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Transforming Growth Factor-β Stimulates Interleukin-11 Transcription via Complex Activating Protein-1-dependent Pathways*

Studies were undertaken to characterize the mechanism by which transforming growth factor-β1(TGF-β1) stimulates epithelial cell interleukin (IL)-11 production. Nuclear run-on studies demonstrated that TGF-β1 is a potent stimulator of IL-11 gene transcription. TGF-β1 also stimulated the luciferase activity in cells transfected with reporter gene constructs containing nucleotides −728 to +58 of the IL-11 promoter. Studies with progressive 5′ deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on 2 AP-1 sites between nucleotides −100 and −82 in the IL-11 promoter. Mobility shift assays demonstrated that TGF-β1 stimulated AP-1 protein-DNA binding to both AP-1 sites. Supershift analysis demonstrated that JunD was the major moiety contributing to AP-1-DNA binding in unstimulated cells and that c-Jun-, Fra-1-, and Fra-2-DNA binding were increased whereas JunD-DNA binding was decreased in TGF-β1-stimulated cells. The sequence in the IL-11 promoter that contains the AP-1 sites also conferred TGF-β1 responsiveness, in a position-independent fashion, on a heterologous minimal promoter. Thus, TGF-β1stimulates IL-11 gene transcription via a complex AP-1-dependent pathway that is dependent on 2 AP-1 motifs between nucleotides −100 and −82 that function as an enhancer in the IL-11 promoter.

Interleukin-11 (IL-11) 1 was originally discovered as a soluble factor in supernatants from transformed stromal cells that stimulated plasmacytoma cell proliferation (1). It has subsequently been shown to be a pleiotropic member of the IL-6-type cytokine family that mediates its biologic activities via binding to a multimeric receptor complex that contains the gp130 molecule (2)(3)(4)(5). Among its many effects are the ability to regulate hematopoiesis, stimulate the production of acute phase proteins, induce the tissue inhibitor of metalloproteinase-1, regu-late bone metabolism, and alter epithelial proliferation (2, 6 -10). Studies from our laboratories and others have also demonstrated that IL-11 can induce tissue fibrosis, regulate tissue myocyte and myofibroblast accumulation, alter airway physiology, and confer protection in the context of mucosal injury of the respiratory and gastrointestinal tracts (11)(12)(13)(14).
In keeping with the biologic importance of IL-11, a number of investigators have studied its sites of production and the regulation of these responses. These studies demonstrated that IL-11 is produced by a variety of stromal cells in response to a variety of stimuli, including cytokines, histamine, eosinophil major basic protein, and respiratory tropic viruses (7,(15)(16)(17)(18)(19)(20). A prominent finding in our studies of fibroblasts (18), epithelial cells (19), and osteoblasts (20) and studies by others of chondrocytes and synoviocytes (7) has been the importance of TGF-␤ moieties in the stimulation of IL-11 production. These studies also demonstrated that TGF-␤ 1 stimulation of IL-11 protein production is associated with proportionate changes in IL-11 mRNA accumulation and, in our studies, IL-11 gene transcription (18).
The IL-11 promoter has been cloned and the cis-elements and trans-acting factors that regulate the levels of basal IL-11 production have been identified by our laboratories (21). Despite the demonstrated importance of gene transcription in the stimulation of IL-11 production, the cis-elements and transacting factors that mediate the transcriptional activation of IL-11 have not been investigated. To further our understanding of the regulation of IL-11, studies were undertaken to characterize the transcriptional elements utilized by TGF-␤ 1 in the stimulation of IL-11. These studies identify two activating protein-1 (AP-1) motifs between Ϫ100 and Ϫ82 in the IL-11 promoter that are essential for TGF-␤-induced IL-11 transcriptional activation. They also demonstrate that this stimulation is associated with complex alterations in the composition of the AP-1 subunits that bind to these sites and that DNA which contains these AP-1 elements confers TGF-␤ 1 responsiveness on a heterologous promoter. Lastly they demonstrate that this mechanism is stimulus-specific since respiratory syncytial virus (RSV) stimulates IL-11 transcription via a different mechanism.

