(Received for publication, January 22, 1997, and in revised form, February 26, 1997)
From the Dermatology Division, In this report, we demonstrate that the AP-1 site
and a distal promoter element regulate transcriptional induction of
collagenase-1 during monocytic differentiation. Chloramphenicol
acetyltransferase expression constructs containing regions of the human
collagenase-1 promoter were stably or transiently transfected into U937
cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated
differentiation. Rapid and strong induction of promoter activity was
lost in constructs with a mutant AP-1 element; however, at 16-96 h
post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent
PMA-mediated transactivation. The AP-1 mutant constructs also showed
delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins
were present in nuclear extracts of PMA-differentiated U937 cells.
Promoter deletion constructs demonstrated the potential role of distal
promoter sequences in regulating collagenase-1 transcription. In
particular, Western, supershift, and promoter deletion analyses
suggested a role for CCAAT/enhancer-binding protein- Remodeling of the extracellular matrix during normal development
and in response to tissue injury and inflammation is thought to be
accomplished, in part, by the properly regulated production of matrix
metalloproteinases (MMPs).1 As a group,
these enzymes can degrade essentially all extracellular matrix
components, and hence, they have been implicated in normal remodeling
processes, such as uterine involution, blastocyst implantation, angiogenesis, and wound healing (for review, see Refs. 1-3). However,
inappropriate expression of these proteinases is thought to contribute
to the pathogenesis of various conditions, such as arthritis (4, 5),
vascular disease (6-8), metastasis (3), and destructive skin diseases
(9). Notably, collagenase-1 has been localized to resident and
infiltrating inflammatory cells in many of these conditions (5, 7,
10-12).
Although extracellular matrix proteins can be degraded by various
proteinases, fibrillar type I collagen, the most abundant protein in
the body, is resistant to degradation by most enzymes. Collagen
degradation is initiated by the catalytic activity of collagenases, a
subgroup of the MMP gene family with the unique ability to cleave
fibrillar collagens type I, II, and III within their triple helical
domain (13). At physiological temperature, cleaved collagen molecules
denature and become susceptible to complete digestion by other
proteinases. Of the three known human metallo-collagenases,
collagenase-1 (MMP-1) seems to be the enzyme that is principally
responsible for collagen turnover in most human tissues. In a variety
of normal and disease-associated tissue remodeling events,
collagenase-1 is expressed by macrophages as well as by epithelial
cells, fibroblasts, endothelial cells, and chondrocytes (14-18).
Collagenase-2 (MMP-8) is found only in neutrophils and chondrocytes
(19, 20), and collagenase-3 (MMP-13), originally cloned from a breast
carcinoma line (21), is also expressed in articular cartilage (22, 23)
and developing bone (24).
Many agents, such as PMA, bacterial endotoxin (lipopolysaccharide), and
proinflammatory cytokines, and events, such as contact with type I
collagen and activated T-cells, induce or markedly stimulate
collagenase-1 transcription in macrophages (25-28). Much of what is
known about the transcriptional regulation of collagenase-1 points to a
critical role for the AP-1 site at We assessed the requirement of the AP-1 site and more distal promoter
sequences to collagenase-1 gene activation during and subsequent to
monocytic differentiation. We used PMA-treated U937 cells as an
in vitro model because they mimic the differentiation of
monocytes into macrophages (33) and because activation of collagenase-1
expression in these cells occurs strictly by a transcriptional mechanism (27, 34). We report that collagenase-1 promoter activity is
induced and maintained in the absence of a functional AP-1 site. We
conclude that although the AP-1 site is required to mediate strong
collagenase-1 transcription, other upstream elements, including a newly
identified CCAAT/enhancer-binding protein- U937 cells (35) were obtained from the
American Type Culture Collection (CRL 1593) and maintained in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with
10% low endotoxin fetal calf serum (Life Technologies, Inc.),
non-essential amino acids, L-glutamine, sodium pyruvate,
100 units/ml penicillin, and 100 µg/ml streptomycin. For induction of
cell differentiation, U937 cells were plated at 5 × 105 cells/ml and exposed to 8 × 10 Total RNA was isolated by
the guanidinium phenol extraction method (36). Conditions for Northern
hybridization and washes were as described (27). Blots were hybridized
with a 2.2-kb human collagenase-1 (37), a 2.0-kb human c-fos
(38), a 1.2-kb human c-jun (39), or a 1.3-kb rat
glyceraldehyde-3-phosphate dehydrogeanse (40) random-primed,
32P-labeled cDNA probe. Filters were washed and then
visualized by autoradiography. Transcription rates of specific
mRNAs were measured using 2.5 × 107 isolated
nuclei as described (41). Nascent RNA transcripts were isolated, and
equivalent counts of 32P-labeled RNA were hybridized to
denatured, gel-purified cDNA inserts slotted on nitrocellulose. As
an indicator of total transcription, a 2.5-kb pair human Alu repeat
fragment derived from the Fig. 1 shows maps of
all collagenase-1 promoter constructs used in this study. pBLCAT2
contains the
The internal deletion construct, p-1197 Heterologous promoter constructs contain collagenase-1 promoter
sequences upstream of U937 cells (107 in
0.5 ml) were transfected with 5 µg of linearized pRSV-Neo and 50 µg
of linearized pBLCAT2, pAPCAT2a, p-72CAT, p-511CAT, p-2278CAT, or
p-2278MCAT. Cells were electroporated at 250 V and 600 microfarads in a
0.4-cm gap cuvette using a BTX 3000 electroporator (Biotechnologies and
Experimental Research, Inc., San Diego, CA), placed on ice for 10 min,
added to 9 ml of culture medium, centrifuged to pellet the cells, and
plated in 10 ml of fresh medium. After 24 h, cells were shifted to
medium supplemented with 400 µg/ml Geneticin® (Life
Technologies, Inc.). After 2 weeks, G-418-resistant cells were
subcloned by limiting dilution or maintained as a pooled population of
clones in medium containing 200 µg/ml Geneticin®. To
minimize insertion effects, two groups of stable clones, one consisting
of 6 clones and the other of 12 clones, were pooled. Southern
hybridization with 32P-labeled CAT cDNA was done on
individual clones and demonstrated that incorporated DNA was roughly
equivalent among clones (data not shown).
