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Induction of the Human Monocyte Chemoattractant Protein (hMCP)-1 Gene in Astrocytoma Cells: Functional Interaction Between an IFN--Activated Site and a GC-Rich Element1
*
Department of Neurosciences, Research Institute, and
Department of Neurology and The Mellen Center for Multiple Sclerosis Treatment and Research, Cleveland Clinic Foundation, Cleveland, OH 44195;
Medical Biochemistry and Neurobiotechnology Center, The Ohio State University, Columbus, OH 43210; and
§
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in astrocytoma cells, because
astroglial cells express chemokines in several central nervous system
inflammatory states. It was found that IFN--induced hMCP-1
transcription was rapid, transient, and mediated by a 213-bp
promoter-proximal regulatory region of the gene. Our studies on both in
vitro and in vivo states of the hMCP-1 regulatory region established
requirement of an IFN--activated site (GAS) and the presence of
IFN--inducible GAS-binding activity involving at least STAT-1
for
IFN--induced hMCP-1 expression. Unexpectedly, in vivo genomic
footprinting of the proximal regulatory region of the IFN--induced
gene revealed protection of a GC-rich sequence (GC box) with the same
temporal pattern as that seen at the GAS; in vitro, this GC-rich
element is associated with nuclear factor Sp1. These observations
suggested a cooperative interaction between the GAS and the GC box
element. Interestingly, site-specific mutations that abolished GC-box
or GAS-element function produced clearly disparate results. Disruption
of the GC box did not affect fold induction by IFN- but reduced
promoter-reporter expression by half. Conversely, GAS mutation
abrogated induction but did not affect the magnitude of expression.
These results establish the importance of the GAS element for induction
of hMCP-1 and further our understanding of IFN--mediated
transcriptional induction by providing the first evidence in vivo for
inducible signaling to the GC box by this cytokine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- is a T cell-secreted proimflammatory cytokine important for
leukocyte recruitment and endothelial activation in inflammation.
Within target tissues, IFN- induces expression of chemokines,
further enhancing endothelial adhesiveness for leukocytes and providing
signals for leukocyte extravasation and tissue invasion. The diverse
functions of IFN- are mediated through an intracellular signaling
mechanism termed the Janus-protein tyrosine kinase
(JAK)3-STAT pathway.
Specifically, binding of IFN- to its receptor leads to
oligomerization of the receptor and the rapid phosphorylation of JAK1
and JAK2 tyrosine kinases and the receptor -chain, which forms a
specific docking site for the SH2 domain of STAT-1 (5, 6).
Phosphorylated STAT-1 monomers assemble in a homodimeric complex,
accumulate in the nucleus, and bind to a palindromic DNA motif, the
IFN--activated site (GAS), the consensus sequence of which is
TTNCNNNAA (7, 8). Although the notion that a single STAT-1 dimer
bound to a single GAS element is sufficient to activate transcription
may still be valid for many genes, recent studies in many laboratories
point to another level of specificity and complexity in
STAT-1-regulated transcriptional activation. As indicated by Xu et
al. (9), specificity of gene activation can be achieved through
cooperative binding of STAT-1 to several adjacent consensus sites.
Guyer et al. (10) have shown that IFN- activates a novel factor
called RF-1, which binds to an imperfect tandem GAS palindrome in
the 5'-flanking region of the mig gene. This RF-1
consists of at least STAT-1 and a 130-kDa protein. Protein-protein
interactions can also occur between STAT1 and other nuclear factors.
Myeloid cell-restricted induction by IFN- of the gene encoding the
human high-affinity Fc receptor (FcR1) requires the cooperation
of STAT-1 and a B cell- and myeloid cell-specific factor,
PU.1/Spi-1 (11).
Recently, it was reported that induction by IFN--mediated
trans-activation of the ICAM-1 gene depended on interaction
of STAT-1 with another transcriptional activator, nuclear factor Sp1
(Sp-1; 12 . Specifically, the functional significance of STAT-1
and Sp-1 binding sites for IFN--responsiveness of the ICAM-1 gene
was evaluated using promoter/luciferase-reporter constructs in
transient transfection assays. Mutations that eliminated binding
activities to either site abolished IFN- responsiveness of the
promoter. Interestingly, the trans-activation domain of Sp-1
in the context of a Gal4/Sp-1 fusion protein functioned fully for
driving IFN--mediated transcription of a synthetic promoter
containing ICAM-1 5'-flanking sequence with a Gal4 binding site in
place of the Sp-1 binding site. This result raised the possibility that
the trans-activation domain of Sp-1 was sufficient for
functional interaction between the two transcription factors. STAT-1
and Sp-1 were coimmunoprecipitated from cell lysates; the significance
of this result was uncertain, because the interaction was only
partially activation dependent.
