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INTRODUCTION |
Cartilage-derived retinoic acid-sensitive protein
(CD-RAP)1 is a small secreted
protein expressed in cartilage throughout chondrogenesis and in mature
chondrocytes (1). Its physiological expression is primarily restricted
to cartilage with transient expression in mammary buds and salivary
glands (2). Pathologically, CD-RAP and its human homologue, melanoma
inhibitory activity (MIA), are also expressed in various tumor tissues,
such as chondrosarcoma, melanoma, and breast cancer (3-5). CD-RAP was
originally cloned as a protein expressed in cartilage and
down-regulated in chondrocytes that had been de-differentiated by
treatment with retinoic acid (6). The protein structure of CD-RAP/MIA
showed that it contains an Src homology 3 domain, which is unique for
an extracellular protein. CD-RAP/MIA can interact with the type III
repeat of fibronectin suggesting a model whereby CD-RAP/MIA binds to
fibronectin and interferes with integrin binding (7). A recent study of
CD-RAP-deficient mice showed that the mice have normal expression and
distribution of type II collagen, aggrecan, and type X collagen but
have increased density, increased diameter, and irregular arrangement
of collagen fibrils (8). Thus, CD-RAP may function to organize highly
ordered ultrastructural fiber architecture. CD-RAP is regulated by
various cytokines or growth factors; insulin and insulin-like growth
factor I stimulate and basic fibroblast growth factor and interleukin 1
(IL-1) suppress CD-RAP expression in bovine articular
chondrocytes (9).
IL-1 is one of the major cytokines that mediate inflammatory
reactions. IL-1 promotes the arachidonic acid cascade resulting in
production of prostaglandin (10). In joint diseases, such as rheumatoid
arthritis and potentially osteoarthritis, IL-1 is thought to
contribute to degradation of matrix proteins not only by the production
of proteases such as matrix metalloproteinases but also by
down-regulation of expression of the matrix proteins such as types II,
IX, and XI collagen and aggrecan, resulting in the loss of the
cartilage structure and prevention of repair (10-13). Because CD-RAP
is thought to be required for formation of the highly ordered
ultrastructural fiber architecture (8), down-regulation of CD-RAP gene
may contribute further to loss of cartilage integrity.
To date, little is known about the mechanism of IL-1-induced
repression of matrix proteins. IL-1 induces or activates many transcription factors, such as nuclear factor-
B (NF-B) (14), Fos/Jun, early growth response-1 (15, 16), and CCAAT/enhancer-binding protein (C/EBP) families in chondrocytes (17). However, direct functions of these factors with respect to repression of matrix protein
gene transcription have not been clearly determined. C/EBP is a family
of basic leucine-zipper transcription factors with six known family
members of C/EBPs as follows: C/EBP
, -, -
, -
, -
, and
-
. Among these, C/EBP and - are known to be activated by
IL-1 and TNF- at the mRNA level (18-22) and protein level (for by phosphorylation and intracellular translocation) (23-28). In response to these proinflammatory cytokines, C/EBP and/or -
activate various genes related to inflammation, such as phospholipase A2 and cyclooxygenase-2 and manganese superoxide dismutase
(17, 20, 21, 29). C/EBP also regulates matrix proteins, such as
pro-1 and -2 type I collagen, matrix Gla protein, and osteocalcin (22, 30-32). C/EBP has two major isoforms, liver-enriched activator protein (LAP) and liver-enriched inhibitory protein (LIP). LAP is
generally considered to be an activator, whereas LIP, which lacks most
of trans-activation domain of LAP, can act as a
dominant-negative inhibitor (18, 33). Thus, C/EBP can directly
activate or inhibit the target gene.
Here, we investigated the factors responsible for down-regulation of
the CD-RAP gene by IL-1, and we show that C/EBP plays a key role in
this regulation. Furthermore, we show that C/EBP can down-regulate type
II collagen gene transcription, thus suggesting that C/EBP might play
an important role in down-regulation of various matrix proteins induced
by IL-1. We provide the first demonstration that C/EBP
down-regulates the expression of a cartilage-specific matrix-associated
gene and that it is a novel pathway mediating repressive effects of
IL-1.
