). The CD11c/CD18 heterodimer is
expressed on NK, mature myeloid, and dendritic cells, as well as on
some cytotoxic T cell clones and some activated B and T
lymphocytes. In addition, CD11c/CD18 is expressed on the surface of
some neoplastic T cells and certain chronic B lymphocytic leukemias and
large cell lymphomas. In hairy cell leukemia, high level constitutive
expression of the CD11c/CD18 heterodimer represents a diagnostic marker
of the disease. Since the CD18 gene is active in all
leukocytes, it is the more selective expression of the CD11c
gene that dictates the specific cell types on which the CD11c/CD18
heterodimer is present.
Expression of the CD11c gene is regulated during cell
activation and differentiation, being induced during B and T cell
activation and the differentiation of progenitor myeloid cells into
monocytes and granulocytes (3, 4). The CD11c/CD18
heterodimer that results on the surface of differentiating
monocytic cells mediates their invasion of the vascular endothelium at
sites of inflammation by interaction with CD54 (ICAM-1). Such
differentiation can be mimicked in vitro by treatment of the
promonocytic cell line U937 with phorbol esters. Using this system, we
recently reported that expression of the CD11c gene is
controlled by the transcription factor PyRo1 (5). While
the identity of PyRo1 remains to be determined by molecular cloning, we
analyzed its DNA-binding characteristics and found that it interacts
preferentially with pyrimidine-rich DNA, which is single-stranded. This
finding suggested that the secondary structure of the CD11c
gene plays an important role in controlling its expression, and that it
might interact with additional ssDNA-binding activities. In this study,
we report the identity of one such activity as being Pur. Unlike
PyRo1, which interacts with pyrimidine-rich ssDNA, Pur interacts
with ssDNA, which is purine rich. However, Pur and PyRo1 are similar
in that both are induced during U937 differentiation. We found that
during this differentiation process, the ability of Pur to activate
the CD11c promoter increases. Furthermore, we found that
during U937 differentiation, the ability of Sp1 to activate the
CD11c promoter also increases. Together, Pur and Sp1
combine to induce CD11c expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The oligonucleotide AA3
(5'-TGCCACTCTTCCTGCAACGGCCCAGGAGCTCAGAGCTCCACATCTG-3'), designed
from the 5' end of a human CD11c cDNA clone
(6), was used to screen a human genomic library (no.
57765; American Type Culture Collection, Manassas, VA). One positively
hybridizing clone, 
CD11c, was isolated and a 16-kb NotI
fragment ligated into the NotI site of
pGEM11Zf+ (Promega, Madison, WI) to generate the
subclone pZ11c-16. A 6.7-kb SacI fragment of CD11c was
ligated into the SacI site of pGEM7Zf+
(Promega) to generate the subclone pZ11c-7.
DNA sequencing
dsDNA was sequenced by the dideoxynucleotide chain termination method (7) using Sequenase (United States Biochemical, Cleveland, OH). A series of specific oligonucleotide primers based on the CD11c cDNA sequence (6) and the CD11c gene sequence, as it became known, was used to sequence the 5' end of the CD11c gene contained in pZ11c-16 and pZ11c-7. This nucleotide sequence was determined on both DNA strands and has been deposited in the GenBank database under accession no. L19440.
Cell culture
The cell line U937 was obtained from the American Type Culture Collection and grown according to their specifications. PMA was obtained from Sigma-Aldrich (St. Louis, MO) and used at a concentration of 100 ng/ml, where indicated. U937 cells were treated with PMA for 12–24 h. These lengths of treatment are sufficient to induce monocytic differentiation as assessed by the production of CD11b mRNA (data not shown).
