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-Stimulated Monocyte Chemoattractant Protein-1 Gene Transcription1
*
Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78284;
Division of Oral Biology, Boston University School of Dental Medicine, Boston, MA 02118; and
Department of Medicine, University of Hamburg, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, a potent
mediator of the immune inflammatory response. Nuclear run-on and
stability studies demonstrated that IFN- stimulated MCP-1
transcription and did not enhance mRNA stabilization. Using MCP-1
promoter/reporter gene constructs, we determined that IFN--enhanced
MCP-1 transcription is regulated by a 29-bp element located at -227
relative to the ATG start codon. This element contains a 13-bp CT-rich
sequence (GCTTCCCTTTCCT) adjacent to a IFN- activation site (GAS).
Since deletion of the CT sequence enhanced both the magnitude and
duration of IFN--stimulated, GAS-mediated transcription, we have
termed it the IFN response-inhibitory sequence (IRIS). The combined
IRIS/GAS sequence is highly conserved in mouse, rat, and bovine MCP-1
genes. In gel-shift assays, nuclear extracts from IFN--stimulated
osteoblastic cells formed two specific inducible bands with labeled
IRIS/GAS DNA. Both bands were supershifted by anti-STAT1 Abs, but
not by Abs to STAT2, p48(ISGF-3), IFN-regulatory factor-1, or
IFN-regulatory factor-2. Formation of one of the bands required the
presence of the IRIS moiety. IRIS/GAS DNA also formed a number of
specific complexes with constitutively expressed factors, none of which
were affected by the above Abs. These studies establish a mechanism for
IFN--stimulated MCP-1 expression and identify a complex element that
regulates MCP-1 gene transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, or bone formation through the production of growth
factors (4, 5). Recent evidence suggests a potential role for the CC
chemokine monocyte chemoattractant protein-1
(MCP-1)3 in the recruitment
of monocytes to bone: 1) MCP-1 is not expressed in normal bone, but is
induced during osseous inflammation (2). 2) The induction of MCP-1 in
inflamed bone is temporally and spatially correlated with the
recruitment of monocytes (2). 3) MCP-1 production is associated with
the recruitment of monocytes to areas of both bone formation and
resorption during developmentally regulated bone remodeling (6). 4)
MCP-1 is the principal monocyte chemoattractant produced by
osteoblastic cells in vitro (3). Thus, MCP-1, through the recruitment
of monocytes, is a likely mediator of bone remodeling.
MCP-1 and other chemokines are typically expressed in tissue
during inflammation and are induced in a variety of cell types in vitro
by the proinflammatory mediators TNF-, IL-1, and endotoxins (7, 8).
Molecular studies from a number of laboratories have identified some of
the cis-regulatory elements and trans-acting
factors involved in this response. Two nuclear factor-
B binding
sites located approximately 2.6 kb from the transcription initiation
site appear to function as the critical elements in MCP-1 induction in
response to IL-1ß and TNF- (9, 10, 11). Binding to these sites by
cytokine-activated B-binding complexes p65/p65 and p65/c-Rel results
in an enhancement of MCP-1 gene transcription. Additional elements
close to these B sites (12, 13) and a 7-bp response element in the
3' UTR of MCP-1 have also been shown to regulate the MCP-1 gene
transcription in 3T3 cells in response to platelet-derived growth
factor (12, 13, 14). The principal trans-acting factors
that mediate this response are reported to be a constitutively
expressed 90-kDa serine/threonine phosphoprotein and a Rel A homodimer
that demonstrate overlapping binding activities to one of the B
sites (13). Thus, this distal group of regulatory elements plays a
significant role in MCP-1 gene transcription in response to both
proinflammatory cytokines and growth factors. A number of functional
cis-regulatory elements has also been identified in the
proximal promoter region. Within a 150-bp segment located 5' to the
translation ATG start codon, two consensus elements for the AP-1
trans-acting factors have been identified in the human MCP-1
promoter, the more distal one of which appears to mediate a response to
the phorbol ester 12-O-tetradecanoylphorbol 13-acetate
(TPA) (15, 16). The more proximal of these consensus AP-1
elements overlaps a consensus site for the Sp1 transcription factor
that appears to be important for the basal transcription activity of
the gene (9, 10, 16, 17). The proximal AP-1-like binding element has
been reported to mediate fluid shear stress-induced regulation of MCP-1
expression in vascular endothelial cells (18, 19).