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EXPERIMENTAL PROCEDURES |
Materials--
The materials used in this work were purchased as
follows: -modified Eagle's minimum medium (MEM), Dulbecco's
modified Eagle's medium (DMEM), DMEM/Ham's F-12 medium, and
Taq polymerase from Invitrogen; fetal bovine serum from
HyClone Laboratories, Inc., Logan, UT; penicillin/streptomycin
solution, dexamethasone, and Nu-Clear Extraction KitTM from
Sigma; Human IL-1 from R & D Systems, Inc., Minneapolis, MN; RNeasy
Mini KitTM from Qiagen, Inc., Valencia, CA;
NorthernMaxTM Northern blot kit from Ambion, Inc., Austin,
TX; Hybond-NTM and -CTM membrane,
[-32P]dATP, and [-32P]dATP from
Amersham Biosciences; FuGENE 6TM Transfection Reagent,
collagenase P, Quick SpinTM-Sephadex G-50 and G-25 column
from Roche Molecular Biochemicals; restriction enzymes, pGL3-basic
vector, Reporter Lysis BufferTM, Luciferase Assay
ReagentTM, oligo(dT)15 and avian
myeloblastosis virus-reverse transcriptase from Promega, Madison, WI;
QuickChangeTM Site-directed Mutanogenesis Kit from
Stratagene, La Jolla, CA; Express GelTM from ISC
BioExpress, Kaysville, UT; anti-C/EBP, C/EBP or Ikaros antibodies
from Santa Cruz Biotechnology, Santa Cruz, CA;
SuperSignalTM West Pico chemiluminescent substrate from
Pierce; non-fat dry milk from Bio-Rad; Pronase from Calbiochem;
SYBRTM Green PCR Master Mix from Applied Biosystems, Foster
City, CA.
Plasmid Constructs--
The CD-RAP promoter 5'-deletion
constructs were made by PCR and subcloned into pGL3-basic vector, as
described (34). Mutagenesis of the C/EBP-binding site in the 
2251-bp
promoter was performed by PCR using the Site-directed Mutagenesis
KitTM according to the manufacturer's instructions. Human
Sox9 expression vector was described before (35). Human Col2A1 promoter
construct and C/EBP expression vectors were the kind gifts provided by
the following: 4-kb promoter and enhancer construct spanning 577 to
+3426 bp of human COL2A1 in the pGL2-basic vector was obtained from Dr.
Mary B. Goldring (36, 37); human C/EBP-full-length in the pCDNA3
vector was from Dr. Erika Crouch (38); the rat pCI-neo-LIP was from Dr.
Cynthia A. Zahnow (39); the rat pCMV-LAP was from Dr. Mina Bissell
(40); and the rat pMSV-C/EBP was from Dr. Alan Friedman (41). The
empty expression vectors were made by excision of cDNAs from the
corresponding C/EBP expression vectors. All the plasmids were confirmed
by sequencing or digestion with restriction enzymes.
Cell Cultures--
Rat chondrogenic cell line, RCJ 3.1C5.18, was
maintained in MEM supplemented with 10% FBS, 10 nM
dexamethasone, and 2% penicillin/streptomycin, as described before
(34). Rat chondrosarcoma (RCS) cells were cultured in DMEM with 10%
FBS and 2% penicillin/streptomycin (34). For RNA or nuclear protein
extraction, RCS cells were plated in 2 × 104/cm2 densities and cultured overnight. Human
IL-1 was then added to the medium at the concentrations indicated.
Primary chondrocyte culture was obtained from human articular
cartilage. Full thickness slices of human cartilage were dissected from
the normal knee joints of donors under aseptic conditions and subjected
to sequential Pronase and collagenase P digestion to liberate
chondrocytes from tissues. Isolate chondrocytes were plated at a
density of 2 × 104/cm2 and cultured in
DMEM/F-12 containing 10% FBS and 2% penicillin/streptomycin for 2 days. IL-1 was then added at a 2 ng/ml concentration, and cells were
cultured for 24 or 48 h as indicated.
Northern Blot--
Total RNA was isolated from cultured cells
using Qiagen RNeasy mini kitTM, and 20 µg of total
RNA/lane were separated on a 1.5% agarose gel and transferred to
Hybond-NTM nylon membrane. The membranes were
pre-hybridized for 5 h and then hybridized at 50 °C overnight
in UltrahybTM hybridization medium with the various probes
labeled with [-32P]dATP by random priming procedure.
To obtain specific probes for C/EBP and -, the sequences that
have low homology with each other were chosen and digested from the
corresponding expression vectors. Probe for CD-RAP was described before
(34). The hybridized membranes were then washed twice with 2× SSC and
0.1% SDS buffer at 50 °C for 15 min and then by twice with 0.1 × SSC and 0.1% SDS buffer in 50 °C for 15 min. The specific
signals from probes were obtained by autoradiography.
Transient Transfection and Luciferase Assay--
DNA
transfections of RCJ3.1C5.18 or RCS cells were performed using FuGENE
6TM transfection reagent. 1 × 105 of
RCJ3.1C5.18 cells or 2.2 × 105 of RCS cells were
cultured on a 12-well plate overnight or for 6 h, respectively.
Transfection mixture containing 3 µl of FuGENE 6TM, 375 ng of various promoter constructs, and 125 ng of pCMV-gal were then
added, and the cells were cultured for 48 h with or without
IL-1 as indicated. For co-transfection assay using C/EBP expression
vector, 500 ng of expression vectors containing empty vector and C/EBP
expression vector in various ratios were added to the transfection
mixture. The cells were then harvested with Reporter Lysis
BufferTM, and the lysate was analyzed for luciferase
activity using Promega Luciferase Assay ReagentTM. The
-galactosidase activities were also measured to normalize variations
in transfection efficiency. Each transfection experiment was performed
in triplicate and repeated at least twice.
