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Molecular and Cellular Biology, February 2000, p. 912-918, Vol. 20, No. 3
Brookdale Center, Department of Biochemistry
and Molecular Biology, Mount Sinai School of Medicine, New York
University, New York, New York 100291;
Laboratory of Biochemistry, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 208922;
and Cellular Biochemistry and Biophysics Program, Memorial
Sloan-Kettering Cancer Center, New York, New York
100213
Received 20 August 1999/Returned for modification 16 September
1999/Accepted 5 November 1999
Extracellular matrix (ECM) formation and remodeling are critical
processes for proper morphogenesis, organogenesis, and tissue repair.
The proinflammatory cytokine tumor necrosis factor alpha (TNF- Tumor necrosis factor alpha
(TNF- Aside from cachexia, TNF- To elucidate the molecular mechanism of TNF-
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Tumor Necrosis Factor Alpha Inhibits Type I Collagen Synthesis
through Repressive CCAAT/Enhancer-Binding Proteins

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ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
)
inhibits ECM accumulation by stimulating the expression of matrix
proteolytic enzymes and by downregulating the deposition of structural
macromolecules such as type I collagen. Stimulation of ECM degradation
has been linked to prolonged activation of jun gene
expression by the cytokine. Here we demonstrate that TNF-
inhibits
transcription of the gene coding for the
2 chain of type I collagen
[
2(I) collagen] in cultured fibroblasts by stimulating the
synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to
a previously identified TNF-
-responsive element. This conclusion was
based on the concomitant identification of C/EBP
and C/EBP
as
TNF-
-induced factors by biochemical purification and expression
library screening. It was further supported by the ability of the
C/EBP-specific dominant-negative (DN) protein to block TNF-
inhibition of
2(I) collagen but not TNF-
stimulation of the
MMP-13 protease. The DN protein also blocked TNF-
downregulation of
the gene coding for the
1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-
-induced signaling pathway that controls ECM formation and remodeling.
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INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
) is a 17-kDa cytokine released by activated macrophages that
locally elicits a wide spectrum of metabolic responses and cellular
activities (20). The pleiotropic properties of TNF-
are
signaled through alternative routes that culminate in diversified
responses, such as cell proliferation, differentiation, or death
(20). These ill-defined intracellular pathways in part
influence gene expression by modulating the activity and synthesis of
several transcription factors. Relevant examples include the activation
of AP1 and NF-
B in the regulation of cell death and cell survival,
the induction of IRF1 in the stimulation of the alpha and beta
interferon genes, and the undefined pathway that inhibits C/EBP
expression in an experimental model of inflammation-induced atrophy of
adipose tissue (3, 6, 17).
is believed to be a key player in
inflammatory disorders such as rheumatoid arthritis and osteoarthritis (20). Extracellular matrix (ECM) degradation is the hallmark of these conditions and an important component in morphogenesis, organogenesis, and tissue remodeling, as well as in wound healing and
tissue repair. TNF-
stimulates ECM degradation by inducing the
production of stromal collagenases. The stimulation is the result of
TNF-
prolonged activation of jun gene expression with the
consequent binding of the AP1 complex to the responsive elements in the
collagenase promoters (4). TNF-
reduces ECM
deposition by inhibiting the synthesis of structural components.
They include elastin, osteocalcin, and type I collagen, the major
structural component of connective tissue (10, 15, 19).
TNF-
also counteracts transforming growth factor
(TGF-
)
stimulation of type I collagen gene expression in cell cultures
(11). The last finding reflects the functionally
antagonistic nature of these cytokines and represents a useful paradigm
to study the complex cellular signals that regulate ECM formation and
remodeling in vivo.
action on ECM
remodeling, we have been studying the cytokine regulation of the human
2(I) collagen (COL1A2) gene. We have shown that the opposing stimuli
of TNF-
and TGF-
converge on the same transcriptional complex
(cytokine responsive complex [CyRC]) that is bound to the
330 to
235 upstream sequence (Fig. 1A)
(8). The
330 to
235 sequence is sufficient to mediate
the antagonistic effects of the cytokines, for it confers TGF-
and
TNF-
responsiveness to the otherwise unresponsive thymidine kinase
promoter. DNA binding assays and cell transfection experiments have
also separated the TGF-
-responsive element from the TNF-
responsive element (Fig. 1A). Indeed, we were able to show that
TNF-
, but not TGF-
, increases protein binding to nucleotides
330 to
306 (box 5A [Fig. 1A]).