Plasmid construction
The activity of the CD11c promoter was assessed using
the expression vector pATLuc (8), which contains a
promoterless firefly luciferase reporter gene. Initially, the PCR was
used to generate one fragment of the CD11c gene representing
nt -128 to +36 relative to the major 5' transcription initiation site
(9). This fragment was then subcloned into the filled-in
HindIII site of pATLuc to generate p11Wt. The construct
p11
Pur again represented nt -128 to +36 cloned into the filled-in
HindIII site of pATLuc. However, this construct was produced
by oligonucleotide-directed mutagenesis (10) and contained
a substitution of the antisense sequence 5'-GGGGAAGGAAG-3' spanning nt
-30 to -40, with the mutant sequence 5'-TTTTAATTAAT-3'. This
substitution abolishes Pur binding (see Figs. 1
and 2). The correct
orientation and nucleotide sequence of all constructs was verified by
DNA sequencing. The Pur expression construct, pHAPur1, was kindly
provided by E. Johnson (Mount Sinai School of Medicine, New York, NY)
(11), and the empty vector equivalent, pHA, was produced
by religation following liberation of the Pur sequence by
RsrII and EcoRI digestion. The Sp1 expression
construct, pCMV-Sp, was produced from the parent plasmid p588
(12). This parent plasmid contains the Sp1 coding region
cloned downstream of the CMV promoter and upstream of a region encoding
the nuclease domain of the restriction endonuclease FokI. To
generate pCMV-Sp, the FokI coding region was removed from
p588 by digesting with the flanking restriction endonucleases
XhoI and HpaI, filling-in these sites, and then
recircularization. The construct, pCMV, is identical to pCMV-Sp, except
that it is empty of Sp1 coding sequences. This construct was produced
by digesting p588 with NotI and HpaI, which flank
the Sp1/FokI coding region, filling-in, and
recircularization. The plasmid p588 was kindly provided by J.
Chung (National Institutes of Health, Bethesda, MD).
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U937 cells were transfected by electroporation
(13, 14, 15) in nonsupplemented medium at 280 V and 960 µF
using a Gene Pulser system and 0.4 cm electrode gap Gene Pulser
cuvettes (Bio-Rad, Hercules, CA). Cells in the early log phase of
growth were harvested by centrifugation, washed once with medium, and
resuspended at room temperature to a concentration of 4 x
107 cells/0.4 ml. Aliquots of 0.4 ml cells were
transfected with 23 µg luciferase test plasmid together with 2 µg
of the plasmid pRSV- (Promega), which contains the lacZ
gene. Each transfection of p11Wt and p11Pur was performed in
parallel with a transfection of the promoterless luciferase plasmid
pATLuc. In those experiments in which monocytic differentiation was
induced, electroporated cells were treated with PMA at 100 ng/ml.
Electroporated cells were incubated at 37°C for 16 h, pelleted,
washed twice with PBS, and lysed in 200 µl reporter lysis buffer
(Promega). One hundred microliters of this cell lysate were used in the
assay of luciferase activity, and 50 µl were used in the assay of
-galactosidase activity. Both luciferase and -galactosidase
activities were assayed using reagents purchased from Promega.
Luciferase activity, assessed as light output, was measured using a
Moonlight 2010 luminometer, which integrated peak luminescence 10
s after injection of assay buffer. The levels of -galactosidase
activity resulting from different transfections were taken as
reflective of relative transfection efficiency and used to correct the
measurements of luciferase activity. Trans activation by
Pur was assessed by transfections in which 8 µg pATLuc, p11Wt, or
p11Pur were mixed with 1 µg pRSV- and 16 µg of either pHAPur1
or the equivalent empty vector pHA (see Fig. 2
). Trans
activation conferred by Pur combined with Sp1 was assessed
by transfections in which 8 µg p11Wt were mixed with 1 µg pRSV-
and: 1) 8 µg both pHAPur1 and pCMV-Sp; 2) 8 µg both pHAPur1 and
pCMV; 3) 8 µg both pHA and pCMV-Sp; or 4) 8 µg both pHA and pCMV
(see Fig. 5B).
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Approximately 250 million cells were collected by centrifugation, washed three times in ice-cold PBS, and resuspended in 4 ml ice-cold buffer 1 (10 mM NaCl, 0.4 M sucrose, 10 mM Tris-HCl (pH 7.8), 0.2 mM EDTA, 0.1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 50 mg/ml antipain, and 1 mg/ml aprotinin). The resuspended cells were then incubated on ice for 30 min and layered over 4 ml ice-cold buffer 2 (buffer 1 containing 1.5 M sucrose and 0.5 mM DTT, but no Nonidet P-40). Nuclei were collected by centrifugation, washed with 4 ml ice-cold buffer 3 (buffer 1 containing 0.5 mM DTT, but no Nonidet P-40), and resuspended to a concentration of 1 x 108 cell equivalents/300 µl in ice-cold buffer 4 (20 mM Tris-HCl (pH 7.8), 300 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 25% glycerol, and the protease inhibitor mixture listed above). Resuspended cells were then incubated at 4°C for 60 min and dialyzed overnight at 4°C against buffer 4. The dialyzed nuclear extract was clarified by centrifugation, frozen in liquid nitrogen, and stored at -80°C. The concentration of protein present in the nuclear extracts was determined using the Bio-Rad protein assay system (Bio-Rad).