Preparation of Nuclear Extracts and Synthesis of Protein in
Vitro--
Nuclear extracts and cytoplasmic protein from RCJ3.1C5.15
or RCS cells were isolated using Nu-Clear Extraction KitTM
according to the manufacturer's instructions. Sox9 and C/EBP-LIP proteins were synthesized using TNT T7 quick-coupled
transcription/translation system with the Sox9 expression vector and
the C/EBP-LIP expression vector, respectively.
Electrophoretic Mobility Shift Assay--
Fragment A (between
2138 and 2037 bp relative to the mouse CD-RAP translation start
site) was amplified by PCR. All oligonucleotides were synthesized by
Invitrogen, and complementary oligonucleotide was annealed to make
double-stranded oligonucleotide. The fragment A or various
double-stranded oligonucleotides were end-labeled using T4
polynucleotide kinase and [-32P]dATP. Bandshifts were
performed by incubating 4 µg of nuclear extracts in the mobility
shift buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM
EDTA, 50 mM NaCl, 0.5 mM dithiothreitol, 1 mM MgCl2, 4% glycerol, 2 µg of poly(dI-dC)
or poly(dG-dC)) with the DNA probe at room temperature for 25 min.
In vitro synthesized proteins were assayed as described
before (35, 42). For the competition studies, the cold DNA fragments
were added at a 100-fold molar excess compared with the probe and
incubated for 15 min at room temperature before adding the DNA
probe. For the antibody interference experiments, the nuclear
extracts and 1 µl of antibody were preincubated in the buffer
for 1 h at 4 °C. The anti-Sox9 antibodies was generous gift from Dr. de Crombrugghe (43). DNA protein complexes were resolved on a 5% polyacrylamide gel at 100 V for several hours. The
gels were dried and autoradiographed.
Western Blot--
Thirty µg of cytoplasmic protein or nuclear
extracts were denatured in SDS sample buffer contains 0.1 M
dithiothreitol at 100 °C for 5 min and separated on a 4-20%
Express GelTM in Tris/glycine/SDS buffer. The gels were
then transferred to Hybond-C extraTM nitrocellulose
membrane in Tris/glycine buffer, pH 8.3, containing 20% methanol. The
membranes were saturated in 7% non-fat dry milk in PBS at room
temperature for 1 h and hybridized with anti-C/EBP antibodies
diluted to 1:2000 in PBS containing 1% dry milk. The hybridized
antibodies were recognized by anti-rabbit IgG antibodies coupled to
horseradish peroxidase, and the secondary antibodies were detected by
autoradiograph using SuperSignalTM peroxidase substrate.
Quantitative Real Time PCR--
Total RNA was isolated from the
primary human articular chondrocytes, and 2 µg of RNA was
reverse-transcribed in the mixture of avian myeloblastosis
virus-reverse transcriptase (1.2 units/µl), oligo(dT)15
(40 ng/µl), dNTP (1 mM each), and RNasin Ribonuclease Inhibitor (1.6units/µl) in 42 °C. Quantitative real time PCR
was performed in the 50-µl reaction mixture containing 25 µl of
SYBRTM Green PCR master mix, 1 µl of RT products, and 300 nM of primers using GeneAmpTM 5700 Sequence
Detection System (PerkinElmer Life Sciences). The primer sequences are
as follows: human GAPDH, 5'-TGGGCTACACTGAGCACCAG-3' (sense) and
5'-GGGTGTCGGTGTTGAAGTCA-3' (antisense); human C/EBP, 5'-CTCGCAGGTCAAGAGCAAGG-3' (sense) and 5'-TCGTCGCTGTGCTTGTCC-3' (antisense); human C/EBP, 5'-ATAGGAGCGCAAAGAAGCTACAG-3' (sense) and
5'-CTCGCAGTTTAGTGGTGGTAAGTC-3' (antisense); human CD-RAP, 5'-CCTGGCAAAGTCGATGTGAA-3' (sense) and 5'-TCACTGGCAGTAGAAATCCCATT-3' (antisense); human COL2A1, 5'-TGGCTGGAGGATTTGATGAAA-3' (sense) and
5'-CCTTGCATTACTCCCAACTGG-3' (antisense). The cycle threshold (Ct)
values for GAPDH and those of genes of interest were measured for each
sample, and the relative transcript levels were calculated as
x = 2
Ct, in which 
Ct = IL C and IL = CtIL-1
-CtGAPDH; C = Ctctl CtGAPDH.