Monocyte chemoattractant proteins (MCP), members of the chemokine ß
subfamily, are implicated in wound healing, the pathogenesis of
atherosclerosis, immune and inflammatory responses, and modulation of
tumor immunity (13, 14). Four human MCP (hMCP) have been identified and
share 
65% amino acid identity (hMCP-1, -2, -3, and -4) (15, 16).
Recent work from the laboratories of A.D. Luster (17) and J.C.
Gutierrez-Ramos (18) sheds unexpected light on the structure and
function of hMCP-1. They have independently identified a novel
chemokine named murine MCP-5 (mMCP-5), which exhibits high sequence
homology to hMCP-1. The mature mMCP-5 protein is 66% identical to the
mature hMCP-1 protein, while JE, another murine homologue of hMCP-1, is
55% identical to hMCP-1. Functional studies revealed that mMCP-5 and
hMCP-1 were more potent agonists of the hMCP-1 receptor (CCR2b) than
was JE, with mMCP-5 a full agonist for CCR2b. hMCP-1 appears likely to
be the functional equivalent of the JE gene product during various
inflammatory responses in vivo, given that hMCP-1 and the JE gene
products are widely and abundantly expressed following varied immune,
traumatic, and toxic insults to nervous system and other tissues
(19, 20, 21, 22, 23).
JE was the first MCP-1 gene cloned (24), and its transcriptional
regulation by mediators including platelet-derived growth factor and
TNF- have been characterized extensively. Both TNF- and phorbol
esters are potent inducers of hMCP-1 expression in various cell types
(25, 26). Transient transfection experiments with hMCP-1 promoter
constructs indicated that two distal 
B sites (27) are important for
TNF--mediated induction, and two proximal activator protein-1 (AP-1)
sites (25, 28) for TPA-induced enhancer activity, respectively. A GC
box between bp 64 and 59 was found necessary for the maintenance of
basal transcription activity (27).
IFN- selectively induces expression of MCP-1 in mesenchymal and
epithelial cells (29, 30). Dexamethasone, an anti-inflammatory
agent, on the other hand, significantly down-regulates MCP-1 transcript
levels (31). In vivo, MCP-1 is expressed by astrocytes in both
immune-mediated and posttraumatic inflammation of the central nervous
system (CNS), and mechanisms that govern MCP-1 expression in astrocytes
have not been defined. To address this issue, we established a
tissue-culture model of astrocyte lineage-specific expression of hMCP-1
and used this system to address IFN--mediated hMCP-1 stimulation.
Our results indicate that IFN- induces hMCP-1 transcription in human
astrocytoma cells in rapid, transient fashion. IFN--induced hMCP-1
transcription is mediated by a 213-bp promoter-proximal regulatory
region of the gene. Studies on both in vitro and in vivo states of this
regulatory region of the hMCP-1 gene before and during exposure to
IFN- provide documentation of the requirement of a GAS and STAT-1
for IFN--induced hMCP-1 expression. In vivo genomic footprinting
(IVGF) of the IFN--induced gene further demonstrated
IFN--inducible occupancy of a GC box that was shown in vitro to bind
specifically to Sp-1. Site-specific mutations in the GAS element
abrogated IFN- hMCP-1 induction, while disruption of the GC box
reduced the efficiency of promoter-reporter expression. Our results
provide insight into the mechanism of IFN- action toward the hMCP-1
gene, which requires cooperativity between a GAS element and a GC box
for optimal expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CRT astrocytoma cell line, as previously described (32), was derived from a grade IV human astrocytoma and is >60% glial fibrillary acidic protein (GFAP) positive. Experiments reported here were done between passages 20 and 40. CRT cells were routinely maintained in RPMI 1640, supplemented with 2 mM L-glutamine and 10% FBS (complete medium; Life Technologies, Grand Island, NY). The CRT cell line has been extensively characterized as a model system for analyzing cytokine regulation of gene expression in human astrocytic cells (33, 34).
Reagents
Purified human recombinant IFN- (1.9 x
107 U/mg protein) was generously provided by Genentech
(South San Francisco, CA). Polybrene (hexadimethrine bromide) and DMSO
were purchased from Sigma Chemical Co. (St. Louis, MO).