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FIG. 1.
Purification and characterization of C1R. (A) Schematic
representation of the TNF-
-responsive element. The
340 to
235
nucleotide sequence of the COL1A2 gene is shown along with the location
of the TGF-
-responsive element (TbRE), TNF-
-responsive element
(TaRE), and box 5A. The C/EBP and Sp1/Sp3 binding sites are highlighted
by the dotted and continuous lines, respectively. (B) C1R is a
heat-resistant protein. Nuclear extracts from NIH 3T3 cells were
incubated at the indicated temperatures for 10 min, followed by EMSA
using box 5A as the probe. (C) Gel filtration chromatography of
denatured nuclear proteins. Aliquots (30 µl) from fractions were
examined for box 5A binding activity by EMSA following renaturation.
The migration of size markers (in kilodaltons) is indicated below the
autoradiograph. U, unfractionated sample.
Based on the above results, we have hypothesized that the antagonistic
signals of the cytokines act on the CyRC by influencing the activity of
the complex in opposite ways and, in the case of TNF-
, by inducing
the binding of a negative factor (C1R) to box 5A (8). Here
we report the characterization of the DNA-binding C1R protein. Our
findings establish a biochemical and genetic link between the C/EBPs
and the regulation of the type I collagen genes by TNF-
.
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MATERIALS AND METHODS |
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|
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Cell culture, plasmids, and reagents.
Fibroblasts were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum (HyClone Inc., Logan, Utah) and antibiotics
(penicillin [50 U/ml] and streptomycin [50 µg/ml]). Human
recombinant TNF-
(Boehringer Mannheim Corp., Indianapolis, Ind.) was
used at a final concentration of 20 ng/ml. Unless otherwise indicated,
cells were placed in medium containing 0.1% fetal bovine serum 12 h before TNF-
addition. The COL1A2 promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid C/EBP/CAT and the A-CREB and
A-C/EBP constructs have been described before (8, 16). A-C/EBP differs from A-ZIP/F in that contains only the C/EBP
leucine
zipper and the acidic extension DPLEQRAEELARENEELEKAEELEQENAE, in
addition to the FLAG epitope (1). Bacterially expressed recombinant proteins were purified over heparin columns as described before (16, 14). Antibodies specific for C/EBP
, C/EBP
,
and C/EBP
and high-affinity recognition sequences for C/EBP, CREB, and CTF/NF-1 were purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.).
Characterization of C1R and DNA binding assays.
The C1R
protein was partially purified by DNA affinity chromatography from
nuclear extracts prepared from 600 150-mm-diameter culture dishes of
NIH 3T3 fibroblasts (9). Southwestern screening was
performed as described by Vinson et al. (22), using
106 independent phage clones engineered in the
gt 11 expression vector by random priming of human fibroblast RNA. Nuclear
extracts were purified from control and TNF-
-treated cells and used
in electrophoretic mobility shift assay (EMSA) with the oligonucleotide corresponding to box 5A of COL1A2 (
330 to
296) or with a
high-affinity recognition sequence for CREB according to published
conditions (8). Unlabeled competitors were added in the
amounts indicated in the figure legends. When appropriate, nuclear
extracts were preincubated with antibodies before addition of the
labeled probe. DNA-protein complexes were separated from unbound
material on a 5% polyacrylamide gel and then visualized by
autoradiography. In some experiments, recombinant A-C/EBP or A-CREB
proteins were mixed at the concentrations indicated in the figure
legends with 5 µg of nuclear extracts obtained from TNF-
-treated
NIH 3T3 cells and used in EMSA with the box 5A or CREB oligonucleotide
as a probe. To estimate the molecular weight of C1R, 500 µg of
nuclear extracts was subjected to gel filtration chromatography using a
Sephacryl S-300 column, and the collected fractions were analyzed by
EMSA using the box 5A probe (9).