U937 cells were transfected by a
modification of the DEAE-dextran method essentially as described (26,
46). Human skin fibroblasts were transiently transfected by calcium
phosphate precipitation. After transfection, cells were allowed to
recover for 24 h prior to treatment with PMA. After recovery,
cultures were divided equally and cells were plated in medium with or
without PMA. After transfection, cells were given fresh medium and
allowed to recover for 24 h prior to treatment with PMA. Hirt
extraction (47) and Southern hybridization with 32P-labeled
CAT cDNA was done to determine transfection efficiency.
At the indicated times, cells were harvested,
washed, and lysed in 200 µl of 250 mM Tris, pH 7.8, by
freeze-thawing. Lysates were incubated at 65 °C for 5 min and then
cleared of debris by centrifugation. Equivalent amounts (25-100 µg)
of cleared lysate, normalized to total protein (Bradford protein assay;
Bio-Rad), were assayed for CAT activity using acetyl-CoA (Sigma) and
[14C]chloramphenicol essentially as described (48).
Reaction products were separated by thin layer chromatography and
visualized by autoradiography. Results were quantified by cutting and
counting the appropriate spots from the chromatography plate. Relative induction was obtained by dividing percent acetylation of treated versus untreated samples.
Nuclear extracts were prepared by the method of Dignam
et al. (49). The integrity of all nuclear extract
preparations was assessed by determining the ability of proteins to
bind a radiolabeled Oct-1 double-stranded oligomer (data not shown).
Oct-1 protein binding was constitutive and, thus, served as an internal
control. Only extracts without apparent protein degradation were used. For AP-1 studies, double-stranded oligomers containing either wild-type
(GATCAAAGCATGAGTCAGACACCT) or mutant (GATCAAAGCAcccgggAGACACCT) human
collagenase-1 promoter sequence were used as probes and competitors.
For the binding analyses to the upstream region at
Supershift reactions were identical to those described above, except 1 µg of appropriate antibody was added to the binding reactions after
addition of the labeled probes and reactions were incubated overnight
at 4 °C prior to electrophoresis. The pan-Jun, pan-Fos, and
pan-C/EBP antibodies were c-Jun/AP-1[D], c-Fos[4-10G], and
C/EBP- Equivalent amounts of
nuclear proteins were prepared for electrophoresis by adding 1 volume
of 2 × sample buffer and We reported that
collagenase-1 transcription is induced in U937 cells at 16-24 h after
exposure to PMA (34). We used various assays to more carefully examine
the kinetics of this differentiation-dependent induction.
Collagenase-1 mRNA was not detected in untreated U937 cells (Fig.
2A). By 12 h post-PMA, collagenase-1
mRNA was detected, increased by 24 h, and remained elevated at
48 h post-PMA. Nuclear run-off assays demonstrated that
collagenase-1 transcription was detectable at 12 h of PMA
differentiation and remained at a constant level thereafter (Fig.
2B). These observations demonstrate that the onset of
collagenase-1 induction occurs earlier than reported previously (34).
CAT activity conferred by the full-length collagenase-1 promoter
construct (p-2278CAT) in transiently transfected, PMA-treated U937
cells paralleled the pattern of induction of the endogenous gene (Fig.
2C). Only slight background CAT activity was seen in untreated cells. By 4 h post-PMA, promoter activity was increased, and maximal and sustained levels of CAT activity were achieved by
8 h post-PMA. Consistent results were obtained in four separate experiments. A similar time course for induction of collagenase-1 promoter activity was observed in stable transformants (Fig.
3,
Stable transformants were created
to determine if a functional AP-1 site is needed for collagenase-1
induction. The proximal AP-1 site in the PMA treatment stimulated activation
of the wild-type collagenase-1 promoter in human skin fibroblasts (Fig.
4,
The kinetics of c-fos and c-jun
expression were assessed by Northern analysis (data not shown). In
contrast to the delayed kinetics of collagenase-1 induction (Fig. 2)
and in full agreement with data from others (50-52), c-fos
and c-jun transcripts were detected as early as 15 min
post-PMA, peaked between 1 and 2 h after PMA addition, and were
sustained at low levels over the next 48 h (data not shown).
Because c-Fos protein expression may not correlate with expression of
its mRNA in U937 cells (50) and because its subcellular
localization is regulated (53), we used an immunoblotting assay to
detect Fos family proteins in nuclear extracts from untreated and
PMA-differentiated U937 cells. c-Fos protein was detected in both 1- and 24-h post-PMA nuclear extracts using pan-Fos or c-Fos-specific
antibodies (Fig. 5). The upward shift in the c-Fos band
seen in the 24-h extract may be due to increased protein
phosphorylation (54). While the presence of c-Fos in nuclear extracts
at 1 h post-PMA was anticipated, the clear abundance of c-Fos
protein in the 24-h extract was not. A previous report indicated that
c-Fos protein could not be detected in PMA-differentiated U937 cells
after 2 h of treatment (50). Because these authors
immunoprecipitated metabolically-labeled protein from whole cell
extracts, they may have underestimated c-Fos protein levels during
periods of low c-Fos protein synthesis. We detected no FosB protein by
immunoblotting nuclear extracts from untreated or PMA-treated U937
cells with a FosB-specific antibody (data not shown). Proteins distinct
from c-Fos were detected by the pan-Fos antibody in the nuclear extract from untreated cells, but not in those from PMA-treated cells. The
identity of these proteins is uncertain, but their sizes are consistent
with the Fos-related proteins Fra-1 (29.4 kDa) and Fra-2 (35.2 kDa)
which are expressed in U937 cells (55). Regardless of the identity of
the bands in basal cell extracts, only c-Fos was detected in nuclear
extracts of PMA-differentiated U937 cells (Fig. 5). The low molecular
weight forms seen in the 1- and 24-h extracts with the c-Fos-specific
antibody are probably c-Fos degradation products.
We also used the immunoblotting assay to detect Jun family proteins in
nuclear extracts from untreated and PMA-differentiated U937 cells.
Using a c-Jun-specific antibody, c-Jun protein was detected in nuclear
extracts of U937 cells treated with PMA for 1 or 24 h (Fig. 5).