Promoter construction and PCR-mediated mutagenesis
The construction of the expression plasmid, pMCP-1(3.5)chloramphenicol acetyltransferase (CAT), was detailed by Li and Kolattukudy (28): the putative promoter region of the hMCP-1 gene was amplified by PCR using the cloned hMCP-1 gene (25) as a template. A 486-bp fragment of 5'-flanking sequence, including the transcription start site, was subcloned into the HindIII/XbaI site of pCAT-basic (Promega, Madison, WI) to generate pMCP-1(486)CAT. An additional 3-kb HindIII fragment of immediately contiguous upstream sequence was then subcloned into the HindIII site of pMCP-1(486)CAT in the appropriate orientation to generate pMCP-1(3.5)CAT. A serial deletion of the 5' region was done by ExoIII digestion from the -378-bp PstI site upstream of the transcription start site (28) to obtain pMCP-1(349)CAT, pMCP-1(292)CAT, pMCP-1(213)CAT, and pMCP-1(147)CAT. pCAT-basic is a promoterless expression vector. A 213-bp fragment with 5'-HindIII- and 3'-NcoI-sites was also generated from pMCP-1(213)CAT DNA by PCR, using 5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3' and 5'-GCGTCTTCCATGGTGGCTTTCTAGAGGCGAGAGTGCGAG-3' as forward and backward primers, respectively. The fragment was excised with HindIII and NcoI and subcloned into the promoterless pGL3-basic vector (Promega) via HindIII/NcoI sites to generate pGL3-213.
Site-directed mutagenesis of GAS and GC box to abolish binding
activities of STAT1 and Sp-1 was achieved by PCR-mediated
mutagenesis as detailed by Aiyar et al. (35), using pGL3–213 as a
template and corresponding primers containing mutated nucleotides. The
primers used to make GAS mutant pGL3-mGAS were
5'-CCTTAAGCTTTGCATGGCAATCCAC-3'
(forward primer, with GAS consensus underlined and mutagenized
nucleotides in bold) and
5'-GCGTCTTCCATGGTGGCTTTCTAGAGGCGAGAGTGCGAG-3' (backward primer).
The PCR product was then digested with
HindIII/NcoI and subcloned into pGL3-basic vector
via HindIII/NcoI sites to generate a full-length
GAS-mutagenized 213-bp fragment. Three rounds of PCR using two sets of
primers were performed to obtain GC box mutant PGL3-mGC. The first pair
of primers comprised 5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3' and
5'-AGGGAGAGAGCTCGGAGTCAAGCAGGAGG-3', with the
GC box consensus underlined and mutagenized nucleotides in bold. The
second pair was made up of
5'-CCTCCTGCTTGACTCCGAGCTCTCTCCC-3' and
5'-GCGTCTTCCATGGTGGCTTT-CTAGAGGCGAGAGTGCGAG, with the GC box
consensus underlined and mutagenized nucleotides in bold. The first two
rounds of PCR produced two PCR products, with 20 bp of overlap covering
the entire GC box motif. These two products were gel purified and used
as the templates for a third round of PCR with
5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3' and
5'-GCGTCTTCCATGGT-GGCTTTCTAGAGGCGAGAGTGCGAG as primers. The PCR
product was then excised with HindIII/NcoI and
subcloned into pGL3-basic vector via HindIII/NcoI
sites to generate a full-length GC-mutagenized 213-bp fragment. A
promoter/reporter construct containing either a 5-bp or 10-bp insertion
between the GC box and GAS element was obtained similarly using
PCR-mediated site-directed mutagenesis. The first pairs of primers were
5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3' and
5'-CGGAGTCAAGGATATCAGGAGGAGGGATCTTCC-3' for 5-bp
insertion and 5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3' and
5'-GAGTCAAGTTTTTGAATTCAGGAGGAGGGATCTTCC for 10-bp
insertion. The inserted sequences are underlined. The second pairs of
primers were 5'-CCTGATATCCTTGACTCCGCCCTCTCTCCC-3' and
5'-GCGTCTTCCATGGTGGCTTTCTAGAGGCGAGAGTGCGAG-3' for 5-bp insertion
and 5'-CCTCCTGAATTCAAAAACTTGACTCCGCCCTCTCTCCC-3'
and 5'-GCGTCTTCCATGGTGGCTTTCTAGAGGCGAGAGTGCGAG-3' for 10-bp
insertion. The inserted sequences are underlined.
B and TPA-response element (TRE) mutants of the hMCP-1 promoter were
generated by two of us (Y-s.L., P.E.K.) at The Ohio State
University (28).
Transient Transfection
The Polybrene (Sigma) method (36) with 50 µg of supercoiled
plasmid DNA was used to transfect human astrocytoma cells (CRT cells).
Briefly, confluent cells (6 x 106) were passed
(1:2) 1 day before transfection on 150-mm dishes and then incubated in
8 ml of transfection medium (10 µg/ml of Polybrene and 50 µg of
plasmid DNA in complete medium). After incubation for 6 h at
37°C with intermittent gentle shaking, cells were subjected to 1.5
min of shock (30% DMSO in incomplete medium, which was RPMI 1640
supplemented with 2 mM L-glutamine), rinsed twice in
incomplete medium, and incubated for 12–16 h in complete medium. Cells
were then pooled and split (1:2) into smaller 100-mm dishes to control
for differential transfection efficiency, incubated for another 5 to
7 h, and reserved as controls or treated with 100 U/ml IFN- for
various periods of time, washed, and rested overnight to allow CAT or
luciferase protein to accumulate.