Cell transfections.
Conditions for the preparation and
transfection of plasmid DNA into NIH 3T3 cells or HepG2 cells by the
calcium phosphate method were described before (8). In the
titration test, 8 µg of the C/EBP
expression vector was
cotransfected in HepG2 cells with 0.3 µg of the C/EBP/CAT reporter
plasmid along with increasing amounts of the dominant-negative (DN)
expression plasmids. A-CREB or A-C/EBP expression plasmids were
cotransfected with 10 µg of the
378COL1A2/CAT reporter constructs
and with 2 µg of pSVLUC, a luciferase-expressing vector under the
control of the simian virus 40 promoter. Eighteen hours after
transfection, cells were washed twice with phosphate-buffered saline,
placed in medium containing 0.5% fetal bovine serum for 2 h, and
then treated with TNF-
for 18 h. Transfections were performed
multiple times in duplicate. CAT activities were normalized against the activity of pSVLUC. The statistical value of the data was evaluated by
the Mann-Whitney U test. Stable transfectants were selected by
culturing the cells for about 3 weeks in the presence of G418 (400 µg/ml).
Northern blot analysis and in vitro transcription assay.
Total RNA was used for Northern blot hybridization to probes for
1(I) and
2(I) collagen, MMP-13, and glyceraldehyde-3-phosphate dehydrogenase (7). Rates of COL1A2 transcription were
determined as previously described (7). Quantitative data
were obtained with the aid of the computer program Adobe Photoshop
(Adobe Systems Inc., Mountain View, Calif.). All experiments were
performed in triplicate. Cells were harvested at various time points
after TNF-
treatment and used to obtain RNA or nuclear extracts as described above. Cell viability, determined by the trypan blue exclusion test, was always higher than 90%.
Western blot analysis.
Nuclear extracts, at the
concentrations indicated in the figure legends, were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on
12.5% gels and transferred onto a nitrocellulose membrane. Membranes
were probed with C/EBP
or C/EBP
antibodies at a final dilution of
1:1,000, followed by incubation with horseradish peroxidase-conjugated
goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) diluted
1:3,000. C/EBPs were detected with an enhanced chemiluminescence system
(Renaissance; NEN Life Science Products, Boston, Mass.), as recommended
by the manufacturer. Conditions used to assess the expression of FLAG epitope in cells stably transfected with the different DN proteins were
described before (14, 16). Proteins resolved by SDS-PAGE were visualized by silver staining.
| |
RESULTS AND DISCUSSION |
|---|
|
|
|---|
C1R was biochemically purified by anion-exchange DNA affinity
chromatography using multimers of box 5A (Fig. 1A). The partially purified protein (~6,000-fold) was found to be a heat-resistant polypeptide with an estimated molecular mass of
40 kDa under denaturing conditions (Fig. 1B and C). These features, and the presence
of the CTTGGA sequence on the coding strand of box 5A (Fig. 1A),
strongly suggested that C1R may be a leucine zipper protein
(13). Parallel Southwestern screening of a fibroblast cDNA
expression library with box 5A provided independent support for this
postulate. The screen identified six positive DNA-binding recombinants.
Three of them code for C/EBP
, -
, and -
; the others code for
non-leucine zipper proteins and are likely to represent false positives.
The results of the biochemical purification and the Southwestern
screening concurred in suggesting that C1R is probably the same complex
as C/EBP. Additional data confirmed this conclusion. First, Western
blot analysis showed a substantial increase of C/EBP
and -
in the
partially purified sample compared to the original nuclear extracts
(Fig. 2A). Second, preincubation of nuclear extracts from TNF-
-treated mouse fibroblasts with
C/EBP-specific antibodies revealed the ability of the C/EBP
and
C/EBP
antisera, but not of the anti-C/EBP
antibody, to interfere
with C1R formation (Fig. 2B). The same results were obtained with
nuclear extracts from human fibroblasts (data not shown). Third, C1R
formation was competed by box 5A and the C/EBP consensus sequence but
not by the recognition site for CTF/NF-1 (Fig. 2B). This last finding is important in view of previous reports that implicated CTF/NF-1 in
mediating TNF-
and TGF-
modulation of collagen synthesis (2,
18).