IFN-, a T and NK cell-derived cytokine, also appears to be important
in bone metabolism. Although IFN- is thought to inhibit osteoclast
formation (20), treatment of osteopetrotic patients with IFN- has
been shown to be efficacious in treatment of the disease (21). IFN-
also has the capacity to affect the tissue recruitment of mononuclear
cells through the induction of chemokine genes. The induction of the
C-C chemokine MCP-1 in response to IFN- stimulation has been
described in a number of cell types, and typically IFN- rapidly and
potently induces MCP-1 mRNA accumulation and MCP-1 protein production
(22, 23, 24, 25).
The secretion of IFN- and other lymphokines from activated T
lymphocytes accumulating at sites of chronic osseous inflammation and
in bone tumors may have a significant effect on the pathogenesis of
these diseases. IFN- may act directly on local tissue to stimulate
the production of MCP-1 and other proinflammatory mediators, and
activate the monocyte-derived macrophages that are subsequently
recruited. To investigate the mechanisms responsible for
IFN--regulated MCP-1 gene expression in bone cells, experiments were
conducted in a human osteoblastic cell line. Our results indicate that
IFN- rapidly increases MCP-1 mRNA and protein expression in these
cells through a transcriptional mechanism, with little or no effect on
MCP-1 mRNA stability. Furthermore, our functional studies suggest that
transcription is regulated by a 29-bp complex element in the MCP-1
promoter located 227 bp from the ATG initiation codon, and does not
require the response elements previously identified in the distal
promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DMEM and newborn bovine serum (NBS) were obtained from Life
Technologies (Gaithersburg, MD). Abs to STAT1/ß, STAT2,
p48(ISGF-3), IRF-1, and IRF-2 were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Human rIFN- was purchased from R&D
Systems (Minneapolis, MN). Oligonucleotides were synthesized by Center
for Advanced DNA Technologies, University of Texas Health Science
Center at San Antonio.
RNA isolation and Northern blot analysis of MG-63 osteoblastic cells
MG-63 human osteoblastic cells were purchased from American Type
Culture Collection (Manassas, VA) (CRL 1427) and grown in DMEM
supplemented with 10% NBS. To determine the effect of IFN- on MCP-1
mRNA stability, cells were incubated with the transcriptional inhibitor
5,6-dichloro-1ß-D-ribofuranosylbenzimidazole (DRB) (20
µg/ml; Sigma, St. Louis, MO), IFN- (100 U/ml), or IFN- (100
U/ml) plus DRB (20 µg/ml) for 0, 0.5, 1, 2, 4, and 6 h. MCP-1
mRNA levels were examined by Northern blot analysis following a
modification of a previously described protocol (26). A full-length
human MCP-1 cDNA probe was generously provided by E. Appella
(Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD).
Autoradiographs were examined by laser-scanning densitometry, and each
band was normalized to the same blots hybridized with a cDNA probe for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was generously
provided by Dr. Judith A. Foster, Boston University (Boston, MA). The
resulting data were expressed as the OD multiplied by the distance
(OD x mm).
Isolation of nuclei and transcriptional analysis
The transcription rate of MCP-1 was determined by nuclear run-on
analysis. Nuclei were isolated from the MG-63 human osteoblastic cell
line incubated with IFN- (100 U/ml) for 6 h. Cells were washed
twice with ice-cold Puck’s saline, lysed by incubation with cold lysis
buffer (10 mM Tris-HCl, pH 7.6, 10 mM NaCl, 3 mM MgCl2, and
0.5% Nonidet P-40), and centrifuged at 500 x g for 10
min at 4°C. Isolated nuclei were stored at -80°C until used. The
transcription assay was performed essentially as reported (27).
pBluescript (Promega, Madison, WI), the vector for MCP-1 cDNA, was used
as a negative control, and a plasmid containing GAPDH served as the
positive control. Equal amounts of radiolabeled transcripts (2–4
x 106 cpm/ml) were hybridized to nylon membranes
containing 10 µg of denatured plasmid DNA per slot. Hybridization was
conducted at 65°C for 72 h. The membranes were exposed to x-ray
film at -80°C for 3 to 7 days. Each experiment was performed at
least twice with similar results.
Sandwich ELISA for MCP-1
mAb hybridoma (E11) was donated by Dr. Edward Leonard (National
Institutes of Health). Polyclonal rabbit anti-MCP-1 has been
described previously (28). ELISAs were performed exactly as described
(29) using a horseradish peroxidase color development kit (Kirkegaard &
Perry Laboratories, Gaithersburg, MD). The MCP-1 ELISAs were sensitive
to 
30 pg/ml.