For the CAT assay, cell lysates were prepared by three freeze-thaw cycles, and protein amount was quantitated by the Bradford method (Bio-Rad, Richmond, CA). CAT assays were performed as described previously (36). For the luciferase assay, cells were lysed and luciferase activity was assayed using a luciferase assay kit (Promega) with a Luminometer (Dynatech Laboratories, Chantilly, VA). Results presented in this article were obtained from two to four separate experiments.
One microgram of a SV40 promoter-ß-galactosidase reporter plasmid, pCH110 (Pharmacia, Piscataway, NJ), was cotransfected with the test plasmids as an internal control to normalize for transfection efficiency. ß-Galactosidase activity was measured in cell lysates by using the ß-galactosidase enzyme assay system kit (Promega). Luciferase or CAT activity of the transiently transfected cells were normalized to ß-galactosidase activity.
RNase Protection Assay
The hMCP-1 probe for the assay protects a 560-bp fragment of
hMCP-1 mRNA and was generated after the pGEM-hJE34 (a generous gift
from Dr. B.J. Rollins at Dana-Farber Cancer Institute) was linealized
with HindIII. The linealized hMCP-1 DNA was then transcribed
with T7 RNA polymerase. Probe for -actin was also used as a control
for mRNA loading (37). Total cellular RNA was isolated from 90%
confluent CRT cells using the TRIzol method (Life Technologies). Total
RNA (10 µg) was denatured and used for hybridization with the
corresponding ribonucleotide probes. The labeling and hybridization
conditions were detailed by Rani et al. (37). Data reported were
obtained from three separate experiments.
Nuclear run-on analysis
For each data point, 5 x 106 cells at 70–80% confluency were washed and scraped in ice-cold 1x PBS and pelleted. Nuclei were isolated by lysing the cells in hypotonic Nonidet P-40 lysis buffer and incubated in a solution containing 148.5 mM KCl; 5 mM MgCl2; 1 mM MnCl2; 10 mM Tris-HCl, pH 8.0; 10% glycerol; 1 mM each of ATP, GTP, and CTP, 2 mM DTT; and 0.144 mCi of [32P]UTP (3000 Ci/mmol; Amersham Life Sciences, Arlington Heights, IL) at 25°C for 45 min. After the reaction, nuclei were lysed with 0.5 ml of RNAzol (Cinna/Biotecx Laboratories, Friendswood, TX) and 100 µl of chloroform-isoamyl alcohol (24:1), and RNAs were pelleted using isopropanol. As hybridization substrates, plasmid DNAs were denatured and spotted onto nitrocellulose membranes (Nitrobind; Micron Separations, Westboro, MA). Labeled transcripts were recovered by hybridization in a solution containing 1 x 107 cpm/ml of 32P-labeled RNA at 42°C for 3 days. Autoradiography was generated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantitated with ImageQuant. Transcriptional activation was calculated as the hMCP-1/ß-actin ratio.
Preparation of nuclear extract and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by a modified method of Dignam et
al. (51), as described by Ohmori and Hamilton (38). For binding
reactions, nuclear extracts containing 10 µg of protein were
incubated with 5 µg of polydeoxyinosinic-deoxycytidylic acid for 15
min at 4°C. The 32P-labeled oligonucleotide probe
(20,000 cpm) in 9 µl of binding buffer (6 mM HEPES, 1 mM DTT, and 6%
glycerol) was then added to the reaction mixture and incubated for 20
min at room temperature. The oligonucleotide probe was labeled using T4
polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN). For
supershift experiments, rabbit antisera against STAT1 (39) or Sp-1
(Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear
extract for 20 min at 4°C before addition of
polydeoxyinosinic-deoxycytidylic acid and oligonucleotide probe. The
reaction products were analyzed by electrophoresis in a 6%
polyacrylamide gel. The oligonucleotide probes for the GAS-binding
assay were 5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC for the
wild-type GAS element and
5'-CCTTAAGCTTTGCATGGCAATCCAC
for the mutagenized GAS element. GAS consensus sequences are
underlined, and mutagenized nucleotides appear in bold. The probes for
Sp-1 were 5'-CCTCCTGCTTGACTCCGCCCTCTCTCCC for the
wild-type GC box and
5'-CCTCCTGCTTGACTCCGAGCTCTCTCCC for the
mutagenized GC box, with the GC box consensus sequences underlined and
mutagenized nucleotides in bold.