|
Northern blot hybridizations established the existence of an inverse
relationship between steady-state levels of C/EBP
mRNA and
accumulation of
2(I) collagen transcripts in TNF-
-treated fibroblasts (Fig. 3). A smaller increase
was also observed for C/EBP
, C/EBP
, and CHOP mRNAs (data not
shown). Two isoforms of C/EBP
(p35 and p20) have been described
(5). The smaller isoform has been shown to act as a DN
inhibitor of C/EBP-mediated transcription by virtue of missing the
transactivating domain, whereas the larger one has been associated with
gene transactivation (5). Western blot analysis confirmed
the role of TNF-
in C/EBP
stimulation by documenting the increase
of both activator and repressor isoforms of the transcription factor
(Fig. 3). It is interesting that the p20 and p35 isoforms showed ~10-
and 6-fold, respectively, increases over the amounts of the same
proteins in the untreated sample. Whether this result indicates the
slight preference of TNF-
to stimulate the repressor as opposed to
the activator isoform of C/EBP
remains to be determined. More
generally, the precise mechanism underlying TNF-
induction of C/EBP
repressive activity on collagen transcription is the focus of ongoing
investigations. Here, we sought to provide functional support for the
DNA binding evidence.
|
Overexpression of DN molecules provides an effective tool with which to investigate the in vivo functions of transcription factors. The design of the construct can allow the study of both induction and repression of transcription and can overcome the problem of functional redundancy among related gene products. The approach has been successfully implemented to create DNs to distinct members of the basic-leucine zipper (bZIP) class of transcription factors. They include A-CREB, which prevents the basic region of CREB from binding to DNA; GBF-F, which changes the specificity of C-EBP binding to DNA; and A-ZIP/F, which inhibits bZIP transcription factors binding to DNA by forming stable heterodimers (1, 14, 16).
In this study we used A-C/EBP, a novel design of the A-ZIP/F type that heterodimerizes promiscuously with all C/EBPs and only with them. The specificity of A-C/EBP was tested by challenging the binding of recombinants C/EBP and CREB to the respective recognition sequences with increasing amounts of recombinant A-C/EBP. The disappearance of the C/EBP, but not the CREB complex, demonstrated the specificity of A-C/EBP (Fig. 4A). As previously reported by Moitra et al. (14) for the prototype A-ZIP/F, A-C/EBP ablated DNA binding at 1-fold molar equivalent, conceivably as a result of preferential formation of heterodimers (14). The same kind of test was repeated to assess the efficacy of A-C/EBP in our system. Crude nuclear extracts were preincubated with recombinant A-C/EBP or recombinant A-CREB, and C1R formation was examined by the EMSA. Preincubation with increasing amounts of A-C/EBP effectively prevented C1R formation but not CREB binding; likewise, increasing amounts of A-CREB acted only on the formation of the CREB complex (Fig. 4B). The results therefore demonstrated the specificity of the DN molecules against the respective bZIP proteins. Most importantly, they ruled out the possibility that C1R was the heterodimerization product of CREB and C/EBP (21).
|
The next set of experiments examined A-C/EBP ability to relieve COL1A2
transcription from TNF-
-induced repression. The inhibitory activity
of A-C/EBP was first titrated in transient cotransfection assays with
the C/EBP
expression vector and a reporter construct harboring the
C/EBP binding site (Fig. 5A). Based on
these results, 5 µg of A-C/EBP was transiently cotransfected with the
378COL1A2/CAT promoter construct in fibroblasts cultured with or
without TNF-
. Controls included the empty CMV-500 vector and the
A-CREB plasmid. Consistent with the in vitro binding test, only A-C/EBP
abrogated TNF-
-induced downregulation of the COL1A2 promoter (Fig.
