(Received for publication, July 10, 1995; and in revised form, October 4, 1995)
From the
The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.
The extracellular matrix provides a controlled environment for
cellular differentiation and tissue development. The integrity of the
extracellular matrix is maintained through a balance between the amount
and activity of matrix-degrading proteolytic enzymes and their
associated activators and
inhibitors(1, 2, 3) . The expression of
tissue inhibitor of metalloproteinases-1 (TIMP-1), ()an
inhibitor of a key regulatory class of matrix-degrading proteinases, is
under strict developmental and tissue-specific control. TIMP-1
expression is largely confined to adult bone and ovary and to tissues
undergoing remodeling or inflammation. In cultured cells its expression
is regulated by serum and growth factors and, in F9 cells, by
differentiation(4, 5) .
The basic structure of the
TIMP-1 promoter has been described previously(6, 7) .
TIMP-1 possesses a TATA-less promoter and is composed of six exons. DNA
sequences conferring transcriptional activation by viruses, serum,
phorbol esters, and transforming growth factor- have been
localized to the 5` region of the gene(6, 8) . This
regulation can be quite dramatic, with serum inducing endogenous TIMP-1
expression by as much as 3 orders of magnitude (6) . (
)Sequences including this region and approximately 2
kilobases more in the 5` direction have also been shown to direct
appropriate embryonic expression of the gene(9) . Furthermore,
TIMP-1 is negatively regulated by the extracellular matrix in primary
mammary epithelial cells, although the sequences conferring regulation
have not been identified. (
)
The TIMP-1 gene is induced in response to the activation of c-Fos(10) , suggesting that in vivo the gene is regulated by AP-1 family members. In fact, inspection of sequences in the TIMP-1 regulatory region has revealed two potential AP-1 binding sites or TPA response elements; however, neither the element that selectively binds AP-1 in vitro nor the sequence that directs AP-1-dependent transcriptional enhancement in vivo has been identified(5, 8) .
Several members of the AP-1 family have been shown to have positive (e.g. JunD) or negative (e.g. JunB) effects on
transcriptional regulation(11, 12) . Each AP-1 family
member is homologous to one of the two prototypes of the family, c-Jun
or c-Fos (13) . The ratio of various Jun and Fos proteins
expressed in cells may be one determinant of their transcriptional
efficacy on target genes. Functionally, AP-1 has been shown to be
necessary for collagenase induction by phorbol esters, oncogenes, and
5
1 integrins(14, 15) and for stromelysin
induction by epidermal growth factor(16, 17) .
The TIMP-1 promoter also has multiple binding sites for Ets transcription factors. One of these sites has been shown to bind Ets in vitro(5) . Like AP-1 and TIMP-1, Ets proteins are serum-inducible. These proteins appear functionally diverse, participating in transcriptional regulation as well as DNA replication and growth control(18, 19) . In Drosophila, Ets-related proteins are critical for cellular differentiation(20, 21) . Ets proteins appear to bind DNA and to activate transcription as monomers or in complexes with other proteins. For example, Elk-1, an Ets-related protein, forms a ternary complex with serum response factor and is phosphorylated in response to growth factors(22, 23) .
It is widely assumed that AP-1 and c-Ets interact to regulate gene expression, because their binding sites are often juxtaposed in cellular promoters, including TIMP-1, and because the proteins act synergistically at a multimerized viral enhancer site(24) . However, such effects have not been documented for cellular promoters, although related proteins such as serum response factor and Elk-1 have been shown to interact(25) . In this study we used TIMP-1 promoter sequences to examine the transcriptional regulatory proteins that control TIMP-1 transcription and to characterize the interaction of AP-1 and Ets in the context of a cellular promoter.
