From the Rheumatology Division, Beth Israel Deaconess
Medical Center and New England Baptist Bone & Joint Institute, Harvard
Institutes of Medicine, Boston, Massachusetts 02115 and the
§ Department of Orthopaedic Surgery, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, September 25, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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Following induction and activation of the early
growth response (Egr)-1 transcription factor in human chondrocytes,
interleukin-1 Type II collagen, encoded by the COL2A1 gene, is the
major collagen of the extracellular matrix of mature articular
cartilage. Together with the other cartilage-specific type IX and XI
collagens, the highly cross-linked fibrils of triple helical type II
collagen molecules form a fibrillar network that confers tensile
strength to articular surfaces (1). Chondrocytes comprise the single cellular component of adult hyaline cartilage and are responsive to a
number of growth factors and cytokines that either stimulate or inhibit
type II collagen synthesis (2). Interleukin-1
(IL-1)1 is an inflammatory
cytokine that plays a critical role in cartilage destruction in
arthritis. IL-1 The early growth response (Egr)-1 transcription factor (also known as
Tis8, Krox24, NHFI-A, and Zif268) is a serum-inducible zinc
finger protein that is a critical upstream regulator of proliferation, differentiation, and apoptosis (7-10). Egr-1 gene expression is rapidly induced by mitogens, hypoxia, shear stress, or mechanical injury in fibroblasts, endothelial cells, and other cell types via
mitogen- and stress-activated protein kinases (see Ref. 11 for review).
These protein kinases, including ERK, JNK, and p38 MAPK, may also
modulate the phosphorylation state of Egr-1 and its ability to bind DNA
and other transcription factors. Egr-1 contains both transactivation
and repression domains and regulates gene transcription either
positively or negatively depending upon other DNA binding sites near
the Egr-1 site (12). In human fibrosarcoma and glioblastoma cells,
Egr-1 directly transactivates the genes encoding fibronectin,
transforming growth factor (TGF)- Induction of Egr-1 mRNA by IL-1 Structural and functional analyses of type II collagen genes from
different species have revealed multiple potential regulatory elements
within both the promoter and first intron regions (29-36). These
regions may interact with positive or negative transcription factors
that determine developmental stage- and tissue-specific expression
during chondrogenesis (37, 38). Sp1 binding motifs were among the first
identified in type II collagen genes (29, 35, 39), and recent work has
shown that Sp3 represses Sp1-mediated transactivation of the promoter
activity (40). E-box sites, which are consensus binding sites for basic
helix-loop-helix proteins, are present in both promoter and enhancer
regions and have been proposed as important regulators of type II
collagen gene expression during chondrocyte differentiation (32, 41,
42). A conserved E-box site (CAGGTG) in the promoter also interacts
with the zinc finger homeodomain protein, In our previous studies, we showed that IL-1 Cell Culture--
The immortalized human chondrocyte cell line
C-28/I2 (20) was cultured in Dulbecco's modified Eagle's medium
(DMEM)/Ham's F12 (1/1, v/v; Invitrogen) containing 10% fetal calf
serum (Biowhitaker) and passaged using trypsin-EDTA solution
(Invitrogen) at >95% confluency every 5-6 days. For experiments,
confluent cultures were changed to medium containing 1% Nutridoma-SP
(Roche Applied Science) for 24 h prior to incubation in the
absence and presence of IL-1 RNA Extraction and Analysis--
Total RNA was isolated by a
one-step extraction procedure using the TRIzol reagent (Invitrogen),
and 0.9 µg was reverse-transcribed in 20 µl containing final
concentrations of 2.4 IU/µl of MuLv Reverse Transcriptase (RT), 2.5 µM of oligo d(T)16, and 1 unit/µl of RNase
inhibitor (all obtained from PerkinElmer Life Sciences), as
described previously (51). The primers for human Egr-1 were 5'-
GCTGCAGATCTCTGACCCGTTCG-3' (sense) and 5'-GCCGCTGGAGATGGTGCTGAG-3' (antisense) derived from the mRNA sequence
(GenBankTM accession X52541). The primers for
COL2A1 and glyceraldehyde-3-phosphate dehydrogenase were as
described previously (51). The PCR amplification mixture contained 5 µl of the RT product in a final volume of 50 µl containing 1 mM MgCl2, 200 µM dNTPs, 0.2 µM of each sense and antisense primers, and 2.5 units of
Taq DNA polymerase (Promega). Semiquantitative PCR was
performed at 30-35 cycles such that all products could be assayed in
the exponential phase of the amplification curve in the MJR Research
PTC-200 Peltier thermal cycler. Following an initial step at 95 °C
for 2 min, each cycle consisted of 30 s of denaturation at
95 °C, 30 s of annealing at 60 °C (COL2A1) or
55 °C (Egr-1), and 30 s of extension at 72 °C, with a final extension at 72 °C for 7 min. The PCR products, 30 µl of PCR
reaction per well, were separated on 1.5% agarose gels.
Western Blot Analysis--
Equivalent amounts of protein (20 µg) in nuclear extracts were resolved on 7.5% SDS-polyacrylamide
gels, electroblotted on 0.45-µm immobilon-P membranes (Millipore),
and immunoblotted with anti-Egr-1 rabbit polyclonal antibody (1:200
dilution; from Santa Cruz Biotechnology, Inc.) for 2 h at room
temperature. Blots were then incubated with horseradish
peroxidase-conjugated anti-rabbit secondary antibody (1:2000 dilution)
for 1 h at room temperature and the Egr-1/antibody complexes were
visualized by chemiluminescence according to the manufacturer's
recommendations (Amersham Biosciences).
Electrophoretic Mobility Shift Assays (EMSA) and Supershift
Analysis--
For preparation of nuclear extracts, C-28/I2 cells were
passaged and grown to confluence, changed to medium containing 1% Nutridoma-SP overnight, and treated with IL-1 Plasmid Constructions and Mutagenesis--
For preparation of
COL2A1 reporter constructs the region spanning
The expression vectors used for cotransfections were pCMV-Egr1wt,
pCMV-Egr1
Expression vectors were constructed to generate recombinant and fusion
proteins for analysis of binding to specific DNA sequences by EMSA. PCR
products for Gal4 (1-95) DBD and the Gal4 (1-95)/Egr1 (1-337) were
made using pCMV-Gal4-Egr1 as template, the forward primer,
5'-gaaatggggaagctactgtcttctat-3', and the reverse primers, 5'-tgttaacaatgcttttatatcctg-3' for Gal4 and
5'-gctctagactaaggggcgttcgtggggggcgtc-3' for Gal4-Egr1 and ligated into
the pEF6/V5-His-TOPO vector (Invitrogen). The PCR product for Sp3 was
made using pCMV-Sp3 as template, the forward primer
5'-gaaatggccggggcccccgccgccgccgg-3', and the reverse primer
5'-ctccattgtctcatttccagaaactgtg-3' and ligated into pCR2.1 (Invitrogen). Proteins were produced by in vitro translation
using the TNT Quick-coupled Transcription/Translation
System (Promega). The Gal4-CBP expression vector was provided by A. E.
Goldfeld (Center for Blood Research, Harvard Medical School) with the
permission of Dr. D. Thanos (Columbia University).
The sequences of all constructs were confirmed by DNA sequencing
performed at the Beth Israel Deaconess Medical Center DNA sequencing
facility using ABI PRISM® BigDyeTM primer cycle sequencing
kit (Applied Biosystems, Foster City, CA) and the Automatic DNA
Sequencer Model 373A (Applied Biosystems).