MCP-1/luciferase plasmids
Heterologous promoter/luciferase constructs were prepared in the pGL2-Basic luciferase vector (Promega). To facilitate subcloning, the XhoI/HindIII fragment from the multiple cloning site of an insert-negative (blue) colony of the T-cloning vector pCRII (Invitrogen, Carlsbad, CA) was inserted into the XhoI/HindIII site of pGL2. This transferred to pGL2 the pair of nonpalindromic BstXI sites that flank the cloning site of pCRII. To prepare the -2910/+21, -248/+21, -227/+21, -214/+21, and -198/+21 MCP-1/pGL plasmids (all numbers refer to nucleotide positions relative to the ATG translation initiation codon and are based on the nucleotide sequence for the human MCP-1 gene, GenBank Accession D26087), the MCP-1 DNA was first synthesized by the PCR. Forward oligonucleotide primers used were 5'-GAG CAT TGA CCC TCA TTT TCC-3' for -2910/+21; 5'-AGT GTC TCG TCC TGA CC-3' for -248/+21; 5'-GCT TCC CTT TCC TAC TTC CTG G-3' for -227/+21; 5'-ACT TCC TGG AAA TCC A-3' for -214/+21; and 5'-CAG GAT GCT GCA TTT G-3' for -198/+21. The oligonucleotide 5'-AAG GGC GGC AGA GAC TTT CAT-3', which corresponds to the reverse and complemented first 21 nucleotides of the MCP-1 coding sequence, was used as the common reverse primer in all reactions. Bacteriophage DNA from a clone containing the human MCP-1 gene promoter region was used as a template. Products from PCR were cloned directly into pCRII (Invitrogen). Plasmid DNA from positive clones was digested with BstXI, and the inserts were gel purified and cloned into the corresponding nonpalindromic BstXI sites of the modified pGL2 vector. Correct nucleotide sequence and orientation with respect to the luciferase gene were confirmed by DNA sequencing using Sequenase (United States Biochemical, Cleveland, OH). Since the -2910/+21 MCP-1 DNA contains an internal BstXI site, the insert was excised from pCRII with BamHI and NotI, blunt ended with DNA polymerase I, ligated with BstXI hemiphosphorylated linkers (Invitrogen), and cloned into the modified pGL2 vector, as above. Orientation and identification were confirmed by partial DNA sequencing. For transfection studies, plasmid DNA was purified using a commercial anion-exchange protocol (Qiagen, Chatsworth, CA).
Cell transfection and luciferase assay
MG-63 human osteoblastic cells (6 x 104
cells/well) were plated in 12-well plates and incubated overnight. The
medium was replaced with 1.5 ml of fresh complete medium, and
transfection was conducted 4 to 6 h later. For transfection, 5
µg of MCP-1/luciferase plasmids was mixed with 0.5 µg of pCMV-RL,
an internal control renilla luciferase expression vector (Promega), and
25 µg of pUC18 as carrier DNA, in a final volume of 0.5 ml of 0.25 M
CaCl2. This was added dropwise to 500 µl of 2x HeBS
(0.275 M NaCl, 8.5 mM KCl, 1.5 mM Na2HPO4, 400
mM glucose, and 0.166 M HEPES, pH 6.9) with constant stirring, and the
mixture was incubated at room temperature for 30 min. A total of 150
microliters was then added dropwise to the cultures and incubated
overnight at 37°C. The medium was removed, and 2 ml of fresh medium
was added, and incubation continued for 48 h. To determine the
effect of IFN- stimulation, the cells were rinsed once with DMEM and
duplicate wells were incubated with human rIFN- (1000 U/ml) in
either 1 ml of DMEM/1% NBS or 1 ml of the medium without IFN-
(unstimulated control). Following incubation for the indicated times,
the cells were rinsed once in PBS, then scraped into 150 µl of 1x
passive lysis buffer supplied with the Dual-Luciferase Reporter Assay
System (Promega). The extracts were vortexed for 10 s, incubated
at room temperature for 10 min, then centrifuged at 12,000 x
g for 5 min. Twenty microliters of the supernatant were
assayed for both firefly and renilla luciferase activity using the
dual-reporter assay system indicated above and a TD-20/20 luminometer
(Turner Designs, Sunnyvale, CA). Firefly luciferase activity was
normalized to the renilla luciferase activity, and the fold stimulation
was calculated as the ratio of the IFN--stimulated to unstimulated
values obtained at each time point.