IVGF
In vivo methylation of cellular DNA and DNA preparation were performed as described by Mueller et al. (40). Ligation-mediated PCR was conducted according to the procedure of Mueller et al. (40), with minor modifications as described by Ping et al. (41). The 213-bp promoter proximal region of the hMCP-1 gene was analyzed by one set of coding-strand and one set of noncoding-strand primers. Primers for the coding strand were 5'-TGTGGTTCAAGGAGAAGAAGAGGG-3', 5'-GCTATGAGCAGCAGGCACAGAAGG-3', and 5'-CAGGCACAGAAGGGCGGCAGAGAC-3'. The annealing temperatures for this set of primers were 59°C, 66°C, and 69°C, respectively. Primers for the noncoding strand were 5'-CCCTCTTAGTTCACATCTGTGGTCAG-3', 5'-CCCATCCTCCCCATTTGCTCAT-T-3', and 5'-TCCCCATTTGCTCATTTGGTCTCAGCAG-3'. The annealing temperatures were 59°C, 66°C, and 69°C, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces hMCP-1 expression
hMCP-1 is produced by a variety of cells on stimulation with
cytokines and bacterial and viral products of mitogens. It plays an
important role in the recruitment of monocytes at sites of inflammation
(14). There is relatively little known, however, of regulation of
hMCP-1 by these inflammatory stimuli. Astrocytes participate in the
pathophysiology of CNS inflammatory diseases. Their expression of
adhesion molecules, chemokines, and MHC antigens may contribute to
these inflammatory processes. Our previous studies showed that
astrocytes were the major source of mRNAs encoding MCP-1 and multiple
other chemokines in the CNS of experimental autoimmune
encephalomyelitis (EAE) mice, in which a transient burst of MCP-1 mRNA
accumulation occurred at the onset of CNS inflammation and neurologic
signs (19, 20, 21, 22). hMCP-1 is also prominently expressed in active multiple
sclerosis lesions by parenchymal astrocytes (52). To initiate our
studies of hMCP-1 gene regulation in an astrocyte-specific tissue model
and to determine the time course of expression of MCP-1 mRNA in human
astrocytic cells, we first examined the effects of proinflammatory
cytokine, IFN-, on hMCP-1 mRNA expression. CRT cells were treated
with IFN- (100 U/ml) for varying times, and mRNA accumulation was
evaluated by RNase protection assay. hMCP-1 mRNA appeared rapidly and
reached maximum by 8 h, decaying significantly by 24 h in the
continued presence of stimulus (Fig. 1
).
This observation indicated that IFN--induced hMCP-1 mRNA expression
was rapid, transient, and down-regulated, consistent with prior in vivo
studies.
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-induced steady-state hMCP-1 mRNA
accumulation was associated with an increase in the transcriptional
rate of the gene, hMCP-1 transcription was evaluated in nuclei isolated
from CRT cells after stimulation with IFN-. hMCP-1 transcription was
not detected in untreated cells; active transcription of the gene was
observed after 2 h of IFN- treatment, with a pronounced
decrease 1 h later (Fig. 2). After
4 h of IFN- treatment, the transcription rate declined to a
barely detectable level (not shown).
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The above observations led to subsequent experiments to test
whether transcriptional induction of hMCP-1 by IFN- was governed by
upstream elements of the hMCP-1 transcription unit. We asked whether
individual plasmids containing upstream elements of the hMCP-1 gene
fused to a reporter, CAT, could be stimulated by cytokines to inducible
expression. pMCP-1(3.5)CAT, a promoter/reporter construct containing
3.5 kb of 5'-flanking sequences driving expression of the reporter gene
CAT, was used for transient transfection experiments.
IFN- induced a 7.5-fold induction of hMCP-1 promoter-driven CAT
activity (Fig. 3, 3.5 kb, lane
1 vs lane 2). This induction was significant
(p < 0.005; t test).
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, lane
3 vs lane 4 and lane 5 vs lane
6). A construct containing 213 bp upstream of the
transcription start site was fully capable of responding to IFN-
stimulation (Fig. 3, 213 bp, lane 7 vs lane 8;
p < 0.001), while an hMCP-1 promoter-reporter
construct truncated at -147 bp failed to respond to stimulation (Fig. 3, 147 bp, lane 9 vs lane 10). The 213-bp
element was previously shown to be competent to direct TPA response in
human glioma cells, while a minimal element comprising 128 bp was
required for basal expression. Consistent with published reports, basal
expression of the 147-bp construct was comparable with that of
constructs containing additional upstream sequence content
(28).