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RESULTS |
IL-1-responsive Element Is Located between 2138 and 2068 bp
of the CD-RAP Promoter--
We have shown previously that the
2251-bp CD-RAP promoter contains elements sufficient for
cartilage-specific expression in transgenic mice (2) and that the
expression of CD-RAP is down-regulated by IL-1 in primary
chondrocyte cultures (9). To identify the IL-1-responsive element
within CD-RAP promoter, three 5'-deletion constructs were transiently
transfected into RCS cells and incubated in the absence or presence of
IL-1 (Fig. 1). Regarding basal
activities of the constructs in untreated RCS cells, 2251-bp promoter
was stronger than 2138 bp, and 2138 bp was stronger than 2068 bp,
suggesting there are activating elements between 2251 to 2138 bp
and between 2138 to 2068 bp. The expressions of the 2251- and
2138-bp constructs were down-regulated by the IL-1 treatment,
whereas the response was lost in the 2068 bp, thus suggesting that
the IL-1-responsive element is located between 2138 and 2068
bp.
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Fig. 1.
IL-1-responsive
element is located between 2138 to 2068 bp of the CD-RAP
promoter. Various 5'-deletion constructs of the CD-RAP promoter
were transiently transfected into RCS cells and incubated for a further
48 h in the absence or presence of IL-1 (10 ng/ml). Luciferase
activities were measured and expressed relative to the activity of
promoterless pGL3b (set as 1). Each bar represents the
mean ± S.D.
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IL-1 Stimulates the Expression of C/EBP and
C/EBP in RCS Cells--
The expressions of C/EBP and
- are known to be stimulated by IL-1 (18, 20). As computer
analysis revealed there was a potential binding site for C/EBPs between
2138 and 2068 bp, we examined whether IL-1 stimulates the
expression of C/EBP or C/EBP in RCS cells (Fig.
2, A and B). The
expression levels of C/EBP and C/EBP were very low in steady
state but were stimulated by IL-1 in 4-24 h increasing up to
48 h. The expression of CD-RAP was repressed by 48 h. Western
blots for C/EBP revealed that in the absence of IL-1 treatment, a
very small amount of C/EBP protein exists in the cytoplasm but not
in nuclei (Fig. 2C). The treatment of IL-1 stimulated the
expression of both of the isoforms of C/EBP, LAP (36 kDa), and LIP
(20 kDa) that were localized in the nuclei. Protein expression of
C/EBP was also stimulated by the IL-1 treatment (Fig.
2C).
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Fig. 2.
IL-1 stimulates the expression of C/EBP
and -. A, RCS cells were treated with 10 ng/ml IL-1
for various times as indicated. B, RCS cells were treated
with various concentrations of IL-1 as indicated for 48 h. The
levels of C/EBP, C/EBP, and CD-RAP mRNAs were analyzed by
Northern blot. C, C/EBP and - proteins were examined
by Western blot of cytoplasmic protein (cyto) or nuclear
extracts (N.E.) from RCS cells treated or without IL-1
(10 ng/ml). IL-1 increased both isoforms of C/EBP:LAP (36 kDa)
and LIP (20 kDa) as well as C/EBP in the nuclei.
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C/EBP Binds to the IL-1-responsive Element of the
CD-RAP Promoter--
To determine whether C/EBP functions within
the IL-1-responsive element, EMSA was carried out using nuclear
extract from the RCJ3.1C5.18 cells. These cells were chosen because
C/EBP is expressed in steady state (Fig.
3A). Fragment A containing the
sequence between 2138 and 2037 bp was used as a probe, and various
cold oligonucleotides were used as competitors (Fig. 3B). Competitor 3, which has a potential binding site for C/EBPs at 2085/2077 bp, competed with the binding of nuclear proteins and the
probe (Fig. 3C). As expected, the mutant oligonucleotide 3, in which the C/EBP-binding site was mutated, did not compete with the
binding, and supershift analysis confirmed that C/EBP bound to the
sequence of native oligonucleotide 3 (Fig. 3D). The antibody
for Ikaros, a transcription factor that is also a potent binding
protein for the sequence of oligonucleotide 3, did not affect the band
shift. EMSA for the nuclear extract of RCS cells treated with 10 ng/ml
of IL-1 to induce C/EBP (see Fig. 2C) was also carried
out using the oligonucleotide 3 as a probe to confirm that the
IL-1-induced C/EBP bound to the element (data not shown).
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Fig. 3.
C/EBP binds the
sequence between 2094 and 2069 bp of the CD-RAP promoter.
A, Western blot of nuclear extracts (N.E.) from
RCJ3.1C5.18 cells for C/EBP. C/EBP-LAP (38 and 36 kDa) and LIP
(20 kDa) are expressed in steady state. B, a diagram showing
DNA fragments used for EMSA. Fragment A from 2138 to 2037 bp and
relative locations of oligonucleotides 1-4 are shown. The
oligonucleotide 3 from 2094 to2069 bp contains C/EBP motif
(underlined). mu, mutant oligonucleotide 3 containing two base pair mutations (AA  CC) is underlined
within the C/EBP motif. C, EMSA for RCJ3.1C5.18 nuclear
extracts using fragment A as a probe and various cold competitors at
100-fold molar excess. The oligonucleotide 3 competes with the binding
of nuclear proteins to the probe. D, EMSA and supershift
analysis for RCJ3.1C5.18 nuclear extracts using oligonucleotide 3 as a
probe. Mutant oligonucleotide 3 does not compete with the binding of
nuclear proteins to the probe. Antibodies against C/EBP or Ikaros
were preincubated with the nuclear extracts. Arrow shows
supershift band of the complex of C/EBP, antibody (Ab),
and probe.