Nuclear extracts and electophoretic mobility shift assay (EMSA)
MG-63 cells were plated in 100-mm dishes and grown in complete
medium until just confluent. The cells were rinsed once with DMEM and
then incubated with 6 ml DMEM/1% NBS, with or without 1000 U/ml
IFN-. At the indicated times, the cells were rinsed twice with
ice-cold PBS, and nuclear extracts were prepared as described by
Schreiber et al. (30), except that in addition to 1 mM PMSF and 1 mM
DTT, all cold extraction buffers contained 1 mM sodium orthovanadate to
inhibit endogenous tyrosine phosphatase activity. Protein
concentrations were determined by the Bradford technique, and the
extracts were stored at -70°C. Complementary oligonucleotides
corresponding to the MCP-1 IRIS/GAS (5'-GCT TCC CTT TCC TAC TTC CTG GAA
ATC CA-3'), truncated IRIS/GAS sequence IRIS/
GAS (5'-GCT TCC CTT TCC
TAC TTC C-3'), the MCP-1 IRIS (5'-GCT TCC CTT TCC TAC-3'), and the
MCP-1 GAS core sequence (5'-ACT TCC TGG AAA T-3') were annealed and end
labeled with [-32P]ATP and T4 polynucleotide kinase.
For the assays, 4 µg of the nuclear extract was incubated for 20 min
at ambient temperature with 7.5 to 10 x 104 cpm of
the labeled DNA probe in 20 µl of binding buffer containing 10 mM
Tris-Cl, pH 7.6, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, 1
µg/µl BSA, and 2 µg poly(dI-dC) (Pharmacia Biotech, Piscataway,
NJ). Samples were separated on 10% polyacrylamide gels at 200 V in 1x
buffer (25 mM Tris, 190 mM glycine, and 1 mM EDTA, pH 8.3). Competition
assays were conducted in the same manner, except the above reaction
mixture was preincubated with competitor DNA for 15 min at 0°C before
addition of the labeled probe. For supershift assays, the binding
reaction was conducted as above, except that up to 2 µl Ab was added
to the reaction mixture following the 20-min incubation, and the
incubation continued for an additional 45 min at room temperature
before separation on PAGE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated osteoblastic cells is
regulated at the transcriptional level
Our initial studies indicated that treatment of normal and MG-63
human osteoblastic cells with IFN- resulted in a rapid increase in
MCP-1 mRNA levels. Since IFN--induced stimulation of chemokine mRNA
levels has been shown in some cell types to be regulated at the
posttranscriptional level (31), mRNA stability studies were conducted
in MG-63 cells. Cells were incubated with either the transcriptional
inhibitor DRB, IFN-, or IFN- plus DRB. Total cell RNA was
isolated, and Northern blot analysis was performed. As shown in Figure 1
A, although the basal levels
of MCP-1 mRNA are high in MG-63 cells, IFN- stimulation resulted in
a marked increase in DRB-untreated cells compared with the level
observed at the zero time point. The increase was observed within
1 h and increased further with longer times of incubation. In
DRB-treated cells, on the other hand, IFN- stimulation did not
increase MCP-1 mRNA levels. Comparison of mRNA levels in DRB-treated
cells with or without IFN- showed that IFN- did not enhance MCP-1
mRNA stability. Under both conditions, the MCP-1 mRNA
t1/2 was approximately 3 to 4 h (Fig. 1B).
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on MCP-1 transcription. Nuclei were isolated from MG-63 cells that had
been incubated with or without IFN- for 6 h. The nuclei were
incubated with [-32P]UTP, and RNA was isolated and
allowed to hybridize with plasmid DNA containing either MCP-1 cDNA or
positive or negative control cDNA. As shown in Figure 2, the resulting autoradiogram indicated
that IFN- increased transcription of mRNA. Results of hybridization
with GAPDH remained the same with or without IFN- treatment,
demonstrating equal loading of samples. These results clearly show that
IFN- modulates MCP-1 mRNA levels in human osteoblastic cells by
increasing the rate of transcription rather than stabilization of
preexisting message.
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was also
accompanied by an increase in MCP-1 protein secretion (Table I). Although the MG-63 cells secreted a
high level of MCP-1 under basal conditions, consistent with the
observed high basal level of MCP-1 mRNA expression, addition of IFN-
increased MCP-1 production in a dose-dependent manner, with an increase
similar in magnitude to that which we have observed previously with
IL-1 and TNF- stimulation in these cells (32). Thus, treatment with
IFN- increases both the transcription and translation of the MCP-1
gene in MG-63 cells.