Figure 4:
AP-1 responsiveness of a TIMP-1 promoter
from which the TRE has been deleted. A, two DNA structures,
one containing the TIMP-1 TRE at -858/-601 and one deleted
of the TRE (-834/-601), were transiently transfected into
F9 cells (-AP-1) or 3T3 cells (+AP-1) in the presence
() or the absence (
) of 0.5 µg of AP-1 as described
in the legend to Fig. 1. Each data point represents an average
of two samples from the same experiment; the values were normalized to
-galactosidase activity as described in the legend to Fig. 1. This experiment is representative of three independent
experiments. B, TIMP-1 promoter fragments
-858/-601 (lanes 1-6) and
-834/-601 (lanes 7-12) were isolated from
the gel, labeled, and incubated with c-Jun and c-Fos protein as
follows: lanes 1 and 7, labeled oligonucleotides
alone; lanes 2 and 8, 100 ng of c-Jun; lanes 3 and 9, 400 ng of c-Jun; lanes 4 and 10,
100 ng of c-Jun and 100 ng of c-Fos; lanes 5 and 11,
200 ng of c-Jun and 200 ng of c-Fos; lanes 6 and 12,
100 ng of c-Jun and 200 ng of c-Fos. The arrows indicate the
positions of the protein-DNA complexes.
Figure 5:
Differential activation by AP-1 family
members of the TIMP-1 promoter containing the TRE and the TIMP-1
promoter from which the TRE was deleted. A and B, the
TIMP-1 promoter containing the TRE (-858/-601) (shaded
bars) and the TIMP-1 promoter minus the TRE
(-834/-601) (open bars) were transiently
transfected into F9 cells as described in the legend to Fig. 1,
except that along with 0.5 µg of c-Jun and 0.5 µg of c-Fos, 2
µg of JunD was included in some experiments. Each data point
represents an average of two samples from the same experiment; values
were normalized to -galactosidase activity as described in the
legend to Fig. 1. This experiment is representative of three
independent experiments. C, DNA binding assays with c-Jun and
c-Fos compared with undifferentiated F9 cell extract. The labeled TIMP
oligonucleotide containing the TRE (see the legend to Fig. 3)
was incubated with no proteins (lane 1), purified c-Fos and
c-Jun (lane 2), or an extract made from undifferentiated F9
cells (lane 3). The protein-DNA complexes are indicated by arrows.
Figure 1:
Activation of TIMP-1 gene transcription
by c-Fos and c-Jun. The drawing at the top represents
a 5` portion of the TIMP-1 gene. Exons 1 and 2 are represented by open rectangles, and all positions are numbered relative to
the translation initiation start site indicated by the arrow.
The position of the TIMP-1 TRE is indicated by the black
rectangle. The regions diagrammed below the map were cloned into a
promoterless CAT vector, pBLCAT3, as described under
``Experimental Procedures.'' The two DNA reporter structures,
-1320/-601 and -858/-601 (2 µg), or pBLCAT3
with no inserted TIMP-1 sequence (vector; 2 µg) were
transiently transfected into F9 cells with 0.5 µg of Rous sarcoma
virus c-Jun and 0.5 µg of Rous sarcoma virus c-Fos (shaded
bar) or without either protein (open bar). A
-galactosidase expression vector (0.25 µg of Rous sarcoma
virus LacZ; (47) ) was cotransfected as an internal control,
and CAT activity was normalized to
-galactosidase activity. Each
data point represents an average of three experiments. The bars indicate S.D.
, with AP-1;
, without
AP-1.
Figure 3:
A 28-bp TIMP-1 TRE transcriptionally
responds to AP-1 in vivo and binds purified c-Jun and c-Fos
protein in vitro. A, see the legends to Fig. 1and Fig. 2for an explanation of the symbols.
Oligonucleotides containing the TIMP-1 TRE were placed upstream of a
tkCAT gene (pBLCAT2) as single (wt 1) or triple (wt
3) copies. These plasmids were transiently
transfected into F9 cells in the presence or the absence of 0.5 µg
of AP-1 as described in the legend to Fig. 1. Each data point
represents an average of two samples from the same experiment; the
values were normalized to
-galactosidase activity as described in
the legend to Fig. 1. This experiment is representative of three
independent experiments. B, a TIMP-1 oligonucleotide
containing the TIMP-1 TRE at -847/-841 binds purified c-Fos
and c-Jun in vitro.