GAS element is required for IFN--induced hMCP-1 gene
activation
A computer-assisted motif search of the proximal promoter between
-213 bp and -147 bp revealed a consensus GAS (5' terminus of GAS at
-212 bp relative to the transcription start site), an NF-B binding
site (5' at -149), and an (AP-1)-binding element (also called the TRE
site, 5' at -156). As a first step in analyzing the proximal
regulatory elements, site-directed mutagenesis was used to disrupt
GAS-212, B-149, and
TRE-156. IFN- responsiveness of the mutant promoters
was assessed by transient transfection experiments. As shown in Fig. 4, in the absence of a functional NF-B
or AP-1 binding site, IFN--induced CAT activity remained intact,
suggesting that these two regulatory elements are dispensable for
IFN- induction. In contrast, disruption of GAS-212
eliminated IFN- responsiveness of the gene.
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-inducible
protein-DNA complex (Fig. 5, complex a),
which reached maximal abundance at 30 min after IFN- treatment and
declined as the treatment continued (Fig. 5, lanes
1–3). The complex was specific for the GAS probe, as
nonlabeled GAS oligonucleotides were able to compete with the probe for
DNA-binding activity (Fig. 5, lane 4). The mutant GAS
motif used in transient transfections was completely inert for factor
binding (Fig. 5, lane 7). Antisera directed against
STAT-1 were also used in EMSA supershift assay. Complex a was
specifically supershifted by antisera to STAT-1, producing complex b
(Fig. 5, lanes 5 and 6).
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-deficient U3A cell line to confirm the
involvement of STAT-1 in IFN--mediated hMCP-1 expression (42). As
expected, no hMCP-1 message was detected in U3A cells after IFN-
treatment, while in wild-type 2fTGH cells, robust hMCP-1 expression was
detected (data not shown). Taken together, transient transfections, in
vitro binding assays, and studies in genetically modified cells provide
the first evidence favoring a requirement of a GAS regulatory element
for IFN--induced hMCP-1 expression. An IFN--inducible GAS-binding
activity containing STAT-1 contributes to this gene activation.
GAS and GC box in the proximal regulatory region of the hMCP-1 gene
are occupied in vivo after IFN- treatment
IVGF permits analysis of protein-DNA interactions in intact cells, by reduction (dimethyl sulfate (DMS) resistance/protection) or enhancement (DMS hypersensitivity) of DMS methylation of guanine residues, which are displayed as alterations in the intensity of specific bands in a guanine sequence ladder. DMS protection results from sequence-specific protein-DNA interactions. Protein-DNA interaction may result in conformational changes in the DNA and therefore increased accessibility of DMS to the DNA, leading to DMS hypersensitivity in neighboring regions. This approach has the advantage of revealing protein-DNA interactions in intact cells; furthermore, all sequences within a given region are examined by IVGF without preconception about the cis elements involved in regulation of a given gene under defined circumstances. It should be emphasized, however, that IVGF does not address the identity of nuclear proteins that interact with protected sites in vivo.
To determine the in vivo state of the regulatory region from -213 to
-147 of the hMCP-1 gene in untreated and IFN--treated cells, IVGF
was conducted. After 30 min of IFN- treatment, a protection on the
noncoding strand of consensus GAS at core residue G-209
and a partial protection on the coding strand at G-207
were observed (Fig. 6). This protection
decayed by 5 h of treatment. The protection on the coding strand
at GAS core residue G-207 was weaker (60% protection) but
highly reproducible through three independent experiments. IFN-
treatment also resulted in DMS hypersensitivity downstream of GAS at
G-201 on the noncoding strand. The in vivo protection
pattern correlated closely with the profile of IFN--induced in vitro
GAS-binding activity, which peaked at 30 min and declined during 5
h of treatment (Fig. 5, lanes 2 and 3).
The time course of factor binding in vivo was also consistent with the
time course of transcription activation of hMCP-1 in IFN--treated
cells (not shown).
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treatment. The GC box of the noncoding strand was very
weakly protected under basal conditions but became highly DMS resistant
(at G-109, G-117, G-119,
G-120, G-121, G-123, and
G-126 of the noncoding strand) upon IFN- treatment
(Fig. 7). An additional region upstream
of the consensus GC box located at -134 to -138 (G-134,
G-135, G-137, G-138 of the
noncoding strand), although weakly DMS resistant under basal
conditions, was found to be inducibly protected as well. No significant
DMS protection was observed at the above region of the coding strand,
in which the footprinting pattern precisely corresponded to that
produced by deproteinized genomic DNA. Upstream of the expanded GC box,
a residue (G-154) at the core of TRE was found to be DMS
hypersensitive, while G-156 was constitutively protected.