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-mediated
transcription
The rapid transcriptional response to IFN- has been shown in
several genes to be mediated through a transcriptional element termed
the GAS. The consensus sequence (TTNCNNNAA) has been defined for the
GAS (33, 34, 35), and inspection of the proximal promoter region of MCP-1
identified a potential GAS element at position -214 relative to the
ATG start codon (Fig. 3A). The
core sequence of this element (TTCCTGGAA) resembles closely the
symmetrical dyad sequence defined as the binding site for the STAT
family of transcription factors (with the exception of STAT2). Adjacent
and 5' to this site in the MCP-1 promoter is a CT-rich element (GCTT
CCCT TTCC TAC) that shows some homology to the consensus sequence for
the IFN-stimulated response element (ISRE) (36) (Fig. 3B),
which has been shown to mediate transcriptional responses to both
IFN- and IFN-/ß.
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rapidly induced MCP-1 through a
transcriptional mechanism, transfection studies were conducted with a
deletion series of MCP-1 promoter/luciferase plasmids to identify and
characterize the IFN--responsive elements. As shown in Figure 4, IFN- stimulation of MG-63 cells
transfected with construct "a" (-2910/+21), the largest construct
used in these studies, resulted in only a 1.7-fold increase in
luciferase activity compared with unstimulated controls. Construct
"a" was prepared to include the B and other regulatory elements
located in the distal enhancer region that recently have been shown to
regulate MCP-1 transcription in response to IL-1ß and TNF- (9, 11). IFN- stimulation of cells transfected with construct "b"
(-248/+21) and "c" (-227/+21), however, in which the functional
B site and the majority of the 5' flanking sequence were eliminated,
resulted in an approximately threefold increase in luciferase activity.
This indicated that the distal enhancer elements in the MCP-1 promoter
do not appear to be obligatory for the mediation of the IFN-
response in MG-63 cells, and that the positive response element resided
within the first 227 bp of the gene flanking the ATG start codon.
Construct "c" (-227/+21) included the promoter sequence up to the
start of the CT element. Since this element shows some homology to the
consensus sequence for the ISRE (Fig. 3
C) and is adjacent to
the potential GAS element, it was possible that either or both elements
may be required for mediating the IFN- response. Therefore, to
investigate the role of the GAS element alone in mediating the response
to IFN-, we prepared construct "d" (-214/+21), which terminated
at the 5' end of the GAS element and eliminated the CT element.
Unexpectedly, this construct produced the greatest increase in the
reporter gene activity observed in these experiments (
10-fold). Thus,
the CT element appears to inhibit the response to IFN- mediated by
the GAS element. When the GAS element was deleted (construct "e,"
-198/+21), responsiveness to IFN- was lost completely. Therefore,
these experiments indicate that in the MG-63 osteoblastic cell line,
the rapid IFN--induced increase in transcription of MCP-1 appears to
be mediated entirely through the GAS element located at position -214
to -198, whereas the adjacent CT element appears to negatively
regulate this GAS-mediated response. We have termed this CT element the
IFN response-inhibitory sequence (IRIS), which describes its observed
functional activity.
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. MG-63 cells
transiently transfected with construct -214/+21
(IRIS-/GAS+) showed a substantial increase in
luciferase activity following 2 h of stimulation with IFN-, and
this increased further between 4 and 8 h. Cells transfected with
the construct -227/+21 (IRIS+/GAS+), on the
other hand, showed only a moderate increase at 2 h and did not
increase further beyond this time point. At the 8-h time point, the
response was approximately 25% of that observed for construct
-214/+21. Thus, the IRIS appears to inhibit both the magnitude and
duration of the IFN--stimulated transcriptional response.
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Nucleotide alignments were conducted using the LALIGN program to
identify potential homologous IRIS/GAS elements in the promoter regions
of other chemokine genes. As shown in Figure 3B, the overall
IRIS/GAS element is well conserved with respect to both sequence (92%
bovine vs human; 79% murine and rat vs human) and location in the
MCP-1 promoters of other species (9, 15, 17, 37, 38, 39, 40). The sequence of
the GAS moiety is perfectly conserved, and the base changes observed in
the IRIS moiety maintain the pyrimidine-rich character of the sequence.
This conservation represents supportive, if indirect, evidence that
this sequence performs an important physiologic function in the
regulation of MCP-1 transcription. Sequences homologous to the IRIS/GAS
described in this work were not identified in the known promoter
sequences of the chemokine genes IL-8, RANTES, IP-10, MIP-1,
MIP-1ß, or MCP-3, suggesting that this particular complex element may
be specific to MCP-1.