The high molecular weight bands detected in all extracts may be due to
ubiquitination (56) or altered phosphorylation of c-Jun (57). Because
c-Jun is not expressed in basal U937 cells, the low molecular mass
bands between 30 and 20 kDa seen in all samples are likely nonspecific
products. No additional bands were detected with the pan-Jun or JunB
antibodies (data not shown).
Electrophoretic mobility shift and supershift assays were
done to confirm the presence of active AP-1 binding Jun/Fos dimers. Nuclear extracts from untreated cells did not support binding to a
double-stranded oligomer containing the native collagenase-1 AP-1 site,
where as extracts from 4, 8, and 24 h PMA-treated cells exhibited
strong binding activity (Fig. 6). The binding activity was competed by
excess unlabeled wild-type AP-1 oligomer (Fig. 6,
24C) but not by excess oligomer containing the mutated AP-1 site or by an unrelated sequence (data not shown). In addition, radiolabeled, double-stranded mutant AP-1 oligomers showed no binding
to nuclear proteins (data not shown).
Supershift analysis demonstrated that JunD and c-Fos were present in
the shifted complexes (Fig. 6). We consistently detected a weak
supershifted complex with the c-Jun antibody and a very weak, if any,
complex for JunB. Neither the relative amounts of shifted complexes nor
their composition changed at any time after the onset of PMA
differentiation. A FosB-specific antibody did not supershift complexes
formed in extracts from PMA-treated cells (data not shown). The lack of
other Fos family proteins agrees with our immunoblotting data (Fig. 5).
Although we cannot definitively determine the identity of the Jun
component, these results suggest that heterodimers of c-Fos and Jun
family proteins contribute to maximal collagenase-1 transcriptional
induction at early and late times of U937 differentiation.
Because the mutant AP-1 collagenase-1 promoter was induced
by PMA, we assessed the influence of regions upstream of the proximal AP-1 site during collagenase-1 induction in U937 cells. Cells were
transfected with the various promoter constructs, and CAT activity was
assessed at 24 h after addition of PMA. Southern hybridization of
Hirt extracted DNA indicated equivalent transfection efficiency among
constructs and that the level of plasmid DNA remained constant up to
72 h after transfection (48 h post-PMA, data not shown). For
presentation, we have divided the collagenase-1 promoter into upstream
( Relative to p-2278CAT (number 1), induction of construct p-511CAT
(number 11) was reduced by 50% in response to PMA differentiation (Fig. 7), and p-511CAT had similar activity in PMA-treated stable transformants (data not shown). Deletion of sequences between
In constructs containing the wild-type AP-1 element, deletion of
sequences To further characterize the upstream regions of the collagenase-1
promoter (
The data with the mutant AP-1 construct (Fig. 3,
To determine if C/EBP- Data presented here, as well as in other studies (27, 34), show
that once collagenase-1 production is induced in macrophages, enzyme
expression remains active for days. In this report, we characterized
regions of the collagenase-1 promoter which are involved in both
activation and maintenance of collagenase-1 transcription during and
subsequent to U937 differentiation. Our findings, in agreement with
others (30, 31), indicate that the proximal AP-1 element is necessary
but not sufficient to confer maximal transcriptional activation of
collagenase-1. We also report that mutation of the AP-1 element reduces
and delays but does not eliminate maintained collagenase-1 promoter
induction in U937 cells or fibroblasts. The sustained nature of the
delayed, AP-1-independent induction of collagenase-1 suggests that the
mediating factor(s) may be important in regulating maintained
collagenase-1 expression by macrophages actively involved in tissue
remodeling events associated with inflammation. As is discussed,
C/EBP- Studies on the collagenase-1 promoter have concluded that the proximal
AP-1 site is a key element necessary for rapid and full stimulation of
gene transcription, regardless of the cell type or stimulus used.
Typically, c-Jun/c-Fos heterodimers are believed to stimulate
collagenase-1 transcription by binding the proximal AP-1 element after
PMA treatment (60). Although Jun family homo- or heterodimers can bind
the collagenase-1 AP-1 site to activate transcription, the affinity of
such Jun dimers for the AP-1 site is far weaker than that of the
corresponding Jun/c-Fos heterodimers (29, 61). We find that c-Fos is
the only Fos family protein expressed in U937 nuclei after PMA
differentiation (Fig. 5) and that c-Fos is a component of the observed
AP-1 binding complexes (Fig. 6). This observation strongly suggests a
prominent role for c-Fos in regulating collagenase-1 transcription in
monocytic cells, but the identity of the corresponding Jun family
member is less apparent. Although our Western data indicate abundant c-Jun protein in PMA-differentiated U937 cells (Fig. 5), our supershift analyses demonstrated weak activity for c-Jun yet strong binding for
JunD (Fig. 6). Angel and Karin (60) showed that expression of c-Jun,
but not JunB or JunD, is necessary for collagenase-1 activation by PMA
in various cell types. Therefore, although a role for JunD cannot be
excluded, we suggest that c-Jun/c-Fos heterodimers are involved in
induction of collagenase-1 expression in U937 cells and stimulated
monocytes.
Comparable to findings in HeLa cells (30), but in contrast to those in
fibroblasts (31), we found that the AP-1 element confers a minimal
response to PMA in U937 cells (Fig. 7B). However, in
association with the AP-1 site, the region between Consistent with the kinetics of c-Fos and c-Jun expression, the AP-1
element is needed for rapid and strong PMA-mediated induction of
collagenase-1 transcription in U937 cells. However, our data indicate
that, distal upstream elements are required to maximize AP-1-dependent induction and can induce and maintain
collagenase-1 expression in an AP-1-independent manner. Most compelling
is that the AP-1 mutant construct, p-2278MCAT, displayed delayed, yet maintained, PMA-mediated activation in U937 cells and fibroblasts (Figs. 3 and 4). While Buttice et al. (63) showed that
mutation of the analogous AP-1 site in the related stromelysin-1
promoter did not fully eliminate PMA responsiveness in fibroblasts, to our knowledge this is the first report of AP-1 independent PMA-mediated collagenase-1 promoter induction in any cell type. We detected no CAT
activity in PMA-treated U937 cells transfected with either a While AP-1 factors are needed for the rapid and strong induction of
collagenase-1 in U937 cells (Fig. 3), our data suggest that cooperation
between distal upstream and downstream factors maintains gene
expression over extended periods. We observed that collagenase-1
promoter activity was suppressed by sequences between Heterologous promoter constructs p-2278TKCAT and p-2010TKCAT were
stimulated by PMA differentiation of U937 cells independent of any
downstream collagenase-1 AP-1 site, with the majority of this response
lost once sequences The C/EBP family consists of five proteins ( We thank Dr. Steven Frisch for the human
collagenase-1 promoter. We also thank Dr. Ulpu Saarialho-Kere for
assistance with the nuclear run-off assays and Dr. Howard Welgus for
many helpful suggestions and discussion.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(C/EBP-
)
binding site between
2010 and
1954 in regulating transcription of
collagenase-1 in monocytic cells. Our findings suggest that distinct
regulatory elements, acting somewhat independently of each other,
control expression of collagenase-1. In addition, our data suggests
that the rapid PMA-mediated induction of collagenase-1 transcription is
controlled by a mechanism distinct from that regulating the sustained
expression of this proteinase in activated macrophages.