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C/EBP Functions as a Repressor for the CD-RAP
Promoter Activities--
The C/EBP-binding site was mutated in the
2251-bp construct using mutant oligonucleotide 3. The promoter
activity of the mutant 2251-bp construct was about 2-fold stronger
than that of wild type 2251-bp construct after transient transfection
into RCJ3.1C5.18 cells, suggesting that C/EBP is acting as a
repressor (Fig. 4). To investigate the
C/EBP function in detail, the expression vectors for C/EBP and
C/EBP were co-transfected with wild type 2251-bp construct into
RCS cells, in which endogenous C/EBP expression is very low (Fig.
5A). Because C/EBP is known
to act in different ways depending on the isoforms, we used the
following three different expression vectors: pCMV-C/EBP-full-length,
pCMV-LAP, and pCMV-LIP. The promoter activity of the 2251-bp
construct was down-regulated in a dose-dependent manner by
all three of the C/EBP expression vectors, no matter what isoform
was expressed. C/EBP also down-regulated the activity of the
2251-bp construct. The presence of each expressed protein was
confirmed by Western blot of lysate after the transfection (Fig.
5B). The C/EBP or empty expression vectors (500 ng) and the 2251-bp construct were also co-transfected into undifferentiated ATDC5 mouse chondrogenic cell line. Interestingly, pCMV-C/EBP-FL and
pCMV-LAP vectors stimulated promoter activity to 3.44 ± 0.57- and
30.2 ± 4.66-fold, respectively, whereas pCMV-LIP did not
(1.03 ± 0.14). These data suggest that the repression induced by
C/EBP expression vectors in RCS cells is neither because of nonspecific reaction nor poor function of these expression vectors.
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Fig. 4.
Mutation of the C/EBP-binding site
up-regulates the promoter activity of the 2251-bp construct.
Site-directed mutagenesis was performed within the C/EBP-binding site
of the 2251-bp construct using the mutant oligonucleotide 3 sequence.
The mutant and wild type 2251-bp constructs were transiently
transfected into RCJ3.1C5.18 cells. The activity of promoterless pGL3b
is set as 1. Each bar represents the mean ± S.D.
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Fig. 5.
The promoter activity of the 2251-bp
construct is down-regulated by co-transfection with C/EBP expression
vectors. A, the 2251-bp construct was
co-transfected into RCS cells with various C/EBP expression vectors.
Expression vectors for C/EBP-full-length (FL),
C/EBP-LAP, C/EBP-LIP, or C/EBP were added to the transfection
mixture as indicated. Total amounts of expression vectors were adjusted
to 500 ng using the empty expression vector in each transfection. The
activity of 2251-bp constructs co-transfected with 500 ng of the
empty expression vector is set at 100. Each bar represents
the mean ± S.D. B, Western blot of the lysate
from the co-transfection assay for C/EBP confirming that the
expression vectors form the protein products.
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Mutagenesis of C/EBP-binding Site Eliminates the
Response to IL-1 Treatment--
To determine whether the
C/EBP-binding site on the CD-RAP promoter was required for the response
to IL-1, the mutant 2251-bp construct, in which the C/EBP-binding
site was mutated, was transfected into RCS cells and incubated in the
absence or presence of IL-1 for a further 48 h. As shown Fig.
6A, the mutant 2251-bp
construct did not respond to IL-1 treatment. Furthermore,
co-transfection of the pCMV-C/EBP-full-length vector did not result in
repression of activity of the mutant 2251-bp construct. These results
indicate that the C/EBP-binding site at 2085/2077 bp of the CD-RAP
promoter is required for the functional response to IL-1 and suggest
that C/EBPs are key factors in this response.
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Fig. 6.
Mutation of the C/EBP-binding site abolishes
the responsiveness of the 2251-bp promoter to IL-1
treatment. A, the mutant 2251-bp
construct, in which the C/EBP-binding site was mutated, was transfected
into RCS cells and incubated with or without IL-1 (10 ng/ml).
B, the mutant 2251-bp construct was co-transfected
into RCS cells with empty or C/EBP-FL expression vector. The promoter
activity of the construct without IL-1 and that co-transfected with
empty vector were set at 100. Each bar represents the
mean ± S.D.
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Sox9 Binds to the Element Overlapping with the C/EBP
Motif--
Because the IL-1-responsive element seems to contain an
activating element as well (see Fig. 1), we hypothesized that C/EBP may
interact with another activator protein conferring an indirect repressive effect. Because both of the flanking sequences of the C/EBP
motif contain an HMG-like motif, the binding of Sox9 to this element
was tested (Fig. 7). EMSA using RCS
nuclear extract revealed the Sox9 binding to the sequence of
oligonucleotide 3 (Fig. 7B). To see the interaction between
Sox9 and C/EBP within this element, EMSA was carried out using Sox9
and C/EBP-LIP protein synthesized by in vitro translation.