IFN- induces binding of nuclear factors to the MCP-1 IRIS/GAS
sequences
Since the above studies demonstrated that deletion of the
IRIS moiety from the IRIS/GAS element significantly altered
transcriptional activity of MCP-1, EMSA studies were conducted to
identify potential nuclear factors affected by this deletion (Fig. 6). Two oligonucleotide probes were
constructed: one contained the IRIS/GAS sequence and the other
contained the core GAS element alone (Fig. 3C). EMSA was
conducted with nuclear proteins extracted from MG-63 cells with or
without IFN- (1000 U/ml) treatment for 30 min. Several DNA-protein
complexes were formed between the IRIS/GAS probe and the nuclear
extracts of unstimulated MG-63 cells (Fig. 6A, bands C).
Stimulation with IFN- resulted in the induction of two major new
bands (Fig. 6A, bands A and B), in addition to the bands
that were observed under basal conditions. However, using an
oligonucleotide probe corresponding to the core GAS element alone, only
a single band was observed corresponding to the faster migrating of the
two major inducible bands that were observed with the IRIS/GAS DNA
probe (Fig. 6, B and C). No DNA-protein complexes
were formed between the labeled GAS probe and nuclear extract from
unstimulated MG-63 cells (Fig. 6C). Thus, the IRIS moiety
flanking the GAS element appears to be necessary for binding both the
constitutive factors and the inducible factor with lower mobility
(band B).
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). The interaction
between nuclear extracts of IFN--stimulated cells and the labeled
IRIS/GAS probe was competed with increasing concentrations of unlabeled
IRIS/GAS and GAS DNA. As shown in Figure 7, the unlabeled IRIS/GAS
probe competed for the two inducible factors (A and
B) and six (C1-C6) of the DNA-protein complexes observed in
unstimulated cells (complex C1 is obscured by band A in Fig. 7, but is
clearly seen in Fig. 8). This competitive
inhibition was dose dependent. The unlabeled GAS probe competed for the
inducible factors, inhibiting the formation of both fast and slow
migrating DNA-protein complexes (bands A and B, respectively). The
unlabeled GAS probe did not compete with any of the DNA-binding factors
that were constitutively expressed. Competition of the slower migrating
inducible complex by the unlabeled GAS probe was surprising, since a
corresponding band was not observed when the labeled GAS probe was
incubated with nuclear extracts from IFN--stimulated cells.
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C, and nuclear extracts from unstimulated MG-63
cells. No DNA-protein complexes were observed in these experiments
(data not shown), suggesting that all or part of the GAS element is
also required. This was further investigated in EMSA assays using
labeled IRIS/GAS DNA as the probe and competing with unlabeled DNA
corresponding to IRIS/GAS, IRIS/GAS, and IRIS sequences (Fig. 3C). As shown in Figure 8, deletion of the 3' domain of the
GAS element from the IRIS/GAS probe (IRIS/GAS) was sufficient to
reduce its ability to inhibit formation of the DNA-protein complexes.
Furthermore, deletion of the entire GAS element to result in a probe
consisting of the IRIS alone resulted in a complete loss of competitive
activity. Thus, these data clearly indicate that nucleotides in both
the IRIS and GAS moieties of the IRIS/GAS element are required for the
binding of the constitutively expressed factors.
A number of factors have been identified that modulate the
transcription of IFN-responsive genes (33, 34, 35, 41, 42, 43, 44, 45). To characterize
the binding proteins forming the DNA-protein complexes observed on
EMSA, supershift assays were conducted with monospecific Abs to
STAT1/ß, STAT2, p48(ISGF-3), IRF-1, and IRF-2 (Fig. 9). Ab to STAT1/ß appeared to cause
a supershift of both IFN--induced bands that formed with the labeled
IRIS/GAS probe. In contrast, Abs to STAT2, p48(ISGF-3), IRF-1, and
IRF-2 had no effect on any of the DNA-protein bands formed with the
unstimulated or IFN--stimulated MG-63 nuclear extracts. Supershift
EMSA was also conducted to identify the proteins in the DNA-protein
complex formed between IFN--stimulated MG-63 nuclear extract and the
GAS probe. As expected, the single band was supershifted by the
STAT1/ß Ab, but not by any of the other Abs used (data not shown).
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treatment is mediated by the
binding of activated STAT1 factors to the MCP-1 GAS element. However,
in the EMSA studies described above, the nuclear extracts were prepared
from MG-63 cells that had been stimulated with IFN- for 30 min, and
the functional studies with the MCP-1 (-214/+21) construct (Fig. 5)
clearly indicated that the reporter gene activity was actively being
produced at later time points. Since a number of the transcription
factors regulating IFN responsiveness are induced with time (44, 45, 46),
it is possible that additional factors may have been induced during
this period and positively regulated the enhanced transcription through
the GAS element. Therefore, EMSA was conducted using the IRIS/GAS
probe, and MG-63 nuclear extracts were isolated after 30 min and 2, 4,
and 8 h of IFN- stimulation. As shown in Figure 10, the STAT1-containing DNA-protein
complexes induced by IFN- treatment (bands A and B) were present
throughout the 8-h period of stimulation, but showed diminishing
intensity after 30 min. The complexes formed with the constitutively
expressed factors, on the other hand (bands C2-C6), appeared to remain
relatively constant. Furthermore, additional bands that might
correspond to newly induced DNA binding were not observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces MCP-1 gene expression
in osteoblastic cells through a mechanism of enhanced transcription.