EMSA
Oligonucleotides were radiolabeled at their 5' ends using T4
polynucleotide kinase and purified through G-25 Sephadex columns. To
generate dsDNA, equimolar amounts of complementary oligonucleotides
were combined. These oligonucleotides were then annealed by adding 5 M
NaCl to a final concentration of 100 mM, heating to 90°C, and slowly
cooling overnight to 4°C. DNA/protein-binding reactions were
conducted in a 20 µl volume. First, nuclear extracts were incubated
with or without a molar excess of unlabeled competitor probe at 4°C
for 15 min in 70 mM KCl, 5 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.5 mM
EDTA, 1 mM DTT, 10% glycerol, and 2.4 µg poly d(I:C)·poly d(I:C).
Radiolabeled probe was then added, and the incubation continued for 30
min. The DNA/protein complexes were resolved by electrophoresis through
7% native polyacrylamide gels and visualized by autoradiography.
Electrophoretic mobility supershift assay analyses were performed in
the same way as the standard EMSA analyses, except that before the
addition of DNA probes, nuclear extracts were preincubated for 15 min
at 4°C with either 1 µl polyclonal Ab, which specifically interacts
with Egr-1, or 1 µl mAb 9C12, which specifically interacts with
Pur (11). The Egr-1 Ab was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), and the Pur Ab was kindly provided
by E. Johnson (Mount Sinai School of Medicine). The oligonucleotides
used in the analyses were: CD11c PWt, 5'-AGGGGAAGGAAGA-3';
CD11c PWt-R, 5'-TCTTCCTTCCCCT-3'; CD11c
PMt, 5'-ATTTTAATTAATA-3'; CD11c P2,
5'-GTGTGGGAGGCCGAGC-3'; CD11c P3,
5'-GAGGGGGCGGGCAGAGT-3'; CD11c P4,
5'-AGAGAGGTGGCCAGGG-3'; GG-Rep,
5'-GGCTGGATGTGGTGGCTCAC-3'; and NS-1,
5'-TATTAATTAAAAT-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human CD11c gene has been cloned and its proximal
promoter region has been shown to direct a pattern of expression in
vitro that mimics that of the endogenous gene in vivo (4).
Recently, we determined that the activity of the CD11c
promoter is controlled in part by a cis-acting element
spanning nt -128 to -110, which interacts with the transcription
factor PyRo1 (5). PyRo1 binds preferentially to
pyrimidine-rich ssDNA, and its binding site within the CD11c
promoter contains repeat sequences capable of forming structures with
such conformations. In addition to the PyRo1 binding site, the region
spanning nt -44 to -29 also contains repeat sequences that might form
single-stranded structures (16). Since it was possible
that the -44/-29 region contains such structures, we tested whether
it could interact with ssDNA-binding proteins. Using single-stranded
oligonucleotides in EMSA analysis, we demonstrated that this was indeed
the case (Fig. 2). Specifically,
this analysis demonstrated that the purine-rich antisense strand of the
region spanning nt -41 to -29 interacts predominantly with a single
nuclear factor. When the antisense strand is annealed with its
complementary sequence to yield a double-stranded probe, no additional
binding activities appear. However, upon the production of dsDNA, the
intensity of the protein-DNA complex is significantly reduced. This
suggests that the nuclear factor detected, while capable of binding
dsDNA, preferentially binds DNA, which is single-stranded.
Identification of Pur as the factor that binds the -41/-29
region of the CD11c gene
The -41/-29 CD11c gene sequence that interacts with
the ssDNA-binding protein detected by EMSA contains two copies of the
sequence GGN, in which N is not G. Repeats of this sequence represent
the recognition element of the transcription factor Pur (17, 18). Pur, like the factor we have identified that interacts
with the -41/-29 region, can bind dsDNA, but exhibits a distinct
preference for DNA that is single-stranded. To determine whether the
factor binding the CD11c gene was Pur, we first attempted
to compete such binding with a single-stranded oligonucleotide
containing GGN repeats. EMSA analysis demonstrated that this
competition was entirely effective (Fig. 2, lane 6).
Therefore, the factor binding the CD11c gene exhibits
DNA-binding characteristics consistent with those of Pur. Next, we
determined whether the factor interacts with the mAb 9C12 that
specifically recognizes Pur (11). This analysis proved
positive, demonstrating that the factor that binds the -41/-29 region
of the CD11c gene is immunologically indistinguishable from
Pur (Fig. 1, lane 7).