Transient Transfections and Luciferase Assays--
Transient
transfection experiments were carried out in C-28/I2 cells using
LipofectAMINE PLUSTM Reagent (Invitrogen). Cells were seeded in 6-well
tissue culture plates at 3.5 × 105 cells/well in
DMEM/F12 containing 10% fetal calf serum 24 h prior to
transfection. For each well, COL2A1-luciferase construct
(maximum of 1 µg of DNA), 6 µl of PLUS reagent and 92 µl of
serum-free DMEM/F12 were mixed and incubated for 15 min at room
temperature. LipofectAMINE+ reagent (4 µl) in 100 µl of serum-free
medium was then added to each reaction mixture, and incubation was
continued for an additional 30 min at room temperature. Finally, the
transfection mixture was combined with 800 µl of serum-free medium
and the lipid-nucleic acid complex was transferred to the washed cell monolayer in each well. After incubation for 4 h at 37 °C, the transfection mixture was diluted with an equal volume of DMEM/F12 containing 2% Nutridoma-SP, IL-1 Time Course of IL-1
Constitutive levels of COL2A1 mRNA increased with time
throughout the 24 h time course, whereas suppression by IL-1
The increase in Egr-1 mRNA levels in response to cycloheximide
alone may be explained by its capacity to activate stress pathways and
act as nuclear signaling agonist to induce the expression of immediate
early genes such as c-Fos and c-Jun mRNA. Thus, we compared the
action of emetine, a protein synthesis inhibitor that does not act as a
signaling agonist (60). As shown in Fig. 2B, emetine alone,
similar to cycloheximide was able to increase Egr-1 mRNA as early
as 15 min and to cause a "superinduction" in the presence of
IL-1 Time Course of IL-1 IL-1
To characterize the critical sequences required for Egr-1 binding, we
performed mutation analysis using double-stranded oligonucleotides containing mutations within (m1 = CtaGGGCGGGCGG) and
outside (m2 = CGGGGGCGGCta) the Egr-1 core-binding
site within the Analysis of Binding Activities of Sp1 Family Members in Nuclear
Extracts from C-28/I2 Cells--
EMSA competition and
supershift experiments using a labeled Sp1 consensus probe confirmed
the presence of Sp1 family members in nuclear extracts from C-28/I2
cells (Fig. 5A). The formation of at least 3 complexes of different mobilities was consistent with the
expected binding patterns of Sp1 family members and identification was
aided by supershift analysis. The Sp1 antibody produced a supershift of
at least one of the bands. The Sp2 and Sp4 antibodies did not produce
discernible supershifts, but decreased the intensities of all of the
bands in this region, especially that of the upper band that was also
recognized by the Sp1 and Sp3 antibodies. The Sp3 antibody produced a
strong supershift and ablated the binding of the three specific
complexes. Addition of the Egr-1 antibody to the binding reaction did
not alter the pattern of binding to the Sp1 probe. However, the excess
unlabeled Egr-1 consensus sharpened or decreased the intensities of the
three Sp1/3-specific bands, suggesting that Egr-1 may be able to
interact nonspecifically with the G-rich Sp1 consensus under the
conditions of the in vitro assay. The Sp1 consensus, which
is recognized by all family members, competed completely for the
binding of all three of the specific complexes (Fig.
5A).
The pattern of binding of nuclear factors to the labeled Mutation Analysis of Egr-1 and Sp1 Binding Sites in the
Functional Analysis of Egr-1 and Sp1 Sites in the COL2A1 Promoter
Region in Transient Transfections--
To determine the functional
consequences of mutations in the Egr-1 and Sp1 binding sites, mutations
were generated in the COL2A1 promoter construct,
pGL2B
The m4 and m5 mutants were the only competitors that were able to
dissociate the binding of Egr-1 and Sp1 to the
Despite the consistencies in the binding and functional analyses of the
mutations within the Overexpression of Egr-1, Sp1, and Sp3 Modulates COL2A1 Promoter
Activity and the IL-1
To determine the roles of the different Egr-1 domains, Egr1
To further substantiate the role of the Egr-1 DBD, cotransfections were
performed using expression vectors encoding the Egr-1 zinc finger
region alone (pEF6-Egr1ZnR1) or with the C-terminal protein interaction
region (pEF6-Egr1ZnR2) (see Fig. 8A). Overexpression of
Egr1ZnR1 had no effect on constitutive activity but reversed the
inhibition by IL-1
To further explore the sequences required for the Egr-1-mediated
IL-1
Analysis of the responses of the m4 and m5 constructs dissociated
responses to Egr-1 and Sp1/3 expression vectors. The m4 reporter did
not respond to the wild type or dominant-negative Egr-1 expression
vectors, whereas the Sp1 expression vector increased m4 activity, but
only by 20%, and Sp3 had no effect. The dominant-negative effects of
Egr1 The Gal4-Egr-1 Chimera Acts as a Transcriptional Activator of
pGL2-COL2/Gal4 and Interferes with Binding of Sp1 or Sp3 to
the Adjacent Site; Reversal of IL-1
Since pGL2-COL2/Gal4 was constructed with the Gal4 site adjacent to
rather than overlapping the Sp1 site as in the wild type promoter, we
performed EMSA analysis of the Gal4-substituted COL2A1 sequence to determine whether Gal4-Egr1 could influence binding of Sp1
or Sp3 to the adjacent site. The results in Fig. 9D show that recombinant Sp1 and Sp3, as well as the fusion protein Gal4-Egr-1, are all capable of binding independently to the COL2/Gal4 sequence. When increasing amounts of recombinant Gal4-Egr1 were added, the binding of constant amounts Sp1 (left panel) or Sp3 alone or
together with Sp1 (right panel) was decreased and
disappeared. However, when the ratio of Gal4-Egr1 to Sp1 or Sp3 was
increased to 2:1 or greater, the binding of Sp1 or Sp3 was displaced.
Recombinant Egr-1 alone did not bind to this sequence (data not shown).
These results suggest that binding of Gal4-DBD with the attached Egr-1 activation domain is able to interfere with Sp1 or Sp3 binding to the
immediately adjacent site.
We showed previously that IL-1 The COL2A1 proximal promoter region studied here is
particularly GC-rich and contains binding sites for zinc finger
factors, such as Sp1, E box/bHLH proteins, and cKrox, which may
positively or negatively regulate constitutive expression in a
cell-type specific manner (39, 42, 43). In this study, we identified two binding sites for another zinc finger protein, Egr-1, one at
Our experiments indicate that a reduction in COL2A1
transcription is associated with increased binding of Egr-1 to the
Our functional analysis of the The cotransfection data further indicate that IL-1 Since residual Sp3 binding activity persists in IL-1 Our finding that the chimeric Gal4-Egr1 protein is capable of
activating the IL-1 The enhanced sensitivity of differentiated chondrocytes to inflammatory
cytokines suggests that indirect regulation of promoter activity may
occur through suppression of chondrocyte-specific constitutive factors
such as Sox9 that bind to the intronic enhancer. A recent report
indicates that IL-1 It has been proposed that Sp1 binding to sites in the COL2A1
core promoter and first intron enhancer regions may result in the
formation of a DNA loop that could mediate protein-protein interactions
at a distance (29, 35). Although Sp1 is also required for constitutive
promoter activities of type I collagen genes (66), whether there are
functional Egr-1 binding sites in those promoters has not been studied.
Thus, in the context of the COL2A1 promoter the role of
Egr-1 as a transcriptional suppressor via interference with Sp1 may be
unique among the interstitial collagen genes. Recent reports suggest
that Sp1 may act in concert with Sox9 and cKrox, another zinc finger
protein and positive regulator of COL2A1 expression that
binds to GC-rich sites overlapping with the Egr-1 sites studied here
(40, 43). The role of the coactivator CBP/p300 in transcriptional
activation by Egr-1 (11, 71), as well as by some SOX proteins (72), has
been established. IL-1 (IL-1
) suppresses the expression of the type II
collagen gene (COL2A1), associated with induction of Egr-1
binding activity in nuclear extracts. The COL2A1 proximal
promoter contains overlapping binding sites for Egr-1 and Sp1 family
members at
119/
112 bp and
81/
74 bp. Mutations that block
binding of Sp1 and Sp3 to either site markedly reduce constitutive
expression of the core promoter. IL-1
-induced Egr-1 binds strongly
to the
119/
112 bp site, and mutations that block Egr-1 binding
prevent inhibition by IL-1
. Cotransfection with pCMV-Egr1
potentiates the inhibition of COL2A1 promoter activity by
IL-1
, whereas overexpression of dominant-negative Egr-1 mutant,
Wilm's tumor-1 (WT1)/Egr1, Sp1, or Sp3 reverses the inhibition by
IL-1
. Cotransfection of pGL2-COL2/Gal4, in which we substituted the
critical residue for Egr-1 binding with a Gal4 binding domain and a
pCMV-Gal4-Egr1 chimera permits an inhibitory response to IL-1
that is reversed by overexpression of Gal4-CBP. Our results indicate
that IL-1
-induced activation of Egr-1 binding is required for
inhibition of COL2A1 proximal promoter activity and
suggest that Egr-1 acts as a repressor of a constitutively expressed
collagen gene by preventing interactions between Sp1 and the general
transcriptional machinery.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
is known to induce or activate members of the
NF-
B, Jun/Fos, C-EBP, and ETS families of transcription factors,
which may then induce or modulate the expression of MMPs, COX-2, and
other genes involved in inflammatory and destructive processes (3).