P-labeled double-stranded
oligonucleotides containing the TIMP-1 sequence
5`-TGGATGAGTAATGCGTCCAGGAAGCCTG-3` were incubated with 100 ng of c-Jun
and 100 ng of c-Fos proteins expressed and purified from E. coli (lanes 2-5). The underlined sequence is
the AP-1 binding site, or TRE. Lane 1, labeled
oligonucleotides alone; lane 2, labeled oligonucleotides with
added c-Jun and c-Fos proteins; lanes 3-5, same as lane 2, except that unlabeled TIMP-1 oligonucleotides were
added in molar excess of labeled oligonideotides as follows: lane
3, 100
; lane 4, 1000
; lane 5,
10,000
. The protein-DNA complex was resolved from unbound DNA by
nondenaturing gel electrophoresis and is indicated by the upper
arrow. The lower arrow indicates DNA not complexed with
protein. The audioradiogram was exposed for 48 h at ambient
temperature.
Figure 2:
AP-1
activates a proximal TRE but not a distal TRE in the TIMP-1 promoter.
The Ets binding sites are represented by open ovals. The
position of the distal TRE is represented by a shaded
rectangle. See the legend to Fig. 1for an explanation of
other symbols. Regions diagrammed below the map were cloned into either
pBLCAT3 (CAT) or pBLCAT2 (tkCAT). These structures (2 µg) were
transiently transfected into F9 cells in the presence () or the
absence (
) of 0.5 µg of AP-1 as described in the legend to Fig. 1. Each data point represents an average of two samples
from the same experiment; the values were normalized to
-galactosidase activity as described in the legend to Fig. 1. The experiment is representative of several independent
experiments.
For chloramphenicol
acetyltransferase (CAT) assays, the cells were washed with 5 ml of PBS,
scraped from the plates in 1 ml of PBS, resuspended in 125 µl of
250 mM Tris-HCl (pH 7.8), and lysed by four cycles of
freeze/thaw (-70 °C/37 °C). Cell debris was pelleted by
centrifugation for 5 min at 10,000 g. CAT activity was
assayed in a 1-h incubation with
C-labeled acetyl coenzyme
A, followed by extraction of the labeled acetylated chloramphenicol
with ethyl acetate (28) . Scintillation fluor was added to the
ethyl acetate supernatant and monitored for radioactivity.
-Galactosidase activity was monitored by adding 50 ml of cell
extract to 450 ml
-galactosidase buffer (60 mM
Na
HPO
, pH 8.0, 1 mM MgSO
,
10 mM KCl, 50 mM
-mercaptoethanol). The reaction
was started by the addition of the substrate, 100 µl of 4 mg/ml O-nitrophenyl-
-D-galactosidase. The samples were
then incubated at ambient temperature until the color began to develop,
and spectrophotometry was performed at absorbance of 420 nm.
The mEts oligonucleotide was designed by changing 5 bp of the Ets consensus binding site in the TIMP promoter shown to bind bacterially expressed c-Ets(5) . This site is also identical to the Ets binding site of the polyoma virus enhancer that was used in the initial studies demonstrating interaction between c-Ets and AP-1(24, 31) .
By sequence inspection of the TIMP-1 promoter, we identified another potential TRE located at approximately -1600. To determine whether this putative AP-1 binding site also participated in AP-1-mediated transcriptional enhancement in vivo, we compared the response of a variety of TIMP-1 promoter constructs to AP-1 by cotransfection into F9 embryonic carcinoma cells. Whereas AP-1 activated (>10-fold) a TIMP-1/CAT reporter construct containing TIMP-1 sequences between -1008/-601, the promoter region containing the putative TRE between -3450/-1008 was not activated (Fig. 2). Furthermore, this sequence was not by itself transcriptionally responsive to AP-1 when placed upstream of either a promoterless CAT gene or a thymidine kinase promoter-driven CAT gene (Fig. 2). Therefore, our functional studies indicate that a TRE located between -858/-601 is a target for AP-1-dependent transcriptional activation, but the TRE at -1600 is not.