The binding of Sox9 protein to the sequence of oligonucleotide 3 was
confirmed by supershift using anti-Sox9 antibody (Fig. 7C).
To screen the binding site, each of two different competitors was
preincubated with the binding reaction mix. The competitor b, which has
the sequence TTCAAAA that is close to the Sox protein motif
(A/T)(A/T)CAA(A/T)G, competed with the Sox9 binding to the probe more
strongly than the competitor a, suggesting Sox9 prefers the motif in b
for its binding (Fig. 7C). When C/EBP and Sox9 proteins
were incubated together with the probe, two major independent bands
were observed to be the same size as Sox9 alone and C/EBP alone (Fig.
7D). The addition of antibodies for either Sox9 or C/EBP
generated supershift of the related band and did not affect the other
band (Fig. 7D). These results indicate that the supershifted
complex did not contain the other protein, suggesting that Sox9 and
C/EBP cannot bind to the probe in the same time probably because the
binding sites partially overlap. Therefore, C/EBP may compete with
Sox9 binding to this element thus eliminating the enhancer effect of
Sox9.
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Fig. 7.
Sox9 and C/EBP bind
to 2094/2069 bp and compete for binding to the probe.
A, the sequences of oligonucleotide 3 and
competitors a and b. The C/EBP
consensus and HMG-like motifs are underlined. B, EMSA
was carried out using oligonucleotide 3 as a probe and RCS nuclear
extract (N.E.). The addition of anti-Sox9 antibody generated
a supershift. The specificity of the anti-Sox9 antibody has been
confirmed before in RCS cell nuclear extract (43) and in
vitro translated protein (35). C, EMSA was performed
using Sox9 protein synthesized by in vitro translation.
Binding of Sox9 was confirmed by supershift using anti-Sox9 antibody
(lanes 1 and 2). Competitor b competes
with the binding of Sox9 to the probe more strongly than a
(lanes 3 and 4). D, EMSA using
Sox9 and C/EBP proteins synthesized by in vitro
translation. When both Sox9 and C/EBP are incubated with the probe
(lane 4), two major independent bands were observed to be
the same size as the bands obtained by Sox9 (lane 1) or
C/EBP (lane 2) alone. The addition of antibody for Sox9
(lane 5) or C/EBP (lane 6) generated
supershift of the related band and did not affect to the other
band.
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Type II Collagen Promoter-Enhancer Construct Is Down-regulated by
C/EBPs--
Because the expression of type II collagen is
also down-regulated by IL-1 and the data base analysis revealed that
the first intron of type II collagen gene has multiple C/EBP motifs, we examined whether C/EBPs also regulate the promoter activity of the type
II collagen gene. Co-transfection of the type II collagen promoter-enhancer construct, pGL2-COL2-577/+3426, into RCS cells with
the expression vectors for C/EBP-LAP, C/EBP-LIP, or C/EBP also
down-regulated the reporter gene activity (Fig.
8). Similar to the response of the CD-RAP
promoter (see Fig. 5A), overexpression of C/EBP-LAP
produced more potent inhibition than C/EBP-LIP or C/EBP. These
results indicate that C/EBP could mediate, at least partially, the
inhibitory effect of IL-1 on the type II collagen gene as well.
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Fig. 8.
The promoter activity of the type II collagen
is also down-regulated by C/EBP and
-. The 4.0-kb construct containing the promoter and
intron enhancer of the human type II collagen gene was co-transfected
into RCS cells with 500 ng of the empty vector C/EBP-LAP,
C/EBP-LIP, or C/EBP expression vector. The activity of
promoterless pGL3b is set at 1. Each bar represents the
mean ± S.D.
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C/EBP and - Are Stimulated in Normal Chondrocytes
from Human Articular Cartilage in Response to IL-1
Treatment--
Our studies have been focused on two chondrocytic cell
lines producing low (RCJ3.1C5.18) and high (RCS) amounts of CD-RAP. In order to examine this response in normal chondrocytes, the levels of
C/EBP and -, CD-RAP, and type II collagen mRNAs were investigated in IL-1-treated chondrocytes isolated from human articular cartilage (Fig. 9). Cells were
treated with 2 ng/ml IL-1 for up to 48 h. By using the real
time quantitative RT-PCR method, the expression of C/EBP and -
were shown to increase at 24 h up to 3- and 2-fold, respectively,
and returned to basal levels by 48 h. Type II collagen mRNA
significantly decreased to 8% within 24 h, whereas CD-RAP
mRNA decreased relatively slowly to 56% compared with the controls
within 48 h. Consistent with the results from cell lines, these
results suggest that C/EBP and - can regulate the expression of
CD-RAP and type II collagen in articular cartilage in the same
manner.