IL-1
also contributes to cartilage depletion by suppressing the
expression of cartilage-specific genes, including types II, IX, and XI
collagens and aggrecan (4-6).
1, and plasminogen activator-1 (13,
14). During vascular remodeling, Egr-1 is involved in transactivation
of multiple genes including platelet-derived growth factor A and B,
tissue factor, TGF-
, and membrane type 1 matrix metalloproteinase
(11, 15, 16). Less commonly, Egr-1 may be involved in transcriptional
repression of genes such as the TGF-
type II receptor (TGF-
RII),
which has a role in restraining vascular repair (17).
, tumor necrosis factor
(TNF)-
, oncostatin M, ionizing, or UV radiation, retinoic acid, and prostaglandin E2 (PGE2) has been demonstrated
in a variety of cell types, including dermal and synovial fibroblasts,
osteoblasts, and chondrocytes (18-25). A recent study identified Egr-1
as a critical transcription factor involved in the induction of CD44 by
IL-1
in human endothelial cells (26). Paradoxically, given the role
of cytokines in cartilage destruction, Egr-1 is differentially expressed at lower levels in osteoarthritic than in normal adult human
articular cartilage (27). In contrast, Egr-1 is overexpressed in
rheumatoid synovium, which is characterized by increased cell proliferation and expression of high levels of inflammatory cytokines (28).
EF1, which represses
constitutive activity of the rat Col2a1 promoter (42). The
zinc finger protein, cKrox, activates Col2a1 transcription
in differentiated chondrocytes but inhibits constitutive activity in
subcultured cells via the
266 bp promoter (43). Sox9, the first
transcription factor to specify the chondrogenic lineage, activates
type II collagen gene transcription by binding to the first intron
enhancer through its high mobility group (HMG) DNA binding domain and
acts cooperatively with L-Sox5 and Sox6 to regulate chondrogenesis
in vivo (44, 45). It has been proposed that down-regulation
of Sox9 expression by IL-1 or up-regulation by fibroblast growth
factors or bone morphogenetic proteins (BMPs) determines the regulation
of type II collagen gene transcription by these cytokines (44, 46, 47).
The homeobox protein, Dlx-2, which is stimulated by BMP-2, also acts
via the intronic enhancer to increase Col2a1 expression (48).
decreases the levels of
type II collagen mRNA by inhibiting COL2A1 gene
transcription in chondrocytes in a manner that did not require the
intronic enhancer (49). We also showed that IL-1
induces the
expression of immediate early gene mRNAs encoding c-Fos, c-Jun, Jun
B, and Egr-1 preceding down-regulation of COL2A1 mRNA
levels and up-regulation of matrix metalloproteinase mRNAs in human
chondrocytes (20). Recently, we reported that the COL2A1
core promoter, which is highly GC-rich and contains a TATA box,
expresses constitutively in chondrocytes and responds to negative
regulation by IFN-
(50). In the present study, we have identified
two potential Egr-1 sites within the proximal COL2A1
promoter spanning
131 to +125 bp that overlap with functional Sp1
binding sites. Since Egr-1 has been shown to regulate the activities of
certain promoters by displacing Sp1 from overlapping binding motifs
(11, 15), we sought to determine whether Egr-1 could interact
functionally with the COL2A1 promoter and account partially
for the inhibitory effect of IL-1
. Our results indicate that Egr-1,
when activated by IL-1
, functions as a transcriptional repressor of
COL2A1 promoter activity in cells that constitutively
express this chondrocyte-specific gene and acts through displacement of
Sp1 family members from at least one of the overlapping binding sites
in the proximal promoter.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
at 200-500 pg/ml for the times indicated.
for 0.5, 1, 4, 14, and
18 h. The cells were lysed in hypotonic buffer with Nonidet P-40
at a final concentration of 0.5%, as described (52). Nuclear proteins
were extracted in buffer C according to the modified method of Dignam
et al. (53), diluted with low salt buffer D, and used
directly for analysis of binding to DNA. Double-stranded synthetic DNA
oligonucleotides were purchased from Operon Technologies, Inc.
(Alameda, CA) and end-labeled using T4 polynucleotide kinase and
[
-32P]dATP. Binding reactions were carried out for 30 min at room temperature using 5 µg of nuclear extract and 0.8 pmol
(~10,000 cpm) of labeled probe in a final volume of 20 µl
containing 12 mM HEPES-KOH (pH 7.9), 0.94 mM
EDTA, 4.65 mM MgCl2, 50 mM KCl, 0.85 mM dithiothreitol, 12.5% glycerol, 0.5 mg/ml bovine
serum albumin, and 1.25 µg of poly(dI-dC). The protein-DNA complexes were separated in low ionic strength 4% polyacrylamide gels using Tris/glycine-EDTA buffer (TGE) or Tris borate-EDTA buffer (TBE, 45, mM Tris borate, pH 8.3, and 1 mM EDTA), as
indicated, and autoradiographed. The wild-type and mutant
COL2A1 promoter sequences spanning
141 to
102 bp (wt1)
and
93/
62 bp (wt2), listed in Fig. 1, and the consensus and mutant
Egr-1 and Sp1 oligonucleotides (Santa Cruz Biotechnology) were used as
labeled probes and as competitors at 50-fold excess. For supershift
analysis, antibodies specific for Egr-1, Egr-2, Sp1, Sp2, Sp3, and Sp4
(Santa Cruz Biotechnology) were incubated with the binding reaction
mixture for 30 min at room temperature before electrophoresis.
577/+3426
bp from pCAT/B4.0 (29, 49) was cloned into the pGL2-Basic luciferase
vector (Promega). The pGL2B
577/+125 construct was prepared by enzyme
digestion of pGL2B4.0 using SmaI and PstI to
remove the +126/+3426 bp fragment followed by religation. The deletion
construct containing
131/+125 bp was prepared by enzyme digestion of
pGL2B
577/+125 using HindIII (cloning site in vector) and
ApaI, followed by ligation. The pGL2B
131/+125 construct
was used as a template to generate point mutations by PCR mutagenesis
employing the QuikChangeTM site-directed mutagenesis kit (Stratagene,
La Jolla, CA). The pGL2-COL2/Gal4 reporter vector was constructed using
pGL2B
131/+125 as template, the primers 5'-gccctccgaggggcgggcggttcaggttac-3' (forward) and
5'-tgtcacccgcggagccccgcctgggccctgc-3' (reverse), to
substitute the Gal4 DNA binding domain (DBD, underlined) (54) in place
of the Egr-1 site (see Fig. 1), and the
ExSite PCR-based site-directed mutagenesis kit (Stratagene). Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).
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Fig. 1.