To determine whether the TRE located within the -858/-601 fragment was the DNA binding site that enabled transcriptional activation by AP-1, we next examined the response to AP-1 of an oligonucleotide containing this TRE upstream of a heterologous promoter (tkCAT). Like many other transcription factor binding sites, a single TIMP-1 TRE was not transcriptionally active. However, when three tandem repeats of this site were placed upstream of tkCAT, significant AP-1-dependent transcriptional enhancement was achieved (Fig. 3A). In addition, this TRE bound bacterially expressed and purified c-Jun and c-Fos in vitro, suggesting that the TRE located between -858/-834 is a site of AP-1 action on this promoter (Fig. 3B).
To test for additional AP-1 binding sites, we used a TIMP-1 reporter construct, -834/-601, in which the TRE had been deleted. Surprisingly, this construct was still AP-1-inducible in F9 cells, albeit to a lesser extent than the -858/-601 construct, suggesting that additional AP-1 binding sites are present in the -834/-601 sequence (Fig. 4).
The possibility that the TIMP-1 TRE between -858/-834 may bind proteins other than c-Fos-c-Jun heterodimers was also supported by binding studies. The incubation of an oligonucleotide containing the TIMP-1 canonical AP-1 binding site with F9 cell extracts resulted in a DNA-protein complex whose electrophoretic mobility was different from that of DNA bound to purified c-Fos and c-Jun (Fig. 5C). We conclude, therefore, that although c-Fos-c-Jun heterodimers are potent transcriptional activators of the TIMP-1 AP-1 binding site between -858 and -834, other proteins, including other AP-1 family members, are capable of activating transcription through this site as well.
Figure 6:
Activation of TIMP-1 by AP-1 and c-Ets-1. A, sequence arrangement of the AP-1 and Ets DNA binding sites
in the TIMP-1 promoter. B, synergistic transcriptional
activation by c-Ets-1 and AP-1. The TIMP-1 promoter structure
-858/-601 was cotransfected with or without c-Ets-1
expression vector (3 or 6 µg) in the presence () or the
absence (
) of 0.05 µg of c-Jun and 0.05 µg of c-Fos.
The values were normalized to
-galactosidase activity as described
in the legend to Fig. 1. This experiment is representative of
three independent experiments.
To determine whether the synergistic transcriptional enhancement observed between c-Ets and AP-1 could be conferred upon a heterologous promoter, an oligonucleotide containing the AP-1 and Ets binding sites was placed upstream of tkCAT. After transfection into F9 cells, the results were similar to those obtained with the larger TIMP-1 promoter fragment. Neither AP-1 nor c-Ets-1 alone had much effect on the reporter plasmid's transcriptional activation potential at the concentrations used (Fig. 7). (Note that even at high concentrations AP-1 alone was able to activate at a multimerized but not a single binding site as shown above.) However, together they showed nearly a 10-fold induction over that of either protein alone (Fig. 7). This synergistic activation was sequence dependent for AP-1, because mutation of this binding site abolished transcriptional activation (Fig. 7). Although Ets has been shown to bind to this Ets site in vitro(5) , mutation of the binding site (see ``Experimental Procedures'') had little effect on transcriptional synergy with AP-1 in vivo (Fig. 7). These results indicate that the interaction between AP-1 and Ets may not require site-specific DNA binding by c-Ets-1 but rather may use protein-protein interactions between AP-1 and c-Ets-1 to achieve synergistic transcriptional regulation.