72 to
66 in the human promoter.
AP-1 elements bind dimers of the Jun (c-Jun, JunB, and JunD) and Fos
(c-Fos, FosB, Fra-1, and Fra-2) families of transcription factors (29).
Angel et al. (30) first demonstrated that the AP-1 site is
necessary and sufficient to confer PMA-mediated induction of the native
collagenase-1 promoter or of a heterologous promoter containing this
element. However, because the level of PMA-mediated induction was
greater with larger collagenase-1 promoter constructs, they concluded
that elements upstream of the AP-1 site might also be important in
regulating collagenase-1. Indeed, the AP-1 site, the polyoma enhancer
A-binding protein-3 site (
91 to
83) and the "TTCA" element
(
105 to
102) are also required for full PMA-mediated induction in
fibroblasts (31, 32).
(C/EBP-
) site,
participate in achieving maximal and sustained PMA-mediated
collagenase-1 transactivation in monocytic cells.
Cell Culture
8
M PMA (Sigma). Human skin fibroblasts were grown in
Dulbecco's modified Eagle's medium, 10% fetal calf serum (Life
Technologies, Inc.) containing the same supplements listed above.
-globin gene (42) was blotted as well.
105/+51 region of the herpes simplex virus thymidine
kinase (TK) promoter fused to a CAT reporter gene (43). pAPCAT2a is
derived from pBLCAT2, and contains a tandem triplet of the
collagenase-1 AP-1 site subcloned 5
of the TK promoter. A plasmid
containing the
2278/+36 region of the human collagenase-1 promoter
(44) was generously provided by Dr. Steven Frisch (La Jolla Cancer
Research Foundation, La Jolla, CA). To eliminate the possibility of
transcriptional differences due to the vector backbone, all
collagenase-1 promoter sequences were subcloned into pBLCAT2. The TK
promoter of pBLCAT2 was removed during the synthesis of the
collagenase-1 promoter deletion constructs (Fig. 1A). The
p-2278CAT, p-2278MCAT, p-511CAT, p-179CAT, p-95CAT, and p-72CAT vectors
were generated by PCR as described (26). Constructs p-2010CAT,
p-1954CAT, p-1689CAT, p-1552CAT, p-1197CAT, and p-997CAT were made by
digestion of p-2278CAT with BsaHI, HpaI, XmnI, EcoRV, BglII, or
BamHI, respectively. The digestion products were blunted
(when necessary) with the Klenow fragment of DNA polymerase, further
digested with XhoI, and the appropriate
blunt/XhoI fragment was subcloned into
blunted-HindIII/XhoI digested pBLCAT2.
Fig. 1.
Human collagenase-1 promoter constructs.
A, 5 deletion and internal deletion (
2010
-1954 and
1197
-997) constructs containing the collagenase-1 TATA box and
transcription start site were made as described under "Experimental
Procedures." The mutant AP-1 construct (p-2278MCAT) contains a
SmaI restriction site in place of the wild-type AP-1
element. B, heterologous constructs contain upstream (
2278
to
997) regions of the human collagenase-1 promoter linked to the
thymidine kinase (TK) promoter of pBLCAT2. pAPCAT2a contains
3 collagenase-1 AP-1 sites in tandem upstream of the thymidine kinase
promoter of pBLCAT2.
[View Larger Version of this Image (21K GIF file)]
-997CAT, was created by
cutting p-2278CAT with BglII and BamHI followed
by ligation of the vector. Internal deletion construct
p-2010
-1954CAT was created by recombinant, whole plasmid PCR (45)
using the 5
(ATAgcatgcACCCTGGAAGAGTCTCAT) and the 3
(CGCgcatgcCTATTAACTCACCCTTGT) primers (deleted/mutated sequences in
lowercase). The PCR product was digested with SphI and
ligated. The resultant construct was digested with HindIII
and BamHI, and the HindIII/BamHI
fragment was subcloned into HindIII/BamHI cut
p-2278CAT. All PCR was performed with either VentTM or Deep
VentTM DNA polymerase (New England Biolabs, Beverly, MA) to
minimize unwanted mutations. All newly created plasmids were sequenced to verify that only the desired alterations were introduced during PCR
steps. Sequencing reactions were done using a
SequenaseTM Kit (U. S. Biochemical Corp., Cleveland,
OH).
997 linked to the TK promoter. These were
constructed by subcloning the BamHI/XhoI TK
promoter fragment of pBLCAT2 into deletion constructs that had been
digested with BamHI and XhoI. For example,
p-2278CAT gives rise to p-2278TKCAT, in which
2278 is the most 5
and
997 (at the BamHI site) is the most 3
nucleotide of the
collagenase-1 promoter fragment (Fig. 1B).