Structure and sequence of the proximal region
of the COL2A1 promoter. A, the
overlapping Egr-1/Sp1 sites, identified by MatInspector
(www.gsf.de/biodv/matinspector.html) and TESS
(www.cbil.upenn.edu/tess), and previously identified E-box and
TATA-box sites are represented schematically. The positions of
nucleotides upstream of the transcription start site (+1) are indicated
above. B, the wild-type sequences, wt1
( 141/
102 bp) and wt2 (
93/
62 bp), the mutants
(m1-m9), and the sequences containing the consensus and
mutant Egr-1 sites and the Sp1 consensus site used in this study are
listed below. The Egr-1 sites are underlined and
the overlapping Sp1 sites are double-underlined.
mt, from which amino acids 331-374 containing the entire
first zinc finger and part of the second were removed, and the
dominant-negative pCMV-WT1-Egr1 chimera, containing the Wilm's tumor 1 (WT1) activation domain and the Egr-1 zinc finger DBD (kind gifts from
Dr. Vikas P. Sukhatme, Beth Israel Deaconess Medical Center, Boston,
MA) (55). The expression vectors for Sp1 and Sp3 were obtained,
respectively, from Dr. J. Madri (Yale University, New Haven, CT) (56)
and Dr. G. Suske (Marburg, Germany) (57). The pCMV-Gal4-Egr1 expression
vector, containing the Gal4 (amino acids 1-95) DBD and the Egr-1
(amino acids 1-337) activation domain, was made by PCR using
pCMV-Egr1wt as template and the primers
5'-cgggatccatggccgcggccaaggccgagatgc-3' (forward) and 5'-gctctagactaagggcgttcgtgggggggcgtc-3' (reverse),
which created, respectively, BamH1 and XbaI sites
(underlined). The PCR products were purified by the Concert Rapid PCR
purification system (Invitrogen), digested with BamH1 and
XbaI, and ligated into pCMV-Gal4. The pEF6-Egr1ZnR1 (amino
acids 337-427) and pEF6-Egr1ZnR2 (amino acids 337-533) expression
vectors were generated using pCMV-Egr1wt as template, the forward
primer, 5'-aagatggcgcccccccacgaacgcccttacgc-3', and the reverse
primers, 5'-tttgtctgctttcttgtccttc-3' for Egr1ZnR1 and
5'-gcaaatttcaattgtcctgg-3' for Egr1ZnR2. The PCR products were ligated
into the pEF6/V5-His-TOPO vector (Invitrogen).
was added 2 h later, and
incubation was continued for 18 h. For cotransfections, the
expression vectors and the empty vectors were first titrated at amounts
ranging from 10 to 200 ng per well; 100 ng was found to be optimal,
whereas 200 ng produced nonspecific effects because of the CMV
promoter. After cotransfection using 750 ng of
COL2A1-luciferase vector and 50 or 100 ng of expression
vector per well, the cells were incubated for 24 h to permit
expression of recombinant proteins prior to treatment with IL-1
for
a further 18 h. Cell lysates were prepared by extraction with 200 µl of Reporter Lysis Buffer (Promega), and the protein content was
determined using the Coomassie Plus Protein Assay Reagent (Pierce
Chemical Company). Luciferase activities were determined by
chemiluminescence assay using the Autolumat LB953 luminometer (EG&G
Berthold, Oak Ridge, TN), normalized to the amount of protein, and
expressed as relative activities against that of untreated
pGL2B-131/+125 in each experiment. Each experiment was repeated at
least three times, and each data point was calculated as the mean of
the results (3-6 wells/experiment) ± S.D. The relative
activities of the wild type and mutant constructs were checked by the
Dual-Luciferase Reporter Assay (Promega) using 750 ng of
COL2A1-luciferase vector and 20 ng of the pRL-TK
Renilla luciferase control vector. The levels of luciferase
activity expressed as RLU/µg of protein ranged from ~1 × 103 to 1 × 105 for COL2A1
promoter activity in untreated C-28/I2 cells.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced Expression of Egr-1 mRNA and
Inhibition of COL2A1 mRNA in Human Chondrocytes--
The C-28/I2
human chondrocyte cell line was selected as a reproducible model for
this study because it expresses chondrocyte-specific matrix proteins,
including type II collagen and aggrecan and responds to IL-1
, as
described previously (20, 58, 59). We first verified that IL-1
stimulates Egr-1 mRNA levels and suppresses COL2A1
mRNA levels in the C-28/I2 cells, as we had reported previously in
primary and immortalized chondrocyte cultures (4, 49). The C-28/I2
cells were preincubated in serum-free medium for 24 h prior to the
addition of IL-1
for periods of time between 15 min and 24 h
(Fig. 2A). Compared with
untreated controls at each time point, IL-1
increased the levels of
Egr-1 mRNA within 15 min, with peak induction by 1 h, and the
IL-1
-stimulated levels declined thereafter. Incubation with the
protein synthesis inhibitor cycloheximide increased the levels of Egr-1
mRNA in either the absence or presence of IL-1
and stabilized
expression beyond 4 h. The apparent increases in Egr-1 mRNA
levels in the untreated controls at each time point were not observed
in every experiment, but could be due to a stress response after 24-48
h in serum-free medium. Higher constitutive levels of Egr-1 mRNA
were observed on Northern blots in a previous study when a medium
change was done at the time of addition of IL-1
(20)
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Fig. 2.
Time course of
IL-1 -induced expression of Egr-1 mRNA in
immortalized human chondrocytes. The C-28/I2 cells were plated in
serum-containing medium and cultured for 5 days, and confluent
cultures were changed to serum-free medium containing 1% Nutridoma
24 h before treatment with IL-1
for the times indicated.
A, cycloheximide (10 µg/ml) or B, emetine (10 µg/ml) was added in the absence (
) or presence (+) of IL-1
.
Total RNA was extracted using TRIzol and Egr-1, COL2A1, and
glyceraldehyde-3-phosphate dehydrogenase mRNA levels were analyzed
by semiquantitative RT-PCR. DNA ladders are shown in the left
lane for each mRNA. PCR products are 500, 359, and 346 bp for
Egr-1, COL2A1, and glyceraldehyde-3-phosphate dehydrogenase,
respectively.
was observed as early as 4 h (Fig. 2A). Cycloheximide
appeared to prevent this inhibition at the earlier time points,
although it had no effect on COL2A1 mRNA levels by
24 h in either the absence or the presence of IL-1
. These
results are consistent with our previous studies, in which
COL2A1 mRNA was found to have a half-life of 17.5 h, and inhibition by IL-1
was ascribed to decreased gene
transcription but not to decreased COL2A1 mRNA stability
(20, 49).
. In this experiment, unlike that shown in Fig. 2A,
constitutive expression of Egr-1 was evident at 15 min and declined
after 1 h; thus, the up-regulation by IL-1
was not evident
until the 1-h time point. This result suggests that the immortalized
cells may constitutively express early growth response genes because
they are in a constant proliferative state and supports the idea that a
stress response may occur in these cells after serum deprivation. The
sustained up-regulation of Egr-1 mRNA by emetine was remarkably
similar to that in the presence of cycloheximide and indicates that
both agents are acting as translational inhibitors in this cellular context.
-induced Expression of Egr-1 Protein and
Egr-1 Binding Activity in Human Chondrocytes--
To determine whether
IL-1
treatment of chondrocytes could also induce activation of
Egr-1, we examined DNA binding activity to the Egr-1 consensus
sequence. Nuclear extracts were prepared after incubation of the
C-28/I2 cells with IL-1
for times between 15 min and 18 h, and
Western blotting analysis was performed using a polyclonal antibody
against Egr-1. As shown in Fig.
3A, Egr-1 protein was present
in untreated nuclear extracts and IL-1
increased the level of Egr-1
protein (82 kDa) by 15 min, which peaked by 1 h and remained
stable up to 18 h. When these nuclear extracts were analyzed in
the EMSA shown in Fig. 3B, binding to the Egr-1 consensus
oligonucleotide appeared within 15 min after addition of IL-1
,
peaked by 1 h, and began to decline by 4 h. Furthermore, the
IL-1
-induced binding activity was supershifted by the Egr-1 antibody, but not by the Egr-2 antibody (Fig. 3C). These
findings indicate that IL-1
increases both the levels of Egr-1
protein and its activation manifested by increased binding to the Egr-1 consensus DNA element.
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Fig. 3.
Time course of Egr-1 protein expression in
IL-1 -treated chondrocytes and activation of
binding to the Egr-1 consensus sequence. Nuclear extracts were
prepared after incubation of C-28/I2 cells in the absence (0) or
presence of IL-1
for the times indicated. A, equivalent
amounts of total protein were fractionated on 7.5% polyacrylamide
Tris-glycine/EDTA gels, transferred to nitrocellulose and probed with
Egr-1 antibody. B, the double-stranded oligonucleotide
containing the Egr-1 consensus site was end-labeled and incubated with
the nuclear extracts shown in A. C, nuclear
extracts from cells incubated in the absence (0) or presence of IL-1
for 1 h were incubated with the Egr-1 consensus (cons)
probe and binding of Egr-1 was identified by supershift analysis using
the Egr-1 antibody (E1). Note that the Egr-2 antibody
(E2) did not produce a supershift.