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Fig. 9.
IL-1 stimulates the
expression of C/EBP and -
in normal chondrocytes from human articular cartilage. The
relative expression levels of C/EBP, , CD-RAP, and type II
collagen were examined in normal chondrocytes from human articular
cartilage treated with IL-1 (2 ng/ml) using quantitative real time
RT-PCR method. Each point represents the mean ± S.D. of fold
change compared with zero time.
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DISCUSSION |
The mechanism by which the proinflammatory cytokines IL- and
TNF- stimulate inflammation and degradative enzymes as well as
repress synthesis of structural matrix is thought to occur via
independent signal transduction and transcriptional mechanisms (44,
45). In this study, we show that C/EBP is a critical regulator for
repression of CD-RAP transcription induced by IL-1. In contrast,
C/EBP is known to activate expression of phospholipase A2, cyclooxygenase-2, and manganese superoxide dismutase in
response to IL-1 (17, 21, 29) and thus promote inflammatory
reactions or catalytic effects in tissues. This is the first report
showing that C/EBP mediates repressive effects of IL-1 for
matrix-associate proteins, including type II collagen, thereby
elucidating a common pathway in chondrocytes for IL-1-induced
activation of degradation and repression of new matrix synthesis.
The mechanism of C/EBP activation in response to IL-1 and TNF-
is complex and still controversial. It is reported that IL-1 or
TNF- stimulate C/EBP expression at the mRNA level in liver,
spleen, and kidney in vivo (18), in MC3T3-E1 osteoblastic cells (19) and in J774.2 macrophage cells (20). In contrast, no change
in the mRNA expression is observed in transformed hepatocyte (25),
in 3T3-L1 adipocyte (28), and in rabbit articular chondrocyte (21).
IL-1, TNF-, or antioxidant activates C/EBP in hepatocytes at the
post-translational level by phosphorylation of serine within the
transcription-activating domain (23) and DNA binding domain (24, 26),
followed by nuclear translocation (25, 26), enhancing transcriptional
efficacy (23, 24). In contrast to the nuclear translocation, it is also
reported that TNF- induces nuclear export of C/EBP by
phosphorylation of serine resulting in repression of albumin gene in
hepatocyte (46). In the current study, the amounts of C/EBP proteins
were very low in cytoplasm and nuclei of RCS cell without IL-1
treatment. IL-1 stimulated both of C/EBP and - mRNA
expression resulting in increase of both proteins in the nuclei. The
mRNA in human articular chondrocytes also responded to the IL-1
treatment. It is likely that the mechanism of C/EBP activation
varies depending on cell types and conditions.
In the current study, the repression of CD-RAP mRNA occurred more
than 24 h later than the activation of C/EBPs in RCS cells and
human chondrocyte. This time lag may be due to the relatively high
stability of CD-RAP mRNA. We performed RNA stability assays for
CD-RAP using the RNA polymerase II inhibitor,
5,6-dichloro-1--D-ribofuranosylbenzimidazole (60 µM) in RCS cell and in bovine articular chondrocyte (9). CD-RAP mRNA was still present at 90% after 6 h of treatment
and at 60 (RCS cell) to 40% (bovine chondrocyte) after 24 h of treatment.
C/EBP is an unusual transcription factor in that it can activate or
repress gene transcription. For example, C/EBP activates the gene
encoding the 1 chain of type I collagen (30), whereas it represses
the gene encoding the 2 chain of type I collagen (22) and albumin
genes (47). We found an HMG-like motif partially overlapping
with the C/EBP motif that can bind to Sox9. As binding of one protein
appears to preclude binding of the other, the balance between C/EBP and
Sox proteins may regulate the transcription of CD-RAP. The interaction
of C/EBPs with other transcription factors has been observed in other
genes, although the mechanisms of binding and the results of binding
are specific to the transcription factor and gene. For example, in the
CD11c and rat cytochrome CYP2D5 genes, C/EBP acts synergistically with
the transcription factor Sp1 to enhance gene expression through a
mechanism whereby Sp1 facilitates the binding of C/EBP to a low
affinity site in the DNA (48, 49). C/EBP also interacts with
retinoblastoma protein and various other transcriptional activators or
co-activators such as c-Myb, PU.1, and ATF-2 (41, 50). In the
osteocalcin gene, C/EBP binds to DNA and, in turn, binds to the
transcription factor Runx2/Cbfa1 to greatly enhance gene expression
(32).