Interaction with Pur is critical to CD11c promoter
activity
Repeats of the sequence GGN are found in all Pur binding sites.
This is also the case for the Pur binding site within the
CD11c gene, which contains the sequence GGGGAAGGAAG. When
this sequence was mutated to TTTTAATTAAT, Pur-binding activity was
lost (Fig. 2, lane 5). These same mutations were introduced
into the -128/+36 promoter to assess the importance of Pur binding
to its activity. Transfection of this mutant promoter into U937 cells
subsequently treated with PMA demonstrated that it directs expression
levels that are over 80% lower than those directed by the wild-type
sequence (Fig. 3A). Therefore,
Pur binding to the CD11c promoter appears to be of
critical importance.
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is capable of activating the CD11c promoter
Mutation analysis suggested that Pur binding is necessary for
the activity of the CD11c gene promoter. However, it
remained possible that the same mutations that disrupt Pur binding
also disrupt promoter topology. In this situation, the effects of the
mutations on promoter function might not truly reflect the importance
of Pur. Therefore, next we sought to determine directly whether
expression of Pur was sufficient to activate the CD11c
promoter. This was achieved by cotransfecting the CD11c
reporter construct p11Wt with the construct pHAPur1, which
constitutively produces Pur driven by the CMV promoter
(11). Under these conditions, in three independent
experiments, p11Wt exhibits a mean expression level in PMA-treated U937
cells that is 3.5 times higher compared with when it is transfected
with the CMV plasmid empty of Pur sequences (Fig. 3B).
Therefore, Pur is capable of activating the CD11c
promoter. Previous studies have demonstrated that in the myeloid cell
line HL-60, the CD11c promoter can be activated by Sp1, Sp3,
c-Jun, c-Fos, and C/enhancer-binding protein
(19, 20, 21). With the exception of Sp3, the degree of
activation conferred by these factors is lower than that conferred by
Pur in U937 cells.
Pur has the ability to interact with the CD11c
promoter at multiple sites
The capability of Pur to trans-activate the
CD11c promoter is significantly reduced by mutations that
prevent Pur binding to the -41/-29 region. Consequently, Pur
influences CD11c promoter activity through this region.
However, mutations within the -41/-29 region fail to completely
abolish Pur trans activation, suggesting Pur can
interact with the CD11c promoter at additional sites. We
analyzed the CD11c promoter for such additional sites and
found that as well as the -41/-29 region, three other regions contain
closely spaced repeats that conform to the consensus sequence
recognized by Pur. These repeats are GGAGGC located between nt -109
and -104, GGCGGGC located between -66 and -72, and GGTGGC present
between nt -22 and -27. The ability of these repeats to compete with
the -41/-29 region for Pur binding was tested in EMSA analysis
(Fig. 4). This analysis demonstrated that
all three repeats effectively competed for Pur binding.
Consequently, Pur has the capacity to interact with the
CD11c promoter at a total of four sites, one located on the
sense strand and three located on the antisense strand.
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increases during U937
differentiation
Previous studies have shown Pur to be subject to
developmental regulation in the mouse brain (22). In this
study, induction of its expression mediates activation of the
myelin basic protein gene promoter at the end of the first
postnatal week. Our trans activation studies presented in
Fig. 3B clearly demonstrate that, in addition to activating
gene expression during brain development, Pur also has the capacity
to activate expression during monocytic differentiation of U937 cells.
Therefore, we were interested in determining whether, as in brain
development, Pur is subject to regulation during U937
differentiation. First, we compared Pur-binding activity in nuclear
extracts prepared from untreated U937 cells with that present in
extracts prepared from U937 cells induced to differentiate along the
monocytic pathway for 24 h with PMA. This comparison demonstrated
that Pur binding to the oligonucleotide CD11c PWt is
induced during U937 differentiation (Fig. 5A). Next, we sought to
determine whether the capacity of Pur to effect trans
activation of the CD11c promoter is also modulated
during differentiation. In transfection studies thatemployed
equivalent amounts of the Pur expression constructpHAPur1, the
transcriptional activity of the CD11c promoter was induced
to a significantly higher degree in PMA-treated U937 cells than in
cells that were left untreated (Fig. 5B). Consequently, our
data indicate that both the expression of Pur, as measured by its
DNA-binding activity, and the ability of Pur to effect
trans activation are induced during U937 monocytic
differentiation.