Cell Culture and TGF-␤ 1 Stimulation
A549 human alveolar epithelial-like cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (22). At confluence, varying concentrations of recombinant human TGF-␤ 1 (1-10 ng/ml) (R & D Systems, Minneapolis) or medium controls were added, and the cells were incubated for up to 48 h. At the desired points in time, supernatants were removed and stored at Ϫ20°C, and nuclei were harvested for further usage (see below). * This work was supported by National Institutes of Health Grants HL-36708, AI-34953, HL-54989, HL-56389 (to J. A. E.), DK-50570, and HL-48819 (to Y.-C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Nuclear Run-on Assay
The relative rates of gene transcription were assessed using modifications of protocols previously described by our laboratory (22)(23)(24)(25). A549 cells were incubated for 16 h under control conditions, with TGF-␤ 1 (10 ng/ml), or after infection with respiratory syncytial virus (RSV) at a multiplicity of infection of 3 as described previously (17)(18)(19). The cells (3 ϫ 10 7 per condition) were then washed twice with ice-cold phosphate-buffered saline, pelleted, and resuspended in lysis buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 , 3 mM CaCl 2 , 3 M dithiothreitol (DTT), 300 mM sucrose, 0.5% Triton X-100). The nuclei were then harvested by centrifugation and resuspended in 100 l of storage buffer (50 mM Tris-HCl, pH 8.3, 5 mM MgCl 2 , 0.1 mM EDTA, 40% glycerol) and stored at Ϫ80°C until further utilized. Nylon membranes were prepared carrying 20 g each of isolated cDNA fragments encoding IL-11 (a gift of Dr. Paul Schendel, Genetics Institute, Cambridge, MA) and pUC18 without a cDNA insert (a control for nonspecific hybridization) using a slot-blotting apparatus (MINI-FOLD II, Schleicher & Schuell) and baked in a vacuum oven (80°C for 2 h). When ready, nuclei were thawed on ice and pelleted in a microcentrifuge at 4°C for 30 s, and in vitro transcription and RNA labeling were carried out in transcription buffer (20 mM Tris-HCl, pH 8.3, 100 mM KCl, 4.5 mM MgCl 2 , 2 mM DTT, and 400 M each of ATP, GTP, and CTP) in the presence of 200 Ci of [␣-32 P]UTP (ϳ3000 Ci/mmol, Amersham Corp.) and 20% glycerol at 30°C for 30 min. The reaction was followed with a cold chase with 1 l of 100 mM UTP for 10 min at 30°C. The reaction was then terminated by incubating with stop buffer (50 mM Tris-HCl, pH 8.3, 500 mM NaCl, 5 mM EDTA) with 200 g/ml RNase-free DNase I and 750 units/ml RNasin (Boehringer Mannheim) at 30°C for 15 min. RNA was extracted with phenol/chloroform, precipitated, and washed with alcohol. Dried RNA pellets were dissolved in equal volumes of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.8), and radioactivity was determined by the mean of duplicate countings of 1-l aliquots. Hybridization was performed by incubating each membrane with equal numbers of counts of radiolabeled RNA. The membranes were then washed at high stringency, and binding was evaluated using autoradiography.

Primer Extension Analysis
A549 cells were incubated for 16 h with TGF-␤ 1 (10 ng/ml). The supernatants were then removed, and poly(A) ϩ RNA was isolated using oligo(dT) affinity based methodology as described (26,27). Primer extension was then performed using a radiolabeled 20-base complementary synthetic oligonucleotide corresponding to oligonucleotides Ϫ11 to ϩ9 with respect to the translation start site. The 5Ј end of the resulting IL-1 mRNA was defined using the Moloney murine leukemia virus primer extension system (Promega, Madison, WI) as described by the manufacturer. In this system the 5Ј end-labeled oligonucleotide hybridized with the IL-11 mRNA and was utilized as a primer by the Moloney murine leukemia virus reverse transcriptase which, in the presence of deoxynucleotides, synthesized cDNA until the 5Ј end of the mRNA was reached. The extended product was then resolved on an 8% urea/ polyacrylamide sequencing gel along with a known DNA sequence ladder.

Plasmid Construction
A 786-bp PvuII fragment of the human IL-11 promoter was previously isolated and cloned in our laboratories (21). This promoter fragment contained the sequences between Ϫ728 and ϩ58 relative to the transcription start site defined above. It was cloned into the SmaI site of the luciferase reporter gene vector pXP2-luc (ATCC) to generate the construct pXP2-IL-11-728.

Preparation of 5Ј Deletion Constructs
Two techniques were used to generate a series of 5Ј deletions of the pXP2-IL-11-728 parent construct. When appropriate restriction sites were present, they were utilized to generate deletion mutants. This approach was utilized with the AvaII site at Ϫ324 and the HinfI site at Ϫ96. In both cases, the Ϫ728 to ϩ58 fragment of the IL-11 promoter was subjected to enzyme digestion, and the desired fragment was recloned into the vector pXP2-luc using standard approaches. When appropriate restriction sites were not available Bal-31 exonuclease digestion was employed to introduce deletions. This technique takes advantage of the fact that Bal-31 degrades both the 5Ј and 3Ј ends of double-stranded DNA without inserting internal cleavages (28). Briefly, BamHI-linearized parent construct pXP2-IL-11-324 was incubated with Bal-31 exonuclease for varying periods. BamHI linkers (New England Biolabs, catalogue number 1071, Beverly, MA) were then added, and the DNA was subjected to BamHI/XhoI double digestion.
The DNA fragments with the various 5Ј deletions were then separated by electrophoresis, electroeluted, and ligated into BamHI/XhoIlinearized pXP2-luc vector. Clones from the subsequent transformation were screened for insert size, and DNA sequencing was used to verify junction sequences for all clones that were chosen for further utilization.
Through the combined efforts of both approaches, a series of constructs were prepared whose 5Ј ends extended from Ϫ728 to Ϫ81. In all cases, the 3Ј end was ϩ58 relative to the transcription initiation site.