This is supported by nuclear run-on experiments and increased reporter
gene activity in transiently transfected MG-63 cells. The rapid
induction of MCP-1 transcription by IFN- is mediated by a
cis-regulatory element located between -214 and -198 of
the 5' flanking sequence. This contains a consensus GAS sequence that
has been shown to confer rapid IFN- responsiveness in a number of
genes. Deletion of this site from the MCP-1 promoter completely
eliminated IFN--stimulated activity, indicating that GAS is the
principal element mediating the IFN--induced MCP-1 gene
transcription in osteoblastic cells.
A regulatory role for nucleotides immediately 5' to the GAS was also
identified. The greatest induction of transcriptional activity was
observed with the deletion construct (-214/+21), which terminated at
the 5' end of the GAS element. Addition of the 13 5' flanking
nucleotides of the promoter (the IRIS moiety) resulted in a major
reduction, but not elimination, in both the magnitude and duration of
the IFN--stimulated MCP-1 transcriptional response. Thus, the
increased transcription observed in the osteoblastic cells reflects a
balance between a positive enhancement mechanism mediated through the
GAS element and an inhibitory mechanism mediated through the IRIS site.
Negative regulatory sequences that modulate responsiveness to the
IFNs have been reported previously in the murine IFN 11 gene and the
9/27 gene (47, 48). In both cases, however, the sequences appear to
modulate an ISRE-mediated rather than a GAS-mediated IFN response, and
are probably unrelated to the negative regulatory sequence that we have
described in this work. Negative regulation may also be mediated by the
transcriptional factor IRF-2, which acts as a repressor of the IFN-
response and competes with the positive transcription factor IRF-1 for
binding to the ISRE (43, 44, 45). However, our supershift EMSA data
indicated that neither of these factors appears to bind the IRIS/GAS
DNA. This is despite the fact that both IRF-1 and IRF-2 can be
identified in the nuclear extract of MG-63 cells (data not shown). The
lack of binding of IRF-2 suggests that the IRIS moiety of the MCP-1 IFN
response element and the ISRE described in other IFN-responsive genes,
although showing some degree of sequence homology, are structurally and
functionally distinct. The IRIS may represent a novel regulatory
sequence in a complex response element that provides a negative
regulatory pathway for IFN--induced gene transcription.
The complex IRIS/GAS element in the human MCP-1 promoter is
highly conserved in both sequence and location in the murine, rat, and
bovine MCP-1 genes. The sequence homology in the IRIS/GAS between
bovine and human and mouse/rat and human is 92 and 79%, respectively.
The conservation of this sequence supports our observations that it
performs an important physiologic role in the regulation of MCP-1
transcription. Using a nucleotide alignment program, we were unable to
identify this same combination of IRIS/GAS sequences in the known
promoter sequences of IL-8, MCP-3, MIP-1ß, or RANTES. This
observation reinforces the concept that among the chemokine genes,
MCP-1 may be induced by IFN- through a unique regulatory pathway.
This is particularly interesting, since the functional evidence to date
seems to indicate that chemokine genes may be regulated by different
mechanisms in response to IFN-. IL-8, for instance, appears to be
negatively regulated by IFN- in a number of cell types (49, 50), but
in those cases in which it has been reported to stimulate IL-8
expression, this has been shown to result from either mRNA
stabilization or increased transcription mediated through a synergistic
induction of AP-1 and nuclear factor-B (31, 51). IP-10, another
member of the C-X-C subfamily of chemokines, is positively regulated by
IFN-. The increase in IP-10 expression has been shown to be mediated
through a consensus ISRE sequence located approximately 235 bases
upstream of the transcription start site (52). In addition, there is
evidence that for full response to IFN-, one or more of the B
binding sites located downstream of the ISRE may also be required (52).
IFN--induced transcription of the chemokine gene mig, on
the other hand, appears to be mediated by a STAT1-containing factor
binding a complex and imperfect palindromic sequence that bears partial
homology to the GAS sequence (53, 54). Our studies reported in this
work indicate that in osteoblastic cells, the positive induction of
MCP-1 by IFN- appears to require only the discrete GAS element
located between -214 and -198 of the proximal promoter, since the
deletion of this element resulted in a complete loss of responsiveness
to the cytokine. The positive response through this element, however,
is partially repressed by the sequence adjacent to it.