Increases Binding of Egr-1 to COL2A1 Proximal Promoter
Elements--
Computer-assisted analysis of the proximal region of the
COL2A1 promoter identified two potential binding sites for
Egr-1, one at position
119 to
112 bp
(CCGGGGGCGGGCGGGCGG) and the other at position
81 to
74
bp (CTGGGGGCAGGGGGCGG), both of which also contain
previously identified Sp1 core binding sites (see Fig. 1) (29). In
initial screens, the fragment spanning
93 to
62 bp bound an
Egr-1-like factor in IL-1
-treated nuclear extracts, but less
strongly than the
141/
102 bp fragment (data not shown). As shown in
Fig. 4 (left panel), nuclear
factors from IL-1
-treated cells formed a major complex with the
labeled Egr-1 consensus sequence that disappeared in the presence of
excess unlabeled self-competitor or the wild-type COL2A1
fragment (
141/
102 bp). When the labeled
141/
102-bp
oligonucleotide was used as probe, a more complex pattern
of binding could be observed. IL-1
-induced binding activity appeared
that was ablated by competition with either the Egr-1 consensus or the
141/
102 bp sequence (Fig. 4, right panel). The identity
of the Egr-1 binding activity was further verified by the supershifts
with Egr-1 antibody on both probes. However, upon competition with the
self-fragment (wt) on the
141/
102 bp probe, several additional
bands disappeared that may be attributable to other factors that
recognize GC-rich sequences, such as Sp1 family members (see below),
cKrox, or Ap2.
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Fig. 4.
Competition and supershift analysis of
nuclear protein binding to the Egr-1 consensus and COL2A1
( 141/
102 bp) promoter sequences. End-labeled probes
containing the Egr-1 consensus (left panel) and
141/
102
bp of COL2A1 (right panel) were incubated with
nuclear extracts from untreated (
) or IL-1
-treated (+) C-28/I2
cells in the absence or presence of 50× excess unlabeled competitor
oligonucleotides, including Egr-1 consensus (cons),
COL2A1 wild-type (wt), or mutants (m) 1, 2, or 3 (see sequences in Fig. 1). The identity of the Egr-1 complex was
confirmed by supershift analysis using a specific antibody
(Ab) against Egr-1 (open arrows).
141/
102 bp fragment, and at both sites (m3 = CtaGGGCGGCta) (see Fig. 1). As shown in Fig. 4, excess
unlabeled m1 and m3 oligonucleotides, which contained the GG to TA
mutation in the Egr-1 site, did not compete for the IL-1
-induced
binding activity attributable to Egr-1 on either the Egr-1 consensus or
the
141/
102-bp sequence. In contrast, the m2 oligonucleotide with a
mutation outside the Egr-1 site produced competition approximately
equivalent to wild type competitor except for a binding activity that
overlapped with one of the nonspecific bands. These results indicate
that the Egr-1 binding activity on the COL2A1 probe migrates
closely with at least one other specific binding activity.
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Fig. 5.
Supershift analysis of DNA binding of Sp1
family members in nuclear extracts from C-28/I2 cells.
A, end-labeled consensus Sp1 oligonucleotide was incubated
with nuclear extracts from IL-1 -treated (+) C-28/I2 cells.
Supershift analysis was performed using antibodies against Egr-1, Sp1,
Sp2, Sp3, or Sp4. Competition analysis with 50× molar excess of Egr-1
or Sp1 consensus sequence confirmed the specificity of binding of the
Sp1 family members. B, the COL2A1 (
141/
102
bp) probe was incubated with nuclear extracts from untreated (
) or
IL-1
-treated (+) C-28/I2 cells. Incubation with antibodies against
Egr-1, Sp1, Sp2, Sp3, or Sp4, decreased specific binding activities
(upper panel) and produced supershifts that are better
observed on the lower panel.
141/
102 bp
COL2A1 probe was more complex than that observed using the
Sp1 or Egr-1 probe (Fig. 5B). The binding activities of Sp1 family members were identified by their relative mobilities compared with those on the Sp1 probe in Fig. 5A and by the loss of
these bands in the presence of antibodies. The major band for Egr-1 (82 kDa) was very close to one of the bands attributable to Sp3 (90 kDa)
(Fig. 5B, upper panel). This may explain our
inability to cleanly dissociate Egr-1 binding on the
141/
102 bp
probe. The presence of Egr-1 binding activity in untreated nuclear
extracts may represent some degree of autoactivation during storage or handling during the assay, since the same nuclear extracts were used at
an earlier time for the experiment in Fig. 4. In the presence of
extracts from IL-1
-treated cells, however, the intensity of the
Egr-1 supershift increased, whereas the Sp1 and Sp3 supershifts decreased, as visualized on an overexposed autoradiograph (Fig. 5B, lower panel). This loss of binding was
probe-specific, since IL-1
did not decrease the amount of Sp1 and
Sp3 recognized by the Sp1 probe (Fig. 5A). Taken together,
these results suggest that IL-1
-induced Egr-1 may interfere with the
binding of Sp1 family members, to the overlapping site in the
141/
102 bp sequence, and that the extent of binding may depend upon
the balance between Egr-1 and Sp1 family members in the nuclear extracts.
141/
102 bp COL2A1 Promoter Fragment--
To dissociate
the binding site for Egr-1 from that for Sp1 family members on the
141/
102 bp fragment, we compared nuclear binding activities on the
Egr-1 and Sp1 consensus probes using a series of mutant
oligonucleotides as competitors. Since the m1 mutation in the Egr-1
binding site (see Fig. 4) might also be expected to affect binding to
the overlapping Sp1 site (underlined), we used additional mutant
oligonucleotides: m4, atGGGGCGGGCGG; m5,
CGGGGGCGatCGG; m6, aGGGGGCGGGCGG; and m7,
CGaGGGCGGGCGG (see Fig. 1). The capacity of each mutant
probe to bind nuclear proteins from IL-1
-treated C-28/I2 cells was
assessed by competition assays using Sp1 or Egr-1 as probe. As shown in
Fig. 6, A and B, m1
and m3 bound neither Sp1 nor Egr-1, whereas m2 and m6 bound both. In
contrast, m4 bound Sp1 but not Egr-1, whereas m5 bound Egr-1 but not
Sp1. However, m7 prevented Sp1 binding, but was a partial competitor
against Egr-1 binding. Comparison of m4 and m6 indicates that the G at
position
119 bp is critical for binding of Egr-1. Comparison of m5
and m7 indicates that the G nucleotides at positions
118 bp and
112
bp are critical for binding of Sp1. EMSA analysis using the
141/
102
bp probe showed similar patterns of competition and confirmed the
identities of the binding activities (Fig. 6C). Egr-1
binding activity was present only in the IL-1
-treated nuclear extracts in this experiment, whereas Sp1-like binding was present in
untreated cells but increased after IL-1
treatment. Thus, these
results lend further support to the notion that both Sp1 family members
and activated Egr-1 bind to this overlapping site but that the extent
of binding depends upon the relative concentrations of the different
factors.
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Fig. 6.
Mutation analysis of binding of nuclear
proteins to the Sp1 and Egr-1 consensus sequences using wild type and
mutant COL2A1 sequences. End-labeled
oligonucleotides containing (A) the Sp1 consensus,
(B) the Egr-1 consensus, and (C)
COL2A1 ( 141/
102 bp) sequences were incubated with
nuclear extracts from untreated (
) or IL-1
-treated (+) C-28/I2
cells. Unlabeled competitor oligonucleotides containing mutations
(m1-7, see Fig. 1) in the
141/
102 bp COL2A1
promoter fragment were added at 50× molar excess, as indicated.
131/+125, and transfections were performed in the C-28/I2 cells.
IL-1
treatment suppressed the activity of the wild-type construct by
40-60% (Fig. 7A). The m1 mutation in the major overlapping Sp1/Egr-1 site decreased constitutive activity of the
131-bp promoter by ~20%, while the extent of inhibition by IL-1
was decreased to 10-35% compared with the untreated mutant promoter (Fig. 7A). The m2 and m3 mutations
were not studied, since m2 bound both Egr-1 and Sp1, and m3, similar to
m1, bound neither (see Figs. 4 and 6). The m4 mutation in the Egr-1
binding site had no effect on constitutive activity, while the response
to IL-1
was reduced (Fig. 7A). In contrast, the m5
mutation, which mutated the Sp1 binding site, decreased constitutive activity, but did not prevent the inhibition by IL-1
. The m6 mutation, which permitted binding of both factors, had no effect on
constitutive promoter activity, nor did it affect the IL-1
response.