2010 to
1954,
double-stranded oligomers containing collagenase-1 promoter sequence
between
2013 to
1990 (TGACGTCTTAGGCAATTTCCTGTC),
1994 to
1968
(CTGTCCAATCACAGATGGTCACATCAC), and
1970 to
1947 (CACATGCTGCTTTCCTGAGTTAAC) were used as probes (1, 2, and
3 in Fig. 10). Competition was done with oligomers 1, 2, or
3, wild-type (TGCAGATTGCGCAATCTGCA) or mutant (TGCAGAgactagtcTCTGCA)
C/EBP consensus oligomers (Santa Cruz Biotechnology, Santa Cruz, CA), or with a wild-type (AGTTGAGGGGACTTTCCCAGGC) NF-
B consensus oligomer (Promega Corp., Madison, WI). Double-stranded oligomers were
radiolabeled with [
-32P]ATP using T4 polynucleotide
kinase or with [
-32P]dCTP using the Klenow fragment of
DNA polymerase. Binding reactions and electrophoresis conditions were
as described (26). Equivalent amounts of nuclear protein (5 µg) and
probe counts were used in all reactions.
Fig. 10.
C/EBP- binds to an upstream element of
the collagenase-1 promoter. Nuclear extracts were isolated from
U937 cells treated with PMA for 0, 4, 8, or 24 h and incubated
with a radiolabeled, double-stranded oligomer containing the
collagenase-1 C/EBP and NF-
B-like sites (region 1 in schematic).
Left panel, free probe (FP) was shifted
(S) by proteins in nuclear extracts from control (0) U937
cells, and binding activity was increased in extracts from PMA-treated
cells. Binding was competed by co-incubating extracts and probe with a
50-fold molar excess of unlabeled, wild-type C/EBP consensus
(W). Binding was not decreased by co-incubation with a
mutant C/EBP sequence (M) or with a wild-type NF-
B
consensus oligomer (not shown). Weak, nonmodulated, and no binding
activity were detected with radiolabeled, double-stranded oligomers to regions 2 and 3, respectively (data not shown). Right panel,
addition of either a pan-C/EBP or C/EBP-
-specific antibody to the
binding reactions resulted in a supershift (SS) of the
shifted probe (S). Identical results were obtained with a
second set of extracts.
[View Larger Version of this Image (65K GIF file)]
[
198], respectively. The antibodies specific to c-Jun, JunB, JunD, c-Fos, FosB, and C/EBP-
were c-Jun/AP-1[N],
JunB[N-17], JunD[329], c-Fos[4], FosB[102], and
C/EBP-
[C-19], respectively. All antibodies were purchased from
Santa Cruz Biotechnology.
-mercaptoethanol to 50 mM. Samples were boiled for 1 min and separated through a
10% SDS-polyacrylamide gel. Gels were equilibrated in 1 × transfer buffer (10 mM CAPS, 10% methanol, pH 11.0) prior
to transferring to polyvinylidene difluoride paper. After transfer,
membranes were blotted according to the procedures suggested by Santa
Cruz Biotechnology. Blots were developed using horseradish
peroxidase-conjugated secondary antibodies and the enhanced
chemiluminescence.
Kinetics of Collagenase-1 Expression
2278). Full induction of the wild-type
collagenase-1 promoter was detected at 6 h after PMA treatment,
and the levels were maintained for up to 96 h (Figs. 2C
and 3). These data indicate that events necessary for maximal induction
of collagenase-1 are activated within 4-6 h post-PMA.
Fig. 2.
Collagenase-1 transcription is induced in
PMA-treated U937 cells. U937 cells were treated with 8 × 108 M PMA for 4-48 h, then processed for:
A, Northern hybridization; B, nuclear run-off
analysis; or C, CAT assay. A, total RNA was isolated from U937 cells at the times indicated and 5 µg were analyzed by blot hybridization with 32P-labeled cDNAs
for collagenase-1 (C'ase) and glyceraldehyde-3-phosphate dehydrogeanse (GAPDH) mRNAs. Autoradiography was for
18 h. B, nuclei were isolated from control U937 cells
(
PMA) and from cells treated with PMA for 12, 24, or
48 h. Nascent transcripts were isolated, and equal amounts of
32P-labeled pre-mRNA were hybridized to an Alu repeat
sequence (Alu) or to full-length cDNAs for collagenase-1
(C'ase) and
-actin. The autoradiograms shown are of a
run-off experiment with nuclei from control (
) and 48 h
PMA-treated (+) U937 cells. Autoradiography was for 6 days. Band
intensity was quantified by densitometry. Background hybridization was
subtracted from gene-specific hybridization signal, and the data in the
histogram are expressed relative to the signal for Alu. Five
run-offs were done for the 0 and 48-h time points, and the results are
shown as the mean ± S.E. Transcription at 12 and 24 h
post-PMA was assessed once. C, U937 cells were transiently
transfected with p-2278CAT and treated with PMA 24 h later. Cells
were harvested and lysed at 4-48 h after the start of PMA exposure,
and CAT activity was assessed using 50 µg of cell lysates. The
results shown are representative of four separate experiments.
[View Larger Version of this Image (54K GIF file)]
Fig. 3.
Collagenase-1 promoters containing wild-type
or mutant AP-1 element are induced in stably transfected U937
cells. U937 cells were transfected with p-2278CAT or p-2278MCAT,
and stable clones were selected and pooled. 107 cells
of each population were treated with PMA for the times indicated, and
CAT activity in cell lysates was assessed. For the lines containing the
wild-type construct (2278), CAT assays were done with 25 µg of total protein for 24 h (left) or 12 h (right). For the mutant construct (
2278M)
lines, CAT assays were done with 100 µg of total protein for 24 h. For all constructs, results shown are representative data of three
experiments each with both pooled populations.
[View Larger Version of this Image (65K GIF file)]
2278/+36 promoter fragment
was replaced with a SmaI recognition site (Fig.
1A). Gel shift analysis with labeled mutant oligomer
demonstrated that the mutated AP-1 site does not bind nuclear proteins
(data not shown). In two groups of pooled clones, mutation of the AP-1
site (p-2278MCAT) eliminated the rapid (i.e. by 4-6 h) and
strong transactivation observed with the wild-type construct
(p-2278CAT) (Fig. 3). Between 0 and 8 h post-PMA, no CAT activity
was detected in U937 cells stably transfected with the mutant AP-1
construct (data not shown). However, transcriptional induction of the
mutant AP-1 collagenase-1 promoter was consistently detected at 16 h post-PMA (Fig. 3,
2278M). Although CAT activity expressed by
2278MCAT was much lower than that conferred by the wild-type
promoter, the level of CAT activity was maintained for up to 96 h
after PMA differentiation, similar to the sustained activity from the
wild-type promoter (Fig. 3). Experiments with individual stable clones
showed the same patterns of induction with both the wild-type and
mutant promoters (data not shown). Southern hybridization demonstrated
that incorporated DNA was roughly equivalent among stable lines (data
not shown).
