Transcriptional Induction of Collagenase-1 in Differentiated Monocyte-like (U937) Cells is Regulated by AP-1 and an Upstream C/EBP-beta Site*

(Received for publication, January 22, 1997, and in revised form, February 26, 1997)

Glenn A. R. Doyle Dagger , Richard A. Pierce and William C. Parks §

From the Dermatology Division, Department of Medicine, and § Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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-beta (C/EBP-beta ) 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.


INTRODUCTION

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 -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).

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-beta (C/EBP-beta ) site, participate in achieving maximal and sustained PMA-mediated collagenase-1 transactivation in monocytic cells.


EXPERIMENTAL PROCEDURES

Cell Culture

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 × 10-8 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.

RNA and Transcription Analyses

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 epsilon -globin gene (42) was blotted as well.

Plasmids and Constructs

Fig. 1 shows maps of all collagenase-1 promoter constructs used in this study. pBLCAT2 contains the -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 (-2010Delta -1954 and -1197Delta -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.
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The internal deletion construct, p-1197Delta -997CAT, was created by cutting p-2278CAT with BglII and BamHI followed by ligation of the vector. Internal deletion construct p-2010Delta -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).

Heterologous promoter constructs contain collagenase-1 promoter sequences upstream of -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).

Stable U937 Transfectants

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).

Transient Transfections

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.

CAT Assays

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.

Electrophoretic Mobility Shift and Supershift Assays

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 -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-kappa B consensus oligomer (Promega Corp., Madison, WI). Double-stranded oligomers were radiolabeled with [gamma -32P]ATP using T4 polynucleotide kinase or with [alpha -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-beta 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-kappa 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-kappa 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-beta -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.
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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-beta [Delta 198], respectively. The antibodies specific to c-Jun, JunB, JunD, c-Fos, FosB, and C/EBP-beta were c-Jun/AP-1[N], JunB[N-17], JunD[329], c-Fos[4], FosB[102], and C/EBP-beta [C-19], respectively. All antibodies were purchased from Santa Cruz Biotechnology.

Western Analysis of Nuclear Proteins

Equivalent amounts of nuclear proteins were prepared for electrophoresis by adding 1 volume of 2 × sample buffer and beta -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.


RESULTS

Kinetics of Collagenase-1 Expression

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, -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 × 10-8 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 beta -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.
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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.
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The AP-1 Site Is Not Necessary for Delayed Induction or Maintenance of Collagenase-1 Transcription

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 -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).

AP-1 Independent Induction of the Collagenase-1 Promoter Is Not Restricted to Monocytic Cells

PMA treatment stimulated activation of the wild-type collagenase-1 promoter in human skin fibroblasts (Fig. 4, -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.
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c-Jun and c-Fos Are Present in Early and Late U937 Nuclear Extracts

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.


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 × 10-8 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.
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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).

Nuclear Proteins c-Fos and c-Jun Bind the Collagenase-1 AP-1 Site

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).


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.
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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.

Transient Transfection of Collagenase-1 Promoter Constructs in U937 Cells

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 (-2278 to -511) and downstream (-511 to +36) regions.

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 -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)]

In constructs containing the wild-type AP-1 element, deletion of sequences -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-1197Delta -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-2010Delta -1954CAT, numbers 4-9).

To further characterize the upstream regions of the collagenase-1 promoter (-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)]

C/EBP-beta Is Present in Nuclear Extracts of U937 Cells and Interacts with Collagenase-1 Promoter Sequences between -2010 and -1954

The data with the mutant AP-1 construct (Fig. 3, -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-kappa 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-beta (Fig. 9). Furthermore, these analyses demonstrated that the relative abundance of C/EBP-beta in U937 nuclear extracts increased with time of PMA treatment. The C/EBP-beta -specific antibody detected a doublet in which the upper band may be the phosphorylated form of the lower band (59).


Fig. 9. C/EBP-beta 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-beta -specific or pan-C/EBP antibodies. Both antibodies detected a band at 42 kDa (arrows), consistent with the size of C/EBP-beta . 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)]

To determine if C/EBP-beta 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-kappa 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-kappa 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-kappa 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-beta binds the collagenase-1 sequence between -2013 and -1990. Both pan-C/EBP and C/EBP-beta -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-beta , but not NF-kappa B, is the factor with increased activity in PMA-differentiated cells that binds the collagenase-1 promoter between -2013 and -1990.


DISCUSSION

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-beta may mediate the AP-1-independent response and maximize AP-1-dependent responses in differentiated monocytes.

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 -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).

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 -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.

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 -1954 and -179 (Fig. 7). This suppression was not seen with p-2278CAT, p-2010CAT, and p-1197Delta -997CAT, nor was it seen with smaller constructs (p-179CAT, p-95CAT, and p-72CAT). Furthermore, the p-2010Delta -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.

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 -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-beta 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-beta 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.

The C/EBP family consists of five proteins (alpha , beta , delta , gamma , 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-beta 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-beta 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-beta may be a necessary factor for normal monocyte/macrophage development and function. Notably, C/EBP-delta , which forms heterodimers with C/EBP-beta 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-beta and C/EBP-delta expression is stimulated or induced in various other tissues by inflammatory mediators such as interleukin-1, interleukin-6, and tumor necrosis factor-alpha (71, 72). Thus, while our data suggest that C/EBP-beta is involved in inducing collagenase-1 expression in monocytes, C/EBP-beta may also be involved in activating collagenase-1 transcription in other cell types once C/EBP-beta expression has been induced by inflammatory factors.


FOOTNOTES

*   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.
Dagger    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-beta , CCAAT/enhancer-binding protein-beta ; kb, kilobase(s); TK, thymidine kinase; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

ACKNOWLEDGEMENTS

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.


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