In contrast, the m7 mutation, which did not bind Sp1 but partially
blocked Egr-1 binding, decreased constitutive activity to a similar
extent as m5 and reduced the inhibition by IL-1
to around 25% (Fig.
7A).
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Fig. 7.
Functional analysis of Egr-1 and Sp1 sites in
the COL2A1 promoter in transient transfections.
A, C-28/I2 cells constructs were transfected with the wild
type pGL2B 131/+125 or mutant construct (m1-m9, see Fig.
1) and IL-1
was added for 18 h prior to harvest for luciferase
assay. The results are expressed as the means ± S.D. derived from
3 (m1, m7-m9) or 6 (m4-m6) separate
transfection experiments, each done in triplicate. B,
double-stranded oligonucleotides containing mutations m4 and m5 and wt1
(
141/
102 bp) were end-labeled and incubated alone (0) or with
nuclear extracts from IL-1
-treated C-28/I2 cells. Note that the wt1
competitor added at 50× molar excess prevented the specific binding of
nuclear proteins. C, end-labeled Egr-1 and Sp1 consensus
oligonucleotides were incubated with nuclear extracts from
IL-1
-treated C-28/I2 cells in the absence (
) or presence of
unlabeled wt2 (
93/
62 bp), m8, or m9 competitors at 50× molar
excess.
141/
102 bp sequence,
as well as to distinguish constitutive activity and the IL-1
response in transient transfections. We, therefore, compared the
binding of nuclear factors to labeled m4 and m5 oligonucleotides to
determine whether they were indeed able to bind Sp1 and Egr-1, respectively. In the EMSA analysis shown in Fig. 7B, binding
activities consistent with Sp1 family members were observed on the m4
probe, whereas m5 bound Egr-1 only, consistent with the competition
analysis shown in Fig. 6. These results also suggest that the loss of
Egr-1 binding activity may account for the lack of IL-1
response by the m4 construct and that loss of Sp1 binding at the m5 site may decrease constitutive activity.
141/
102 bp site, it was of interest to examine
the other potential Egr-1/Sp1 site at
83/
73 bp. Transient
expression analysis showed that COL2A1 promoter constructs
containing deletions at either
(
121/
108) or
(
83/
73) expressed at 20-50% of the level of wild type constructs (data not
shown), suggesting that both regions are required for full constitutive
activity of the COL2A1 promoter. Deletion of the intervening
sequence
(
98/
88) also decreased constitutive activity (not
shown), possibly because it shortens the distance between the two
Sp1-binding regions or because it contains a nonconserved E-box site
(CAGCTG at
92/
87 bp). To determine the contribution of the
downstream site at
81/
74 bp to either constitutive activity or the
IL-1
response, we examined the consequences of the m8 and m9
mutations (see Fig. 1), which analogous to m4 and m7 were predicted to
affect Egr-1 and Sp1 binding, respectively. However, both mutations
decreased constitutive activity and the response to IL-1
to a
similar extent (Fig. 7A). These functional results were
consistent with the EMSA analyses using nuclear extracts, in which both
m8 and m9 were ineffective competitors against Sp1/3 and Egr-1 binding
to consensus probes (Fig. 7C), as well as the binding of
recombinant Egr-1, Sp1, or Sp3 to the COL2A1 (
93/
62 bp)
probe (data not shown). These findings may also account for the failure
of mutations in the upstream Egr-1/Sp1 site to completely block
constitutive activity or the response to IL-1
, since the
83/
73
bp site would still be available for binding to Egr-1 and Sp1.
Response in Cotransfection Assays--
To
determine whether overexpression of recombinant Egr-1, Sp1, and Sp3
proteins could modify constitutive COL2A1 promoter activity
or the response to IL-1
, cotransfection experiments were performed
using the expression vectors shown in Fig.
8A. The expression vectors
driven by the CMV promoter and the empty vector (pCMV) were first
titrated at amounts ranging from 10 to 200 ng per well to determine the
optimal amount that would avoid nonspecific effects. In the experiments
shown in Fig. 8, the C-28/I2 cells were cotransfected with
pGL2B
131/+125 and 50 ng of expression vector. Cotransfection with 50 ng of pCMV-Egr1wt did not change constitutive activity of the
COL2A1 promoter, but the inhibitory effect of IL-1
was
potentiated compared with cotransfection with the empty vector (Fig.
8B). These results suggest that IL-1
is required to
activate the overexpressed Egr-1 protein.
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Fig. 8.
Effects of Egr-1, Sp1, and Sp3 expression
vectors on COL2A1 promoter activity and
IL-1 responses in cotransfection assays.
A, wild type and truncated Egr-1 proteins and the WT1-Egr1
fusion protein expressed by the vectors used in this study are shown
schematically. B, C-28/I2 cells were cotransfected with
pGL2B
131/+125 and the empty vector, pCMV, or the expression vector
for Egr1wt, Egr1(
331-374), WT1-Egr1, Sp1, or Sp3 at 50 ng of vector
per well. C, cotransfections were performed with
pGL2B
131/+125 and 100 ng of pEF6 (empty vector), pEF6-Egr1ZnR1, or
pEF6-Egr1ZnR2. Cotransfections were performed with the mutant
constructs pGL2B-131/m1 (D), pGL2B-131/m4 (E),
and pGL2B-131/m5 (F) together with the Egr1wt,
Egr1(
331-374), WT1-Egr1, Sp1, and Sp3 expression vectors. After
transfections, the cells were incubated for 24 h prior to further
incubation for 18 h in the absence (solid bars) or
presence (hatched bars) of IL-1
, followed by luciferase
assay. The results show the means ± S.D. of triplicate wells from
one representative of 5 (B, C) or 3 (D-F) separate transfection experiments.
mt, and
WT1-Egr-1 were overexpressed and found to have no effect on
constitutive activity of pGL2B
131/+125. The pCMVEgr1
mt vector, containing the transactivation domain and one-half of the zinc fingers
in the DBD, reversed completely the inhibitory effect of IL-1
(Fig.
8B). The pCMV-WT1-Egr-1 vector, a chimera of the WT1
activation domain and the entire Egr-1 zinc finger DBD, which acts in a
dominant-negative manner against Egr-1 activity in other systems (55),
also reversed the suppression by IL-1
without affecting constitutive
activity (Fig. 8B). In contrast, overexpression of Sp1
increased constitutive activity of the
131-bp promoter by 1.5-fold.
Overexpression of Sp3 had no effect on constitutive activity in this
experiment, although we frequently observed inhibition of up to 50%.
However, overexpression of Sp1 or Sp3 reversed the inhibitory effect of
IL-1
. These results confirm the suggestion from the binding data in
Fig. 5 that IL-1
-induced Egr-1 may decrease constitutive activity by
competing with endogenous Sp1 family members, and further suggest that
increasing the ratio of Sp1 or Sp3 to Egr-1 may attenuate the action of
IL-1
-induced Egr-1.
. In contrast, overexpression of Egr1ZnR2 decreased constitutive activity by 30%, and almost completely reversed
the inhibition by IL-1
(Fig. 8C). These results confirm that binding of Egr-1 to the COL2A1 core promoter is
essential for the inhibitory effect of IL-1
.
response, we performed similar cotransfections using the mutant
reporters, pGL2-131/m1 (Fig. 8D), pGL2-131/m4 (Fig. 8E), and pGL2-131/m5 (Fig. 8F). Overexpression
of the Egr1wt, Egr1-
mt, or WT1-Egr1 expression vector down-regulated
the activity of the m1 construct to a similar extent, around 25%, and
the extent of inhibition by IL-1
remained similar to that in
presence of the empty pCMV vector. In contrast, overexpression of Sp1
increased m1 activity by 2.5-fold, whereas addition of IL-1
decreased the Sp1-stimulated expression to the pCMV control level. Sp3,
on the other hand decreased m1 activity to a slightly greater extent than Egr1wt either in the absence or presence of IL-1
.
mt and WT1-Egr1 suggest that the 24-30% inhibition by IL-1
in the presence of empty vector, Sp1, or Sp3 may be mediated via the
downstream Egr-1/Sp1 site. On the other hand, in the absence of the
upstream Sp1/3 binding site, the wild type and mutant Egr-1 expression
vectors seemed to be permissive for increased constitutive m5 activity,
and WT1-Egr1 had the expected dominant-negative effect on the IL-1
response. Sp1 only slightly increased m5 activity, Sp3 had no apparent
effect, but both Sp1 and Sp3 reversed the inhibitory effect of IL-1
.