2278). Basal activity of the wild-type
collagenase-1 promoter was high in these cells, likely due to
constitutive c-Jun expression (data not shown), but at 8 and 24 h
post-PMA, CAT activity increased. Mutation of the AP-1 site eliminated
the high basal activity seen with the wild-type collagenase-1 promoter
(Fig. 4,
2278M). However, similar to that observed in differentiated
U937 cells, p-2278MCAT conferred transcriptional induction in
transiently transfected fibroblasts at 8 and 24 h post-PMA (Fig.
4,
2278M). Thus, the collagenase-1 promoter can be transcriptionally
induced by PMA in the absence of a functional proximal AP-1 element.
Time matched controls incubated without PMA had the same level of CAT
activity for p-2278CAT or p-2278MCAT as did the 0 h cells (data
not shown).
Fig. 4.
The mutant AP-1 collagenase-1 promoter is
stimulated in fibroblasts. Human skin fibroblasts were transiently
transfected with either p-2278CAT or p-2278MCAT, and treated with PMA
24 h later. At the times indicated, cells were harvested and
lysed, and equal amounts of extract (50 µg) were assessed for CAT
activity.
[View Larger Version of this Image (50K GIF file)]
Fig. 5.
Jun and Fos family proteins are found in
nuclear extracts of U937 cells. Nuclei were isolated from control
U937 cells and from cells treated with 8 × 108
M PMA for 1 or 24 h. Nuclear proteins were extracted
and resolved by SDS-polyacrylamide gel electrophoresis, and Fos and Jun
family proteins were detected by immunoblotting. Both pan-Fos and
c-Fos-specific antibodies detected c-Fos protein (arrow). A
c-Jun-specific antibody detected c-Jun protein in 1- and 24-h extracts
(arrow). The numbers next to the gels indicate
the migration of molecular weight standards. The results shown are
representative of three experiments.
[View Larger Version of this Image (35K GIF file)]
Fig. 6.
AP-1 binding activity of Fos and Jun family
proteins in U937 nuclear extracts. Nuclear extracts from control
U937 cells and from cells treated with PMA for 4, 8, or 24 h were
incubated with a radiolabeled, double-stranded AP-1 probe. No binding
activity was seen in nuclear extracts of untreated U937 cells (0). With nuclear extracts from PMA-treated U937 cells, free probe
(FP) was shifted (S). The shifted band was
effectively competed by co-incubation with a 50-fold molar excess of
unlabeled probe (24C). Addition of antibodies to Fos family
(pan-Fos and c-Fos-specific) and Jun family proteins (pan-Jun, c-Jun,
and JunD, but not JunB) resulted in a supershift (SS)
species of the bound probe (S). Results shown are
representative of four experiments.
[View Larger Version of this Image (76K GIF file)]
2278 to
511) and downstream (
511 to +36) regions.
511 and
179 (p-179CAT, number 12) resulted in no further decrease in PMA
responsiveness relative to p-511CAT (number 11). However, PMA
responsiveness was further reduced when sequences between
179 and
95 were deleted (
95CAT, number 13). Weak, yet reproducible transcriptional induction was observed with the smallest AP-1 containing collagenase-1 promoter construct, p-72CAT (number 14). CAT
activity from this construct was also stimulated to a similar degree in
PMA-treated stably transfected U937 cells (data not shown). Thus, the
AP-1 site, in the absence of upstream sequences, was sufficient for
minimal PMA responsiveness. However, because the level of induction
observed with p-72CAT (number 14) is extremely low compared with most
other constructs, other upstream elements are needed for full
activation of collagenase-1 transcription by PMA differentiation.
Although mutation of the AP-1 site, in the context of the full-length
promoter (p-2278MCAT, number 2) resulted in a loss of detectable PMA
responsiveness in transiently transfected U937 cells, this construct
was induced in stably transformed cells (
2278M; Fig. 3). Hirt
extraction (47) verified that p-2278MCAT entered transiently
transfected cells with the same efficiency as other constructs (data
not shown). The seemingly contradictory transfection data with
construct p-2278MCAT (Figs. 3 and 7) can be reconciled
by the greater sensitivity inherent in the use of stable lines
versus transient transfections.
Fig. 7.
Deletion analysis reveals that upstream
promoter elements are needed to mediate full induction of collagenase-1
in response to PMA-differentiation. A, shown are
representative CAT assays for each deletion and mutant construct used.
U937 cells were transiently transfected with various constructs (see
Fig. 1), and half of the cells were treated with PMA for 24 h. CAT
activity in lysates (50 µg) was assessed for untreated () and
PMA-treated (+) cells. All procedures and manipulations were identical
for each construct series. In this figure, each construct is numbered
1-16 corresponding to the number below the histograms. AP
is pAPCAT2a, which contains 3 tandem repeats of the human collagenase-1
AP-1 site upstream of the thymidine kinase promoter, and BL
is the parental plasmid pBLCAT2. B, for each CAT assay, the
percent acetylation was determined by scintillation counting, and these
data are shown in the upper histogram. The relative stimulation
(fold change) of promoter construct activity in response to
PMA is shown in the lower histogram. A fold change equal to 1.0 indicates no difference in CAT activity between untreated and
PMA-treated samples. The results shown are the mean ± S.E. of
four to six determinations for each construct.
[View Larger Version of this Image (64K GIF file)]
2278 to
997 (p-997CAT, number 9) diminished both baseline
and PMA-induced transcription (Fig. 7), and the fold induction was
decreased about 2-fold relative to the wild type (p-2278CAT, number 1)
construct (Fig. 7B). Deletion of sequences between
2278
and
2010 (p-2010CAT, number 3) and between
1197 and
997
(p-1197
-997CAT, number 10) had only a minor effect on promoter
activity in basal U937 cells or induction in PMA-differentiated cells
(Fig. 7). In contrast, promoter activity was markedly reduced and fold
induction decreased relative to p-2278CAT (number 1) upon deletion of
sequences between
2010 and
1954 from the collagenase-1 promoter
(p-1954CAT to p-997CAT, and p-2010
-1954CAT, numbers 4-9).