Interestingly, IFN--induced protection at the GC box displayed the
same temporal pattern of protection as seen at the GAS. Protection
peaked at 30 min of IFN- treatment but decreased with longer
treatment. This IFN--induced concerted binding to several proximal
regulatory sites is novel and suggests that events beyond STAT-1
homodimer binding to GAS element may be implicated in transcriptional
induction of responsive genes.
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, lane 3).
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The functional significance of GC box and GAS was further studied
to investigate the nature of cooperation between Sp-1 and STAT-1 for
IFN- responsiveness of the hMCP-1 gene. An hMCP-1 (213
bp)/luciferase-reporter construct containing mutations of core residues
at the Sp-1-binding GC box was used in transient transfection assays.
This mutation, which blocked Sp-1 binding to the GC box (Fig. 8, lane 5), failed to block IFN- inducibility of the
promoter/reporter construct in transient transfection (Fig. 9). Fold induction of hMCP-1 mGC
promoter-reporter mutant by IFN- was equivalent to that observed for
the wild-type -213 hMCP-1/luciferase plasmid. Basal expression and
maximal IFN- responsiveness, however, were proportionally reduced by
more than 50% in the GC mutant.
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binds the hMCP-1 GAS and
activates suboptimal transcription in the absence of a functional GC
box/Sp-1-binding element and therefore argue against an obligatory
physical interaction between Sp-1 and STAT-1 for inducing hMCP-1 by
IFN- in human astrocytoma cells. Consistent with the lack of
association between the two factors under these conditions,
immunoprecipitation experiments with whole-cell extracts from untreated
or IFN--treated cells showed that anti-Sp-1 antisera failed to
coprecipitate STAT-1 (data not shown).
To determine whether the cooperation between STAT-1 and Sp-1 was
affected by spacing between GAS and GC box, insertions of 5, one-half
turns of the DNA helix, or 10 nucleotides, were introduced between the
two sites. Neither insertion significantly affected efficiency or
IFN- inducibility of the promoter/reporter construct in transient
transfection assays (data not shown). Taken together, results described
in this section favor indirect cooperation between the GAS and GC-rich
element of the hMCP-1 gene in responding to IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated hMCP-1 gene. Their functional analysis
extends our understanding of the mechanism by which IFN- induces
hMCP-1 gene expression. In particular, it is demonstrated that
IFN--induced hMCP-1 transcription is mediated through a 213-bp
upstream region of the gene, containing a GAS and GC-rich element.
Furthermore, the GAS and GC box are shown by site-specific mutagenesis
to mediate separable functions in the induction of the gene: GAS was
required for response to IFN-, while the GC box was essential for
efficient gene expression. In vitro factor-binding studies showed that
STAT-1 complexes bound inducibly to the GAS, while Sp-1 was capable
of binding to the GC box. Although the binding of Sp-1 in vitro was not
regulated by IFN-, mutations that disrupted factor binding also
abrogated efficient gene expression. Finally, our results provide the
first demonstration in vivo that IFN- signals to the GC box, as well
as the GAS. Taken together, these observations indicate that IFN-
treatment orchestrates cooperation between the GAS and the GC box in
inducing efficient transcription of the hMCP-1 gene.
IFN- induction of hMCP-1: molecular mechanism
IFN-, alone or in combination with other proinflammatory
cytokines, induces hMCP-1 gene expression and secretion in multiple
cell types. Transcriptional regulation by IFN can be achieved in
several ways: binding of single STAT to a single cis
element, cooperative binding of STATs to several adjacent consensus
sites, and interaction of STATs with other nuclear factors. In this
report, the role of a single GAS element was established through the
analysis of the GAS-deficient promoter in a transient transfection
assay. EMSA subsequently identified STAT-1 to be the GAS-binding
factor. More compelling was the observation that the GAS element in the
hMCP-1 promoter was occupied in vivo after IFN- treatment.
Unexpectedly, IFN--induced protection was also found at the GC box,
a sequence constitutively involved in basal transcription of many
TATA-less "housekeeping" genes. EMSA identified Sp-1 as a potential
GC box-binding factor that was abundant in nuclei of resting and
IFN--stimulated cells. In vivo binding activities at both GAS and GC
box peaked 30 min after IFN- treatment and declined at 5 h. The
observation that IFN- promotes factor binding at the GC box of the
hMCP-1 gene is novel and suggests that this event is pertinent for gene
expression. Further support for this interpretation came from
disrupting the GC site in the context of the intact hMCP-1
promoter-reporter; in transient transfection assays, this GC-deficient
construct was inducible by IFN- but was expressed at >50% reduced
efficiency. These observations raise the possibility that there are
functional interactions between the GAS and GC box in the hMCP-1 gene.
Because the best established DNA-binding factor in the IFN-
signaling pathway is composed of STAT-1 homodimers, one potential
mechanism of such an interaction would suggest that STAT-1 binding
may perturb DNA and render the GC site accessible to Sp-1 binding.