Figure 7:
Transcriptional activation of a 34-bp
oligonucleotide containing the TIMP-1 AP-1 and c-Ets binding sites
upstream of tkCAT. Oligonucleotides containing an Ets binding site (open oval) and an AP-1 binding site (black
rectangle) (WT), a wild-type Ets site and a mutant AP-1
site (mAP-1), or a wild-type AP-1 site and a mutant Ets site (mEts) were cotransfected with or without c-Ets expression
vector (4 µg) in the presence () or the absence (
) of
0.05 µg of c-Fos and 0.05 µg of c-Jun. Each data point
represents an average of three samples from the same experiment; the
values were normalized to protein concentration. This experiment is
representative of three independent
experiments.
Figure 8:
In vitro binding of AP-1 and
c-Ets-1. [S]Methionine-labeled c-Ets-1 protein
was produced by in vitro transcription and translation in
reticulocyte lysates (Promega). The labeled product was mixed with
unlabeled bacterially expressed and purified c-Jun and c-Fos and
incubated at ambient temperature for 30 min. The mixture was precleared
with an excess of protein A/G-agarose beads before the addition of
fresh beads and antibody against the amino terminus of c-Jun. The beads
were washed extensively before the proteins were subjected to gel
electrophoresis on a 10% SDS-polyacrylamide gel. Lane 1,
[
S]methionine-labeled c-Ets-1 product from in vitro translation; lane 2, labeled c-Ets-1 with no
added c-Jun or c-Fos (this lane represents the nonspecific association
of labeled c-Ets-1 with the c-Jun antibody-protein A/G-agarose resin or
interaction of c-Ets-1 with endogenous AP-1); lane 3, labeled
c-Ets-1 with c-Jun and c-Fos. The arrows indicate the c-Ets-1
protein translation products. This experiment is representative of
three independent experiments.
The TIMP-1 gene is precisely regulated in vivo, with high expression restricted to adult bone and ovary(9, 40, 41) . TIMP-1 is also induced in development (4, 9) and in areas of inflammation and during tissue remodeling (3, 5) when AP-1 expression is high(40, 42) . To elucidate some of the regulation of the TIMP-1 gene, we used F9 teratocarcinoma cells, which do not contain demonstrable AP-1 when they are undifferentiated. Upon differentiation to parietal endoderm, F9 cells up-regulate their endogenous TIMP-1 expression (4) and AP-1 activity(37, 38) . In these experiments we used TIMP-1 promoter structures in conjunction with the transcriptional regulatory proteins c-Ets-1 and AP-1 to investigate their roles in TIMP-1 transcriptional regulation. We functionally characterized three response elements necessary for TIMP-1 transcriptional enhancement (Fig. 9A). The first element consists of a high affinity canonical AP-1 site located at -858/-834 of the TIMP-1 promoter. Although this site was highly responsive to c-Jun and c-Fos heterodimers in vivo and bound purified AP-1 in vitro, it also responded to other AP-1 family members, including c-Jun homodimers, c-Fos, and JunD, as well as c-Fos and undetermined partners resident in F9 cells. Next to this canonical AP-1 site lies an Ets binding site that is entirely dependent on the activity of AP-1 at the adjacent binding site for Ets-dependent gene activation. Moreover, this Ets/AP-1 element in the form of an oligonucleotide transferred the AP-1-dependent Ets response to a heterologous promoter.