Although difficult to interpret, the combined results using the m1, m4,
and m5 promoters suggest that in the absence of the binding site for
Egr-1 or Sp1/3 in the
141/
102 bp region, the binding sites in the
92/
62 bp region may take over the functional responses to Sp1 and
Sp3. However, the absence of regulation by the Egr-1 wild type and
mutant expression vectors is consistent with the weak Egr-1 binding
activity in the
92/
62 bp region and suggests that the IL-1
response is also mediated by other regulators that interact directly or
indirectly with the COL2A1 proximal promoter.
-induced Inhibition by
CBP--
To determine the nature of the interaction between Egr-1 and
Sp1, the Egr-1 binding site was "knocked out" by substituting the
critical G for Egr-1 binding at
119 bp in the pGL2B(
131/+125) construct with 18 bp of nucleotides encompassing a Gal4 binding site
(Fig. 9A). We also constructed
an expression vector for a chimeric protein containing the Gal4 (1-95)
DBD and the Egr-1 activation domain (1-337) (Fig. 9A).
Neither the empty pCMV vector nor pCMV-Gal4 had any effect on
pGL2-COL2-Gal4 activity in either the absence or presence of IL-1
(Fig. 9B). Thus, binding of Gal4 to the substituted Gal4
binding site was, by itself, not sufficient to induce promoter
activity. However, overexpression of the chimeric Gal4-Egr1 protein
increased pGL2-COL2-Gal4 activity in a dose-dependent manner (Fig. 9B), but to a level that was 25% of the
activity of the wild-type pGL2-COL2-131/+125 reporter. Furthermore,
IL-1
inhibited this expression by more than 50% (Fig.
9B). These results suggest that the presence of the adjacent
Sp1-binding site is not sufficient for full activation of this
artificial promoter, which also contains the downstream Sp1 site at
81/
74 bp. Furthermore, Egr-1 (amino acids 1-337) appears to serve
as an activator in this context, but requires induction by IL-1
to
produce an inhibitory response. This is similar to the response of the
m5 promoter (Fig. 8F), where Egr-1 is an apparent activator
in the absence of the Sp1 site. Additional support for the validity of
this mechanism is provided by the experiment in Fig. 9C, in
which cotransfection with the CBP expression plasmid reversed the
IL-1
-induced inhibitory response.
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Fig. 9.
The Gal4-Egr-1 chimera acts as a
transcriptional activator of pGL2-COL2/Gal4 and interferes with binding
of Sp1 or Sp3 to the adjacent site. A, pCMV-Gal4-Egr1
expression vector contains the Gal4 DBD fused to the Egr-1 activation
domain (1-337). The pGL2-COL2/Gal4 sequence contains the 131/+125 bp
promoter with a Gal4 binding sequence (underlined) inserted
in place of the nucleotide at
119 bp and adjacent to the Sp1 site
(double-underlined). B, C-28/I2 cells were
cotransfected with the pGL2-COL2/Gal4 reporter and pCMV (50 ng),
pCMV-Gal4 (50 ng), or pCMV-Gal4-Egr1 at 25-150 ng, as indicated. Note
that cotransfection with 25, 100, or 150 ng of pCMV-Gal4 produced
activity similar to that shown for 50 ng of this vector. C,
cotransfections were performed using 25 ng of each expression plasmid,
including the vector expressing full-length CBP fused to the Gal4 DBD
(Gal4/CBP). (D) For EMSA analysis, the COL2/Gal4
oligonucleotide shown in A was labeled and incubated alone
(0) or with the Gal4-Egr1 fusion protein at 0.5 µg (+) or increasing
amounts (left panel, 0.5, 1, 1.5, 2, 2.5 µg, or
right panel, 0.5, 1, 1.5 µg). In vitro
translated Sp1 was added at 0.5 µg (left panel) or 1 µg
(right panel) and Sp3 was added at 1 µg (right
panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
inhibits COL2A1
mRNA expression and synthesis of type II collagen, resulting in
accelerated dedifferentiation and loss of phenotype in primary cultures
of human chondrocytes (4). Treatment of chondrocytes with IL-1
also
increases the levels of the immediate early gene mRNAs, including c-Fos, c-Jun, and Jun B, as well as Egr-1, preceding down-regulation of
COL2A1 mRNA (20). However, binding sites directly
responsive to nuclear protein complexes containing Fos/Jun proteins or
other IL-1
-induced transcription factors have not been identified
previously in the COL2A1 promoter. Our present results show
that Egr-1 mediates, in part, the inhibition by IL-1
of expression
of the proximal COL2A1 promoter by interacting with an
overlapping binding site for Sp1 family members and thereby interfering
with constitutive gene transcription. Egr-1 is coregulated with c-Fos
during skeletal development in the perichondrial interface and
interstitial cells of opposing cartilaginous elements and at sites
destined for endochondral ossification (61). However, the absence of
skeletal phenotype in Egr-1 knockout mice indicates that this
transcription factor is not essential for normal cartilage and bone
development (62). Thus, these findings are relevant primarily to
chondrocyte function in adult cartilage. Similar to other immediate
early genes, Egr-1 is induced by serum and acts primarily as a
transcriptional inducer of gene expression. Certain growth factors
present in serum may suppress the expression of
COL2A1-reporter gene activity (49). Thus, the ability of
Egr-1 to repress transcription in the context of the strongly expressed
COL2A1 promoter may reflect decreased expression of this
matrix gene observed in proliferating chondrocytes that are undergoing
dedifferentiation in vitro.
119/
112 bp and the other at
81/
74 bp, both of which also contain the previously identified Sp1 core motifs (29, 39, 40). Many
previous studies have shown that Egr-1 can activate gene transcription
by displacing Sp1 in promoters where Sp1 serves as a weaker activator.
However, interaction of Egr-1 with an Ets-like site was shown to serve
a negative regulatory function in the TGF
RII promoter
(17). Mechanisms by which Egr-1 could suppress transcription include
direct repression of promoter activity via DNA binding (12, 17) or
"squelching" transcription through interactions with Sp1,
independent of Sp1 binding to DNA (63).
119/
112 bp site and displacement of Sp1, rather than binding of
these factors to adjacent sites in a noncompetitive manner. The extent of binding depends upon the balance of Egr-1 and Sp1 family members in
the nuclear extracts. The results of the mutation analyses indicate
that the Egr-1 and Sp1 binding sites overlap and encompass the core
Sp1-binding site, GGGGCG. Egr-1 binding requires the sequence,
GGGGGCG, with the critical G at the 5'-end. Both 5' and 3'
nucleotides are critical for Sp1 binding, since mutation to GGGGGCGat
(m5) or to GaGGGCGGG (m7) prevents competition for binding to the Sp1
consensus. The interaction of Egr-1 with the overlapping Sp1 site is
consistent with known binding activities of these zinc finger
transcription factors (11, 64).
131/+125 bp promoter supports the
findings of the binding studies, which indicate that Egr-1 binding to
the overlapping site at
119/
112 bp mediates IL-1
-induced suppression of promoter activity and that Sp1 binding is important for
constitutive expression. In fact, all mutations except m4 and m6
decrease constitutive activity, corresponding with the loss of Sp1
binding activity. The results of experiments using m4 and m5 as
competitors, where the binding activities of Sp1 and Egr-1 are
dissociated, also correlate with the functional data. The constitutive
activity of
131/+125-bp promoter is sensitive to the m5 mutation,
which does not permit Sp1 binding, whereas the IL-1
response is
sensitive to the m4 mutation that blocks Egr-1 binding. Furthermore,
the m7 reporter construct is less responsive to IL-1
possibly
because it binds Egr-1 less strongly. Thus, in the absence of elements
upstream of
131 bp, binding of Egr-1 to the
119/
112-bp site is
required for the IL-1
response. However, both the m8 and m9
mutations in the downstream site at
81/
74 bp, although they are
analogous to m4 and m5, respectively, decrease both Egr-1 and Sp-1
binding and their functional activities are similar. Thus, the binding
of Sp1 to the downstream site at
81/
74 bp is required for full
constitutive activity of the proximal promoter, but the upstream site
at
119/
112 is required for the full response to IL-1
, as also
suggested by the relative nonresponsiveness of the m4 promoter to Egr-1
overexpression (Fig. 8E). The different responses of the
mutant promoters to Sp3 overexpression indicate that this Sp1 family
member may act as a repressor, especially in the absence of Egr-1
binding to the upstream site.