Nuclear extracts from IFN--treated cells formed two specific
inducible DNA-protein complexes with the IRIS/GAS DNA probe. Their
formation was inhibited by competition with both unlabeled IRIS/GAS and
GAS DNA, indicating that both bands were dependent on the binding of
nuclear factors to the GAS sequence. Supershift assay indicated that
both bands contained the STAT1 protein, but not STAT2, p48(ISGF-3),
IRF-1, or IRF-2. However, when the EMSA was conducted using labeled
MCP-1 GAS as the probe, only the faster migrating of the inducible
bands (band A) was observed. Taken together, these data indicate that
the rapid IFN- induction of MCP-1 transcription is mediated by STAT1
binding to a well-defined GAS element, as has been shown in IFN-
induction of other genes. However, the formation of an additional
slower inducible band on EMSA also suggests that a second larger
complex may be formed between the IRIS/GAS element and DNA-binding
factors. This must include the activated STAT1 factor, since the band
was supershifted with specific anti-STAT1(/ß) Ab. It may also
include other factors that bind to both STAT1 factors and the adjacent
IRIS moiety.
This pattern of DNA-protein complex formation observed with the
MCP-1 IRIS/GAS is analogous to the multiple DNA-protein complexes
formed between the IFN--responsive region of the gene for a high
affinity receptor for IgG (FcRI) and nuclear extracts of
IFN--treated monocytic cells (55, 56). There, like the MCP-1
IRIS/GAS, the formation of two DNA-protein complexes on EMSA was
absolutely dependent on the presence of an intact GAS element located
in the 3' domain of the DNA probe, whereas the formation of a second
slower migrating complex required in addition the presence of the 5'
domain. However, the influence of these 5' sequences on function is
opposite. The 5' domain of the FcRI response element was shown to
enhance GAS-mediated transcriptional enhancement in response to IFN-
(56), whereas as we have shown in this work, the presence of the 5'
IRIS moiety in the MCP-1 IRIS/GAS results in inhibition of induced
transcription.
Nuclear extracts from unstimulated osteoblastic cells formed several
specific DNA-protein complexes with the IRIS/GAS DNA. These could be
competitively inhibited with unlabeled IRIS/GAS, but not by GAS or IRIS
alone. In addition, they were not observed on EMSA with labeled GAS or
IRIS probes. Thus, their formation required the presence of both IRIS
and GAS sequences. We did not identify these constitutively expressed
factors, but Abs to STAT1, STAT2, p48(ISGF-3), IRF-1, and IRF-2 all
failed to supershift or inhibit their complex formation on EMSA, making
it unlikely they are immunologically related to these known factors.
Since transcriptional studies indicated that the IRIS moiety of the
IRIS/GAS element inhibits IFN--stimulated MCP-1 expression, it is
possible that one or more of these DNA-binding proteins is responsible
for inhibiting the GAS-mediated transcription through constitutive
occupation of the IRIS/GAS binding site. This could represent a
mechanism for limiting MCP-1 expression under basal conditions or in
response to inflammatory stimuli.
Physiologically, the diverse regulatory pathways displayed by the
chemokine genes in response to IFN- may reflect their particular
roles in IFN--mediated immune events. Temporal patterns of chemokine
expression have been noted in a number of pathophysiologic states of
immune etiology, including tissue rejection and wound healing (57, 58).
Differential regulation of chemokine genes by IFN-, which results in
the ordered trafficking and activation of immune cells, may be an
important component of these immune processes. An understanding of the
molecular mechanisms that mediate IFN- responsiveness will help
clarify the role of the chemokines in immune and inflammatory events.
Note added in proof.
During submission of this paper, Zhou et al. (59) showed IFN-
stimulated MCP-1 transcription in astrocytoma cells through the same
GAS promoter element described here.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anthony J. Valente, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7870. E-mail address:
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; AP-1, activating protein-1; DRB, 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole; EMSA, electophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS, IFN- activation site; IP-10, inflammatory protein-10; IRF, IFN-regulatory factor; IRIS, IFN response-inhibitory sequence; ISRE, IFN-stimulated response element; MIP, macrophage-inflammatory protein; NBS, newborn bovine serum; p48(ISGF-3), the 48-kDa component of the IFN-stimulated gene factor-3 complex.
Received for publication October 1, 1997. Accepted for publication May 28, 1998.
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