2278 to
997), we constructed a series of heterologous promoter constructs containing various fragments of the distal collagenase-1 promoter linked to the TK promoter of pBLCAT2 (Fig. 1B). CAT activity from the TK promoter of pBLCAT2 was not
changed by PMA treatment of U937 cells (Figs. 7 and 8).
Constructs p-2278TKCAT and p-2010TKCAT clearly responded to PMA (Fig.
8). Like construct p-2278MCAT, these constructs conferred PMA
responsiveness in the absence of the proximal collagenase-1 AP-1
element. However, once sequences between
2010 and
1954 were
deleted, the heterologous constructs (p-1954TKCAT to p-1197TKCAT)
responded weakly or not at all to PMA treatment (Fig. 8).
Fig. 8.
Upstream collagenase-1 promoter regions
respond to PMA in the absence of the AP-1 element. Heterologous
constructs containing upstream (2278 to
997) regions of the human
collagenase-1 promoter linked to the thymidine kinase promoter of
pBLCAT2 (see Fig. 1). pAPCAT2a contains 3 tandem repeats of the human
collagenase-1 AP-1 site upstream of the thymidine kinase promoter of
the parental plasmid pBLCAT2. A, shown are representative
CAT data for each heterologous construct. U937 cells were transiently
transfected, and half of the cells were treated with PMA for 24 h.
CAT activity in cell lysates (50 µg) was assessed for untreated (
)
and PMA-treated (+) cells. All procedures and manipulations were
identical for each construct series. B, the percent
acetylation was determined by scintillation counting and these data are
shown in the histogram. The results shown are the mean ± S.E. of
at least four to six determinations for each construct.
[View Larger Version of this Image (46K GIF file)]
Is Present in Nuclear Extracts of U937 Cells and
Interacts with Collagenase-1 Promoter Sequences between
2010 and
1954
2278M)
and the drop in PMA-mediated transactivation between p-2010TKCAT and
p-1954TKCAT (Fig. 8) suggest the existence of functional
AP-1-independent element between
2010 and
1954 of the collagenase-1
promoter. We inspected this region of the promoter for known
transcription factor DNA-binding elements. A putative C/EBP-binding
site (TTAGGCAATT) and NF-
B-like site (GGCAATTTCC) were identified
between
2013 and
1990. Because the C/EBP family of transcription
factors can regulate cellular differentiation (58), we looked for the
presence of C/EBP proteins in U937 nuclear extracts. Immunoblotting of nuclear extracts with a pan-C/EBP antibody detected only one band of
about 42 kDa (Fig. 9). Detection with a specific
antibody verified that this band was C/EBP-
(Fig. 9). Furthermore,
these analyses demonstrated that the relative abundance of C/EBP-
in
U937 nuclear extracts increased with time of PMA treatment. The
C/EBP-
-specific antibody detected a doublet in which the upper band
may be the phosphorylated form of the lower band (59).
Fig. 9.
C/EBP- is in nuclear extracts of U937
cells. U937 cells were treated with 8 × 10
8
M PMA, and nuclear extracts were prepared from control
cells (0) and from cells at 4, 8, and 24 h post-PMA. Proteins were
separated by SDS-polyacrylamide gel electrophoresis, and the gels were
processed for immunoblotting with C/EBP-
-specific or pan-C/EBP
antibodies. Both antibodies detected a band at 42 kDa
(arrows), consistent with the size of C/EBP-
. The
numbers between the gels indicate the migration of molecular
weight standards. Identical results were obtained with extracts from
cells in three separate experiments.
[View Larger Version of this Image (31K GIF file)]
could bind the sequences between
2013 and
1990 of the collagenase-1 promoter, we performed gel shift and
supershift analyses with a double-stranded oligomer encompassing this
region (Fig. 10). Gel shift analysis demonstrated that
a nuclear factor in untreated and PMA-differentiated cells bound this
sequence (Fig. 10, left). In agreement with the
immunoblotting data, the quantity of shifted probe increased with time
after PMA treatment. To identify which site (C/EBP or NF-
B-like) in
this region bound the nuclear factors, we competed binding with
wild-type (W) or mutant (M) C/EBP consensus
oligomers or with a wild-type NF-
B consensus oligomer. The wild-type
C/EBP consensus oligomer competed nuclear factor binding to the
collagenase-1 sequence probe, whereas mutant C/EBP (Fig. 10,
left) or wild-type NF-
B oligomers did not (data not
shown). In addition, double-stranded oligomers to other regions of the
sequences between
2010 and
1954 (probes 2 and 3 in Fig. 10) did not
shift when incubated with nuclear extracts from control or PMA-treated
cells (number 3, data not shown) or had only weak binding activity
which was not modulated by PMA treatment (number 2, data not shown).
Supershift analysis confirmed that C/EBP-
binds the collagenase-1
sequence between
2013 and
1990. Both pan-C/EBP and
C/EBP-
-specific antibodies caused a supershift of the bound probe,
and the intensity of the super-shifted band increased after PMA
treatment (Fig. 10, right). Thus, these results suggest that
C/EBP-
, but not NF-
B, is the factor with increased activity in
PMA-differentiated cells that binds the collagenase-1 promoter between
2013 and
1990.
may mediate the AP-1-independent response and maximize
AP-1-dependent responses in differentiated monocytes.