During U937 differentiation, Pur combines with an
increase in the trans activation capacity of Sp1 to
effect induction of the CD11c promoter
The functional activity of Pur has been shown to be influenced
either in a positive or negative manner by its interaction with a
number of cellular proteins, including calmodulin, Sp1, YB-1, Pur,
and the retinoblastoma protein Rb (11, 22, 23, 24, 25, 26). Of these
proteins, only Sp1 has been reported to influence CD11c gene
expression (4, 19, 20, 21, 27). Therefore, we sought to
determine whether in the context of the CD11c promoter, Sp1
and Pur combine or compete to induce transcription. Trans
activation experiments performed in U937 cells either untreated or
treated with PMA demonstrated that recombinant Pur and Sp1 combine
to induce CD11c promoter activity (Fig. 5B). In
addition, we found that, as with Pur, expression of Sp1 was more
effective in activating the CD11c promoter in PMA-treated
U937 cells than in cells that were left untreated. These increases in
trans activation capacity resulted in the combination of Sp1
and Pur being over 2.5-fold more effective in inducing the
CD11c promoter in PMA-treated U937 cells than in untreated
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
Pur binds to repeats of the sequence GGN. Therefore, these
were mutated within the -41/-29 region to determine the functional
significance of Pur interaction with the CD11c promoter.
This analysis demonstrated that when mutations are introduced into the
-41/-29 region, promoter activity in PMA-treated U937 cells is
reduced by over 80%. Consequently, the binding of Pur appears
critical to CD11c promoter activity. The binding site for
Pur overlaps sequences that in HL-60 and HeLa cells have been shown
to interact with the Ets family of transcription factors
(28). This family recognizes DNA in its double-stranded
conformation. However, when the single-stranded Pur site is made
double-stranded, Pur binding is inhibited, but no additional
complexes are observed in PMA-treated U937 cells. Therefore, in
contrast to HL-60 and HeLa cells, in PMA-treated U937 cells, Ets
factors appear not to bind the -41/-29 region. As a result, the
mutations within the -41/-29 region that we have tested in
PMA-treated U937 cells appear to reflect only the contribution of
Pur to CD11c promoter activity.
Mutation of the -41/-29 region indicates that Pur binding is
necessary for the functional activity of the CD11c promoter.
Next, we sought to determine whether Pur binding is sufficient to
induce such activity. This proved to be the case with Pur
expression, causing a 3.5-fold induction of promoter activity in
PMA-treated U937 cells. This degree of trans activation
compares favorably with that conferred by the four other proteins that
individually have been shown to be able to trans-activate
the CD11c promoter in myeloid cells. Within the
myelomonocytic cell line HL-60 Sp3 causes a 4.3-fold induction, while
Sp1 induces 3.4-fold, c-Fos 2.5-fold, and c-Jun 1.8-fold (19, 21). In addition, in HeLa epithelial cells, C/enhancer-binding
protein can induce the CD11c promoter by a factor of two
(20). Whether Pur contributes to the expression of the
CD11c gene in cell types such as HeLa that are not monocytic
in origin remains to be determined. The ability of Pur to activate
the CD11c promoter in U937 cells was significantly reduced
by point mutations that abolish its binding to the -41/-29 region.
Therefore, it appears that this region participates in the induction of
CD11c expression by Pur. However, since disruption of the
-41/-29 region failed to completely abolish Pur trans
activation, other regions most likely also mediate its function.
Indeed, in support of this hypothesis, we have identified three other
regions within the CD11c promoter with the capacity to bind
Pur. While it remains to be determined whether these sites function
independently or in concert, it is of interest that Pur has the
capacity to form homodimers. Consequently, it seems plausible that a
Pur protein bound at one site within the CD11c promoter
may interact with another bound at a different site. Such interaction
could impart a particular architecture to the CD11c promoter
facilitating its activation.
In addition to homotypic interaction, Pur has been shown to
interact with a range of different cellular proteins (11, 22, 23, 24, 25, 26). Such interactions may account for the complex nature of
the supershift obtained upon analysis of the Pur complex that
interacts with the -41/-29 region (Fig. 2, lane 7).