5B). This result functionally links C/EBP binding with TNF-
activity. To provide further support for this correlate, we repeated
the analysis with cells stably transfected with the DN plasmids and examined the activities of several endogenous genes, including the
genes coding for
1(I) and
2(I) collagen and the metalloproteinase MMP-13.
|
Steady-state levels of
2(I) collagen mRNA remained virtually
unaltered in TNF-
-treated cells that express A-C/EBP (Fig. 6). By contrast,
2(I) collagen mRNA
levels were reduced after TNF-
treatment in cells stably transfected
with CMV-500 or A-CREB (Fig. 6). A run-on transcription assay showed
that A-C/EBP block of COL1A2 inhibition by TNF-
occurs at the
transcriptional level (Fig. 7A). The EMSA
confirmed the specificity of A-C/EBP to inhibit C/EBP binding to box 5A
(Fig. 7B). A-C/EBP had also an effect on the coordinately expressed
1(I) collagen (COL1A1) gene. As for COL1A2, stable expression of
A-C/EBP, but not A-CREB, led to block of TNF-
-induced downregulation
of COL1A1 (Fig. 6). Additionally, COL1A1 expression was reproducibly
less in the TNF-
-untreated A-C/EBP transfectants than in the CMV-500
counterparts (Fig. 6). This result is opposite what is observed with
the COL1A2 gene; together, the data may suggest that distinct
mechanisms underlie C/EBP regulation of the type I collagen genes.
Consistent with the established involvement of AP1 in the upregulation
of metalloproteinases (4), A-C/EBP had no effect on TNF-
stimulation of MMP-13 (Fig. 6). This last result and previous work by
Brenner et al. (4) thus indicate that the opposing effects
of TNF-
on collagen downregulation (matrix deposition) and
metalloproteinase upregulation (matrix degradation) are apparently
transduced through distinct signaling pathways.
|
|
In summary, this study provides evidence for involvement of C/EBPs in
ECM remodeling and implicates specifically this small group of bZIP
proteins in mediating TNF-
inhibition of matrix accumulation. Our
conclusion is based on the following observations. First, TNF-
induces production of C/EBP
. Second, the cytokine induction
translates into increased C/EBP
binding to the TNF-
responsive
element of the COL1A2 gene. Third, A-C/EBP relieves the COL1A2 gene
from TNF-
inhibition by sequestering all C/EBPs from DNA binding. It
is therefore plausible to conclude that TNF-
downregulates
COL1A2 transcription at least in part by inducing C/EBP molecules with
repressing activity. C/EBP-induced repression could be the
result of higher binding affinity of the p35-p20 heterodimer or
could be dictated by protein interactions taking place within the
context of the CyRC. Although only suggestive, the greater abundance of
p20 than of p35 after TNF-
treatment seems to favor the former possibility.
The precise mechanism responsible for the generation of the C/EBP
isoforms is still controversial. Two recent reports have raised the
possibility that these C/EBP isoforms are the products of proteolysis
rather than translational initiation, as previously thought (5,
12, 23). C/EBP
participation in TNF-
-induced inhibition of
type I collagen expression is not indispensable, in that other C/EBPs
compensate for its loss in c/ebp
/
fibroblasts (data not shown). Our study demonstrates two additional new
points which are of relevance to the understanding of ECM remodeling
and the etiopathogenesis of diseased conditions. First, TNF-
downregulates the type I collagen genes coordinately through the action
of the bZIP proteins. Second, the opposing effects of TNF-
on ECM
deposition and degradation are transduced through distinct nuclear
signals which utilize C/EBPs and AP1, respectively.
| |
ACKNOWLEDGMENTS |
|---|
P. Greenwel and S. Tanaka made equal contributions to this work.
We thank K. Calame, P. Johnson, D. Ron, U. Schibler, and S. Shapiro for generously providing invaluable materials for this study and V. Vinson for critical review of the manuscript. We also thank K. Johnson for typing the manuscript and R. Ni and C. Else for excellent technical assistance.
This work was supported by grants AR38648 and AA12196 from the National Institutes of Health and by the Arthritis Foundation.
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Brookdale Center in the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York University, New York, NY 10029. Phone: (212) 241-7984. Fax: (212) 722-5999. E-mail: ramirf01{at}doc.mssm.edu.
Present address: Unité INSERM 441, 33600 Pessac, France.
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