We have found that C/EBP also repressed the transcription of the type
II collagen gene. There are multiple C/EBP motifs in the first intron
of type II collagen gene. Interestingly, analysis revealed that there
is a C/EBP motif within the 48-bp core enhancer element defined by
Lefebvre et al. (42, 43) that regulates cartilage-specific
expression of type II collagen gene; the C/EBP motif is next to the
Sox9-binding motif in the element. A zinc finger transcription factor,
A-crystallin-binding protein 1 (CRYBP1), that is expressed in a
reciprocal pattern compared with that of type II collagen is reported
to bind to the sequence overlapping the C/EBP motif and compete with
Sox9 binding thereby repressing gene expression of type II collagen
(51). C/EBP may participate in IL-1-induced repression of type II
collagen by taking place of the CRYBP1 binding and competing with Sox9
binding in a similar manner. It has been reported that Sox9 is
down-regulated by IL-1 and TNF- (52). In that report, the
expression of Sox9 was decreased within a few hours via direct
interaction of IL-1-induced NF-B with the Sox-9 promoter, whereas
the decrease of type II collagen takes much longer. Because no further
Sox9 is then expressed but the existing Sox9 remains bound to the
enhancer of type II collagen gene, C/EBP might be involved in this
regulation by displacing Sox9 from the element. Further studies will be
performed to ascertain the involvement of C/EBP in the regulation of
type II collagen gene.
One of the unique observations of CD-RAP gene is that its physiological
expression is primarily restricted to cartilage. Various transcription
factors have been shown to function in regulating the CD-RAP promoter
activity. For example, AP-2 binds to the sequence from 463 to 456
bp and regulates transcription in a biphasic manner, activating at a
low concentration of AP-2 and repressing at a high concentration (34).
Sox9 binds at 410 to 404 bp (a different site from the one shown in
this study) and activates transcription (35). Upstream
stimulatory factor and delta-EF1 were also found to function in
an E box located at 488 to 482 bp and activate or repress
CD-RAP depending on the relative population of upstream
stimulatory factor and delta-EF1 in the nuclei (53). Studies of
transgenic mice, which harbor various lengths of CD-RAP 5'-flanking
sequence linked to -galactosidase gene (lacZ), revealed that 2251-bp promoter was sufficient to direct cartilage-specific expression of reporter gene, but 2068 bp was not (2). These results
in transgenic mice indicate that the regulation of tissue-specific expression of the CD-RAP gene cannot be explained completely by the
factors found binding to the sequence surrounding 400 bp and that
there may be important elements between 2251 and 2068 bp needed to
control tissue-specific expression.
In the current study, we show that there is a C/EBP-binding site within
the domain located on 2091 to 2066 bp, which functions as a
repressor, and the HMG-like motif, adjacent to the C/EBP site, which
could be a potent activator domain by binding of Sox proteins. Another
HMG-like motif at 2152 to 2245 bp has been shown from our
laboratory (54), which is consistent with the presence of another
activating element between 2251 and 2138 bp. These positive and
negative regulators within the tissue-specific domain may exert the
tissue-specific regulation of the CD-RAP gene. C/EBP expression is
negatively correlated with CD-RAP synthesis. For example, in
undifferentiated RCJ3.1C5.18 cells C/EBP is present, and expression
of CD-RAP is very low, whereas in RCS, whose expressions of C/EBP
and are very low, CD-RAP expression is very high. In skeletal
tissue, C/EBP is reported to be expressed in the germinal cell but
not in mature chondrocytes of the growth plate (55). C/EBP and -
are expressed in osteoblasts and activate osteocalcin gene
transcription in cooperation with Runx2 (32). The reciprocal expression
of C/EBP and CD-RAP suggests that C/EBP may also participate in
tissue-specific regulation of CD-RAP by repressing its expression in
undifferentiated chondrocytes, osteoblasts, or other tissues. In
preliminary studies, we have generated transgenic mice harboring the
3345-bp promoter of CD-RAP linked to lacZ gene. This
promoter directs cartilage-specific expression of the reporter gene. In
another transgenic line, in which the sequence from 2251 to 2068 bp
(including the C/EBP-binding site) was removed from the 3345-bp
construct, the reporter gene was expressed widely.2 These results
support the hypothesis that C/EBP may be responsible for
tissue-specific repression of CD-RAP gene expression.
In summary, the data presented here show that C/EBP is a critical
factor for IL-1-induced repression of CD-RAP gene. This effect may
be coordinated with binding competition between C/EBP and the enhancer
protein Sox9. C/EBP also down-regulated the type II collagen gene
transcription. This is the first report that has shown that C/EBP
regulates a cartilage-specific gene. These results strongly suggest
that C/EBP is the common factor accounting for the disparate effects of
IL-1: transcriptional repression of chondrocyte extracellular matrix
production in addition to the promotion of inflammatory cascade and
catabolic processes in joint diseases, such as rheumatoid arthritis and osteoarthritis.
and 
Mediate the
Repression of Gene Transcription of Cartilage-derived Retinoic
Acid-sensitive Protein Induced by Interleukin-1
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of Orthopaedic
Surgery, Washington University School of Medicine at Barnes-Jewish Hospital, Mail Stop 90-34-674, 216 S. Kingshighway, St. Louis, MO
63110. Tel.: 314-454-7800; Fax: 314-454-5900; E-mail:
sandelll@msnotes.wustl.edu.