Heterotypic interactions can either promote or inhibit Pur
functional activity. Of the proteins known to interact with Pur, the
only one reported to control CD11c expression is Sp1. This
factor binds the CD11c promoter at three sites (4, 19, 20, 21, 27). Interestingly, each of these three Sp1 binding
sites overlaps or is adjacent to one of the four Pur binding sites
that we have identified (Fig. 1). Such
close proximity of all the identified Pur and Sp1 binding sites
suggested to us that these factors could either combine or compete to
activate the CD11c promoter. Our trans activation
experiments demonstrated that Sp1 and Pur in fact act together to
induce CD11c expression. However, this finding raised a
potential paradox since Sp1 binds dsDNA, while Pur exhibits a marked
preference for binding ssDNA. Consequently, Pur and Sp1 binding to
the same DNA sequence would appear to be mutually exclusive events that
compete, not combine, to induce the CD11c promoter. Two
answers to this paradox appear possible. The first is that at any given
time, Pur and Sp1 bind the CD11c promoter at different
sites. The second answer stems from the observation that in the mouse
brain, the interaction of Pur and Sp1 has only been observed in the
absence of DNA. Such interaction was not seen on a DNA target
containing overlapping Sp1 and Pur binding sites (22).
Rather, under these circumstances, the interaction of Sp1 with Pur
enhanced the ability of Pur to interact with this target. If this
situation occurs in the context of U937 cells, then it suggests a
mechanism by which Sp1 and Pur cooperate in driving CD11c
expression. In this mechanism, Sp1 and Pur would interact away from
their overlapping target sequences, causing an increase in the ability
of Pur to interact with single-stranded structures within the
CD11c promoter. If such a mechanism exists, it must be
subject to induction during monocytic differentiation. This must be the
case, since we have found that, in combination, Sp1 and Pur are much
more effective in activating the CD11c promoter in
differentiating U937 cells than in nondifferentiating cells. Two
observations indicate how such regulation during U937 differentiation
might occur. First, we have demonstrated that the expression of Pur,
as assessed by its ability to bind DNA, increases during U937 monocytic
differentiation. Second, it has been reported that Pur binds a
phosphorylated form of Sp1, not Sp1 that is unphosphorylated.
Expression of this phosphorylated form is induced upon both
megakaryocytic differentiation of K562 cells and monocytic
differentiation of HL-60 cells (29, 30). Consequently, a
mechanism can be envisaged during U937 differentiation in which Sp1
becomes phosphorylated, and this phosphorylated form of Sp1 then
interacts with induced levels of Pur, causing an increase in the
ability of this factor to bind and activate the CD11c
promoter.
The expression of a number of mammalian genes is known to be controlled
by Pur. This list is composed of the genes encoding myelin basic
protein, TGF-1, smooth muscle actin, c-Myc, neuropeptide Y, the
4 subunit of the nicotinic Ach receptor, the
neuron-specific protein FE65, and CD43 (18, 25, 31, 32, 33, 34, 35, 36, 37, 38).
The CD11c and CD43 genes are the first described
to be controlled by Pur which are expressed specifically in
leukocytes and which produce molecules directly responsible for
leukocyte adhesion during inflammation. CD43 is a large, highly charged
mucin-like transmembrane molecule that maintains resting leukocytes in
the circulation by preventing intercellular interaction
(39, 40, 41, 42, 43, 44, 45). During inflammation, there is a dramatic
down-regulation of CD43 expression mediated by proteolytic cleavage
events at the cell surface and repression of transcription directed by
the CD43 gene promoter (38, 46, 47, 48, 49, 50). This
down-regulation of the antiadhesion molecule CD43 coupled to
up-regulation of proadhesive molecules such as CD11c is critical to the
inflammatory process, since these changes help leukocytes acquire the
adhesive phenotype capable of extravasation. Recently, we have shown
that Pur represses expression of the CD43 promoter during
U937 differentiation (38). Consequently, Pur is capable
of both inducing expression of the proadhesion molecule CD11c and
repressing the expression of the antiadhesion molecule
CD43. Therefore, it appears that Purrepresents a means by
which leukocytes coordinately regulate the expression of proadhesive
and antiadhesive forces. A role for Pur in inflammation has
previously been implied by its control of TGF-1
expression and its increase in infiltrating eosinophils and activated
endothelium during allergic reactions in the rat lung
(51). Calcium mobilization is characteristic of the
cellular activation that occurs during inflammation. In this regard, it
is worth noting that the DNA-binding activity of Pur is increased by
its interaction with the calcium-binding protein calmodulin
(25).
In conclusion, we have demonstrated that during monocytic
differentiation of U937 cells induction of the CD11c
promoter is mediated by the combined action of Pur and Sp1. Since
Pur binds preferentially to ssDNA, our data suggest that the
secondary structure of the CD11c promoter plays a critical
role in its regulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Simon Shelley, Renal Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail address: shelley{at}receptor.mgh.harvard.edu
3 Current address: Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02115.
4 Current address: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Received for publication October 30, 2001. Accepted for publication February 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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