Figure 9: Schematic representation of the TIMP-1 promoter and its regulatory elements. A, The TIMP-1 promoter contains two AP-1 sites, a high affinity sequence (black rectangle) and a low affinity sequence (shaded rectangle). Next to the high affinity site is a c-Ets-1 binding site (open oval). Sequence inspection of the promoter revealed additional c-Ets-1 and AP-1 sites farther upstream, but they appear functionally inactive. The large hatched boxes represent TIMP exons. B, Model for TIMP-1 transcriptional regulation. In each panel, the solid line depicts DNA surrounding the TIMP-1 promoter. The various sized arrows represent relative levels of transcriptional activity. Large bisected rectangles marked J and F represent the Jun-Fos complex, and the large open oval marked ets represents c-Ets protein. Top panels, in the absence of AP-1 activity, Ets domain proteins fail to interact functionally with the promoter, and no transcriptional activity is observed. Middle panels, at low levels of AP-1 activity, the c-Jun-c-Fos complex binds and activates the promoter; c-Ets-1 interacts with both DNA and AP-1, producing a stable complex that synergistically enhances promoter function. Bottom panels, at high levels of AP-1 activity, both AP-1 sites are occupied and strongly enhance promoter function; under these conditions, c-Ets may not contribute substantially to transcriptional enhancement.
A third response element, between -834/-601 of the TIMP-1 promoter, also responds to AP-1 and was revealed when the canonical AP-1 site between -858/-834 was deleted. Although c-Fos and c-Jun heterodimers appear to be capable of binding and activating transcription through this site in the TIMP-1 promoter, other AP-1 family members are not and activate transcription exclusively through the high affinity TRE between -858/-834. These results suggest that AP-1 family members bind to different sites in the TIMP-1 promoter with different affinities, resulting in distinct patterns of TIMP-1 expression.
Both Ets and AP-1 families of transcription factors play important roles in differentiation and development(19, 20, 24, 40, 42) . Through cotransfection experiments with AP-1 and c-Ets expression vectors, as well as through coimmunoprecipitation experiments with AP-1 and Ets proteins, we have demonstrated that these transcriptional regulatory proteins functionally and physically interact. This mode of TIMP-1 regulation may be analogous to the complex formed at the serum response element by the interaction of the ubiquitous transcription factor, the serum response factor with the ternary complex factor, an Ets domain protein that cannot bind the serum response element by itself(43) . The ternary complex factor is recruited to the serum response element after phosphorylation in response to activation of the mitogen-activating protein kinase pathway by growth factor stimulation. Like ternary complex factor, perhaps phosphorylation of c-Ets-1 in response to growth factors results in the Ets-AP-1 complex formation and transcriptional regulation.
The role of Ets in transcriptional regulation is similar to that of proteins such as adenovirus E1a protein. Both E1a and Ets possess internal negative regulatory domains(44, 45, 46) . This inhibition is relieved by interaction with other transcription factors. Similarly, our results indicate that Ets activates TIMP-1 only in the presence of AP-1. It is likely that such interactions cause a conformational change in the Ets protein, enabling it to interact with components of the general transcriptional machinery and affect transcription. In fact, there is evidence that Ets may play a role in the formation of an initiation complex at minimal core promoters lacking the TATA sequence, such as TIMP-1 (reviewed in (18) ). This mode of action may be similar to the ability of E1a to regulate transcription of multiple genes that lack a common promoter element(46) .
On the basis of our results, we propose a simple model that accounts for both basal and induced TIMP-1 expression (Fig. 9B). According to this scheme, TIMP-1 expression would not be evident in the absence of AP-1; hence, TIMP-1 would be inactive in cells that lack AP-1 activity. Our results also indicate that the Ets-dependent activation of TIMP-1 is entirely dependent on the activity of AP-1 at the adjacent binding site; thus, c-Ets-1 alone would not be sufficient for TIMP-1 gene expression. The model further envisions that in the presence of low levels of Jun and Fos proteins, a functional AP-1-TIMP-1 complex is formed, resulting in low level basal expression; under these conditions, TIMP-1 can be induced by Ets proteins, which functionally interact synergistically with AP-1, thus leading to increased TIMP-1 expression. Finally, at high AP-1 concentrations, multiple AP-1 sites are occupied and cooperate to induce TIMP-1 transcription; Ets protein, under these conditions, would not substantially increase TIMP-1 expression. We conclude that TIMP-1 gene expression is controlled by both Ets and AP-1 family members, suggesting that multiple signal transduction pathways coordinate and regulate TIMP-1 expression.