Alternatively, STAT-1 could facilitate recruitment of Sp-1 through
direct protein-protein interaction.
Functional interaction between GAS and GC-rich element in
IFN- induction of hMCP-1
Our data suggest in two respects that functional interaction
between the GAS element and the GC box for IFN--inducible hMCP-1
transcription in astrocytoma cells may be indirect: first, the promoter
is insensitive to the spacing between the GAS and the GC box; second,
coimmunoprecipitation experiments failed to show an association of Sp-1
and STAT-1 in IFN--activated nuclear extracts.
A direct protein-protein interaction between STAT-1 and Sp-1 was
proposed to mediate IFN- activation of the ICAM-1 gene promoter (12)
in which the GAS element is 7 nucleotides upstream of GC box. Arguing
somewhat against this interpretation, the trans-activation
domain of Sp-1 was found to be sufficient to mediate a functional
interaction with STAT-1 and stimulate ICAM-1 transcription (12).
Most importantly, disruption of either the GAS or the Sp-1 site in the
ICAM-1 promoter had similar effects and completely disabled the
promoter. This result stands in direct contrast to the consequences of
corresponding mutations in hMCP-1 gene promoter (Fig. 9). These
disparate results suggest that detailed mechanisms of GAS/GC box
cooperativity may be governed in large part by spacing between cognate
binding sites.
We observed a discrepancy between EMSA and IVGF for analyzing factor
binding to GC box of the hMCP-1 promoter, since Sp-1 binding to the GC
box was constitutive in vitro, but protection of the GC element was
IFN--inducible in vivo. Such an observation is not without precedent
in studies of GC box function. In particular, the myeloid-specific
CD11b gene promoter contains a GC element that bound Sp-1 in vitro in
extracts prepared from multiple cell types. In vivo, the CD11b GC box
was protected only in myeloid cells that expressed the gene, and not in
HeLa cells in which the gene was silent (43). A compatible observation
was made in studying the transformation-dependent expression of the
TGF- promoter. These investigators found that Sp-1 mRNA and protein
were present at similar levels in normal and transformed cells that
expressed TGF- at markedly different levels. Occupancy of potential
Sp-1-binding GC-rich sites occurred only in cells actively transcribing
TGF- (44). The putative interaction between GAS and GC-rich elements
of the hMCP-1 gene in the IFN- response is likely not a universal
phenomenon: in the IFN regulatory factor-1 promoter, in which a GAS
element is 56 bp upstream of GC box, IFN--induced in vivo protection
at GAS was demonstrated, while occupancy of the GC box was
constitutive (45).
It is shown in this report by site-directed mutagenesis that the sequence (5'-CCCGCC) of the GC box is required for optimal hMCP-1 promoter activity. In vivo footprinting revealed an Sp-1 binding footprint that covered a region longer than the previously described consensus binding site. Similar observations were made in studies of the CD11b promoter (43). No DMS protection at GC box on the coding strand was observed; this pattern is consistent with binding in vivo of Sp-1, a zinc finger protein that binds in the major groove of B-DNA and contacts principally the guanine-rich strand of the binding site (46). As we note above, however, IVGF does not address the nature of DNA-bound components.
Sp-1 is universally expressed and has long been thought to regulate
basal levels of transcription, primarily for housekeeping genes.
However, recent evidence suggests that its activity can be modulated
through regulation of its level of expression (47), binding affinity
(48), and posttranslational modifications such as phosphorylation and
glycosylation (49, 50), to confer gene or tissue specificity. The
present study is the first to document IFN--inducible protection of
a potential Sp-1 binding site in vivo and suggests a possible role for
Sp-1 in this induction event. hMCP-1 gene expression is up-regulated in
multiple pathologic and physiologic states of the CNS and PNS,
presumably through distinct cis elements and
trans-acting factors (21). It will be interesting in the
future to determine whether interactions between cytokine-regulated
cis elements and the GC-rich motif provide a general
mechanism by which transcription of hMCP-1 is up-regulated by multiple
independent stimuli.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard M. Ransohoff, Department of Neurosciences, Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.
3 Abbreviations used in this paper: JAK, Janus protein tyrosine kinase; MCP, monocyte chemoattractant protein; hMCP, human MCP; mMCP, murine MCP; GAS, IFN--activated site; Sp-1, nuclear factor Sp1; GC box, GC-rich sequence; TRE, 12-o-tetradecanoylphorbol 13-acetate-response element; Ap-1, activator protein-1; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; IVGF, in vivo genomic footprinting. CNS, central nervous system; DMS, dimethyl sulfate.
Received for publication September 8, 1997. Accepted for publication December 19, 1997.
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