-induced Egr-1 can
suppress COL2A1 transcription when overexpressed in chondrocytes. This effect requires activation of the overexpressed Egr-1, since suppression of
131/+125 bp promoter activity is not
observed in the absence of IL-1
. Additional support for the involvement of Egr-1 as a transcriptional suppressor is provided by the
results showing reversal of the IL-1
-induced suppression by
overexpression of pCMV-WT1-Egr1. This fusion protein, which contains
the Egr-1 zinc finger DBD, has been shown to act in a dominant-negative
manner in other systems (55, 65). The Egr1
mt, with deletion of 1.5 of the zinc fingers, also acts in a dominant-negative manner, possibly
due to its ability to interfere with activation of the endogenous
Egr-1. Neither construct, however, has any effect on constitutive
activity. Further support for the DNA-binding requirement for the
Egr-1-mediated IL-1
response is provided by the results showing
reversal of IL-1
-induced inhibition by overexpression of Egr1ZnR1
and Egr1ZnR2.
-treated nuclear
extracts and overexpression of Sp3 decreases expression of the
131 bp
promoter, Sp3 may play a negative role among the Sp1 family members in
chondrocytes. In contrast, overexpression of Sp1 increases
COL2A1 promoter activity. However, overexpression of either
Sp3 or Sp1 blocks the inhibition by IL-1
. These results suggest that
the Sp1/Sp3 ratio determines the level of constitutive activity of the
COL2A1 promoter, but that increasing the ratio of either Sp1
or Sp3 to Egr-1 may prevent Egr-1 binding. Our results contrast
somewhat with those from previous studies in which Sp1 binding
activities to the COL2A1 promoter either decreased (43) or
increased (39) in dedifferentiated chondrocytes that expressed low
levels of COL2A1. However, our findings agree with those of Ghayor et al. (40), who showed that Sp1 transactivates the
Col2a1 promoter (
266/+121 bp) independent of the
differentiation state of chondrocytes and that Sp3 blocks Sp1-induced
activity. Thus, increasing the ratio of Sp3 to Sp1 may promote
chondrocyte dedifferentiation manifested by decreased
COL2A1 promoter expression. Although the presence of
endogenous Sp1 and Sp3 proteins may obscure the interpretation of the
cotransfection experiments, the lack of any effect after a 6-h
expression time following transfection (data not shown) compared with
the significant changes observed when the expression time is extended
to 24 h prior to IL-1
treatment indicates that the observed
changes are specific. Our findings are consistent with the general
concept that, in mammalian cells, Sp3 is a DNA binding-dependent repressor that may abrogate Sp1-driven
transcription from promoters containing GC boxes by displacing Sp1 and
preventing its interaction with the general transcription machinery
(57). Similarly, Sp1 acts as a transcriptional activator of the type I
collagen gene, COL1A2, whereas Sp3 overexpression blocks the promoter activity induced by TGF-
(66). Constitutive nuclear factor
binding to Sp1 sites in the TGF-
RI promoter, for example, could then
represent an additional level at which the TGF-
response is mediated
(67).
131/+125 bp promoter with the Egr-1 site knocked out
by substitution with a Gal4 binding site, suggests that Egr-1 does not
mediate repression of COL2A1 via direct interaction with Sp1
or Sp3. The Gal4 substitution only removes the upstream G critical for
Egr-1 binding leaving the Sp1 site intact, such that it is possible to
observe binding of recombinant Sp1 and Sp3 proteins in the presence of
the Gal4-Egr1 fusion protein when added at equivalent concentrations.
However, increasing the relative concentration of Gal4-Egr1 to
Sp1and/or Sp3 prevents binding of the latter proteins. Nevertheless,
the Gal4-Egr1 fusion protein is able to mediate an inhibitory response
to IL-1
on the artificial COL2/Gal4 promoter construct, possibly due
to the presence of the transcription activation domain (TAD) of Egr-1.
This model suggests that Egr-1 could serve as an activator of the
COL2A1 promoter by cooperating with Sp1 or Sp3 bound to the
adjacent site, but in the presence of IL-1
, Egr-1 serves as a repressor.
induces many cellular responses via the stress-activated
kinases, p38 MAPK and JNK, which have been shown to modify the
phosphorylation state of preformed Egr-1 protein and activate DNA
binding (11, 68). Induction of Egr-1 promoter activity is also mediated
by these kinases through activation of the ternary complex factor,
Elk-1 (68). On the other hand, casein kinase II, which also mediates
phosphorylation of Sp1, may act as a negative regulator of DNA binding
activity of Egr-1 and also modify protein-protein interactions between
Egr-1 and Sp1 (63, 69). Further work will be required to determine the
stress-activated signaling pathways involved in inhibition of
COL2A1 expression by IL-1
, as suggested for p38 MAP
kinase in our previous study (51).
decreases Sox9 mRNA and protein levels via
stimulation of NF-
B, which then binds to the Sox9 promoter and
inhibits its activity (46). However, the levels of L-Sox5,
Sox6, and Sox9 mRNAs, which are all expressed by the C-28/I2 cells
used in this study (50), are not decreased by IL-1
treatment and the
response of the COL2A1 promoter to IL-1
occurs in the
presence or absence of the intronic
enhancer.2 The early time
course of the response in our study and the lack of dependence on
protein synthesis for the later suppression of COL2A1
mRNA levels also argue against a primary effect via inhibition of
Sox9 expression, although sustained suppression in vivo
would require inhibition of further Sox9 protein synthesis. Similarly, we found that interferon-
causes a rapid decrease in activity of the
proximal COL2A1 promoter without affecting Sox9 expression (50). Thus, our studies are consistent with the roles of Sox9 and
related HMG factors as architectural proteins that function to bend and
unwind DNA and thereby maintain an open chromatin network surrounding
the constitutively active COL2A1 promoter (70).
has been shown to stimulate
CBP-dependent histone H4 acetylase activity associated with
stimulation of GM-CSF promoter activity (73). Our results showing that
CBP overexpression reverses the inhibitory effect of IL-1
suggest
that coactivators such as CBP play a positive role in maintaining
constitutive activity of the COL2A1 promoter. Thus, Egr-1
may prevent interactions among CBP, Sp1 and TATA-binding proteins and
thereby permit transcriptional repression by other IL-1
-induced
factors that bind to upstream promoter sequences. Candidate factors are
C/EBP and ETS family members that are known to be involved in cytokine
regulation of other genes (74-77). Our results support the notion that
there are multiple pathways and transcription factors mediating the effects of IL-1
on COL2A1 gene expression. These findings
provide new insights into a mechanism whereby Egr-1, when activated by IL-1
, can serve as a transcriptional suppressor of a constitutively expressed collagen gene by preventing interaction of positive regulators such as Sp1 with the general transcriptional machinery.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. V. Sukhatme, J. Madri, G. Suske, and A. E. Goldfeld for generously providing expression vectors.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AR45378 and AG22021 (to M. B. G.) and CA68544 (to P. E. A.) and a Biomedical Science Grant from the Arthritis Foundation (to M. B. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Harvard Institutes of Medicine, Room 246, New England Baptist Bone & Joint Institute, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0742; Fax: 617-975-5299; E-mail: mgoldrin@bidmc.harvard.edu.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M301676200
2 R. Yamin, L. Tan, M. Osaki, B. K. Choy, L. J. Sandell, and M. B. Goldring, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: IL-1, interleukin-1; BMP, bone morphogenetic protein; Egr-1, early growth response transcription factor; TGF, transforming growth factor; TNF, tumor necrosis factor; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; DBD, DNA-binding domain; WT1, Wilm's tumor 1; HMG, high mobility group; CBP, CREB-binding protein; CMV, cytomegalovirus.
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