179 and
95 of
the collagenase-1 promoter was needed for strong PMA responsiveness in
U937 cells (Fig. 7B). In fibroblasts, PMA- and
oncogene-mediated transactivation of collagenase-1 is enhanced when a
complete "12-O-tetradecanoylphorbol-13-acetate/oncogene responsive unit," polyoma enhancer A-binding protein-3, and AP-1 elements in tandem, is present in promoter constructs (32). In
monocytic cells, the polyoma enhancer A-binding protein-3 site (
91 to
83) may not be critical for up-regulating collagenase-1 expression
since only a small difference in promoter induction is seen between
constructs p-95CAT and p-72CAT, with both responses being relatively
weak (1.5-2-fold; Fig. 7B). Indeed, other studies have
shown that the 12-O-tetradecanoylphorbol-13-acetate/oncogene responsive unit is not sufficient to maximally stimulate collagenase-1 transcription in response to PMA (30, 31). In contrast to the weak
induction of p-72CAT and p-95CAT, p-179CAT confers much of the PMA
responsiveness (5-fold) observed in U937 cells (Fig. 7B). In
the analogous region of the rabbit collagenase-1 promoter, a "TTCA"
element (
105 to
100) and less characterized sequences at
182 to
161 are necessary to confer strong PMA responsiveness in fibroblasts
(31, 62). There is extensive homology (95-100% identical in the areas
mentioned) between the human and rabbit promoters within this
downstream region. This fact, together with the decreased PMA-mediated
induction observed when sequences
179 to
95 are deleted (Fig.
7B) suggests the possibility that factors binding these
sequences may play some role in activating collagenase-1 expression in
monocytic cells. Nonetheless, although this downstream region conferred
much of the PMA responsiveness, distal upstream promoter elements were
needed to achieve a maximal response (Fig. 7B).
511 or
179 deletion construct containing the AP-1 mutation (26). Similarly,
Jonat et al. (64) found no evidence of PMA-mediated induction of a
517/+63 collagenase-1 promoter fragment containing an
AP-1 mutation when assayed in HeLa cells. Thus, the delayed AP-1-independent response is likely controlled by a regulatory element(s) upstream of position
517.
1954 and
179
(Fig. 7). This suppression was not seen with p-2278CAT, p-2010CAT, and
p-1197
-997CAT, nor was it seen with smaller constructs (p-179CAT,
p-95CAT, and p-72CAT). Furthermore, the p-2010
-1954CAT internal
deletion construct had diminished transcriptional activity relative to
p-2278CAT. Thus, we suggest that a suppressive element is located
between
511 and
179 and that factors bound between
2010 and
1954 overcome this suppression to enhance AP-1 dependent responses.
Imai et al. (65) have proposed a model in fibroblasts in
which the collagenase-1 promoter is brought into an active conformation
by the interplay of regulatory factors which bind elements between
1705 and
1595. Similarly, we speculate that factors bound between
2010 and
1954 might help maintain collagenase-1 expression in
PMA-differentiated U937 cells by interacting with downstream factors to
overcome the potential inhibitory effect of the intervening
sequences.
2010 to
1954 were deleted (Fig. 8). The
proserpine-binding site located at
1704 to
1689 (65) may function
in monocytic cells because weak PMA responsiveness is lost when this
sequence is deleted from heterologous promoter constructs (Fig. 8).
However, this effect is minimal compared with the decreased PMA
responsiveness caused by deletion of sequences between
2010 and
1954 from heterologous promoter constructs. Notably, no PMA
responsiveness from heterologous constructs containing upstream
collagenase-1 promoter regions was observed in HeLa cells (30). In the
context of the wild-type collagenase-1 promoter, sequences between
2010 to
1954 were necessary to achieve maximal collagenase-1
promoter induction in U937 cells (Fig. 7B). In addition, deletion of this region caused decreased collagenase-1 promoter activity in untreated and PMA-differentiated U937 cells (Fig. 7B). In contrast, deletion of
2010 to
1954 did not
decrease basal collagenase-1 promoter activity when assayed in
fibroblasts (65). We identified a putative C/EBP-binding site (
2006
to
1997) within this distal promoter region. Importantly, C/EBP-
protein capable of binding this collagenase-1 promoter site is present
in untreated U937 nuclear extracts, and levels are increased in
PMA-differentiated U937 cells (Figs. 9 and 10). Together, these data
suggest that C/EBP-
may mediate, in part, the observed
AP-1-independent activation and maintenance of collagenase-1 expression
and specifically enhance AP-1 dependent responses in
monocyte/macrophage cells.
,
,
,
, and
CRP-1) which are bZIP transcription factors that form homo- and heterodimers to bind the consensus sequence (NT(T/G)NNGNAA(T/G)) (66).
Although C/EBP-
is found in many tissues (67), it seems to play a
prominent role in activating and regulating gene expression in
monocytes and macrophages (59, 68, 69). C/EBP-
expression is induced
during later stages of monocytic, but not granulocytic differentiation
(69), is constitutively low in monocyte/macrophages (68, 70), and is
strongly stimulated in macrophages by inflammatory mediators, such as
lipopolysaccharide (71). Therefore, C/EBP-
may be a necessary factor
for normal monocyte/macrophage development and function. Notably,
C/EBP-
, which forms heterodimers with C/EBP-
to synergistically
activate transcription, is also strongly induced in monocytic cells by
lipopolysaccharide (72), an agent that potently stimulates
collagenase-1 expression in macrophages (27). In addition, C/EBP-
and C/EBP-
expression is stimulated or induced in various other
tissues by inflammatory mediators such as interleukin-1, interleukin-6,
and tumor necrosis factor-
(71, 72). Thus, while our data suggest
that C/EBP-
is involved in inducing collagenase-1 expression in
monocytes, C/EBP-
may also be involved in activating collagenase-1
transcription in other cell types once C/EBP-
expression has been
induced by inflammatory factors.
*
This work was supported by Grants HL29594, HL48762, and
HL54049 from the National Institutes of Health and by a grant from the
Council for Tobacco Research.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.
Current address: Dept. of Oncology, McArdle Laboratories for
Cancer Research, University of Wisconsin, Madison, 1400 University Ave., Madison, WI 53706-1599. Tel.: 608-263-4767; Fax: 608-262-2824; E-mail: doyle{at}oncology.wisc.edu.
¶
To whom correspondence should be addressed: Div. of
Dermatology, Jewish Hospital, 216 S. Kingshighway, St. Louis, MO 63110. Tel.: 314-454-7543; Fax: 314-454-5372; E-mail:
bparks{at}imgate.wustl.edu.
1
The abbreviations used are: MMP, matrix
metalloproteinase; PMA, phorbol 12-myristate 13-acetate; C/EBP-,
CCAAT/enhancer-binding protein-
; kb, kilobase(s); TK, thymidine
kinase; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain
reaction; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.