Preparation of IL-11/tk/luc Constructs
A 156-bp BamHI/XhoI fragment from the herpes simplex virus thymidine kinase (tk) minimal promoter/chloramphenicol acetyltransferase reporter gene construct ptk-CAT (31) was obtained from Dr. Anuradha Ray (Yale University, New Haven) and subcloned into the BamHI and XhoI sites of the pXP2-luc reporter gene construct to generate ptk-XP2. Two oligonucleotides (5Ј-GAT CCG AGG GTG AGT CAG GAT GTG TCA GGC CGA AGC TT-3Ј and 5Ј-GAT CAA GCT TCG GCC TGA CAC CTG ACT CAC CCT CG-3Ј) were then synthesized and annealed to form a 38-bp DNA duplex with sticky ends compatible with the BamHI site (5Ј of herpes simplex virus tk promoter) in ptk-XP2. This insert contains a 27-bp DNA sequence of the IL-11 promoter (Ϫ103 to Ϫ77 relative to transcription start site) that contains both the 5Ј and 3Ј AP-1 sites. This double-stranded DNA sequence was then cloned into ptk-XP2 in the correct (sense) (pIL11(ϩ)tk-XP2) and reverse (antisense) (pIL11(Ϫ)tk-XP2) directions. DNA sequencing was performed to verify the sequence and orientation of DNA insertion.

Cell Transfection and Reporter Gene Assay
A549 cells were seeded at 40 -50% confluence and incubated overnight in DMEM with high glucose and 10% fetal bovine serum. Transfections were performed using the DEAE-dextran method as described previously by our laboratory (22). The cells were then incubated for 24 h in serum-free DMEM alone or in DMEM supplemented with TGF-␤ 1 (10 ng/ml). In experiments where RSV was utilized the A549 cells were incubated for 90 min with RSV (multiplicity of infection ϭ 3) or appropriate medium control, washed, and then incubated for 24 h in serumfree DMEM. At the end of these incubations, cell lysates were prepared, and luciferase activity was assessed on a Lumat luminometer using the Luciferase Assay System from Promega (Madison, WI). In all transfections the construct pCMV-␤-gal (CLONTECH, Palo Alto, CA) was also included to control for transfection efficiency. The ␤-galactosidase activity in unstimulated and stimulated cell lysates was characterized using the CPRG method as described previously by this laboratory (22). The ␤-galactosidase levels were then used to standardize the measurements of luciferase activity.

Electrophoretic Mobility Shift Assay (EMSA)
Preparation of Nuclear Extracts-Nuclear extracts were prepared using modifications of the techniques of Schreiber et al. (32). Unstimulated, TGF-␤ 1 -stimulated, and RSV-infected A549 cells were prepared as noted above. At the desired points in time, the cells (10 7 per condition) were mechanically detached, suspended in Tris-buffered saline freshly supplemented with protease inhibitors (1 g/ml leupeptin, 5 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), pelleted at 4°C, and resuspended, and swollen in solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT with freshly added protease inhibitors as above) for 15 min on ice. Membrane lysis was accomplished by adding 25 l of Nonidet P-40 followed by vigorous agitation. The nuclei were collected by centrifugation, resuspended in 50 l of solution B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and freshly added protease inhibitors as above), and agitated vigorously at 4°C for 15 min. The membrane debris was discarded, and the nuclear extracts were snap-frozen in small aliquots and stored at Ϫ80°C. The protein concentrations of the nuclear extracts were determined using the DC Protein Assay System (Bio-Rad).
Electrophoresis-EMSAs were performed using the techniques of Schreiber et al. (32). Radiolabeled double-stranded oligonucleotide probes were prepared by annealing complementary oligonucleotides and end-labeling using [␥-32 P]ATP and T4 polynucleotide kinase (New England Biolabs). The labeled probes were purified by push-column chromatography, diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to the desired concentration, and incubated with equal aliquots of nuclear extract (2-5 g) and 2 g of poly[dI-dC]⅐poly[dI-dC] in a total volume of 20 l at room temperature for 1 h. Resolution was accomplished by electrophoresing 10 l of the reaction solution on vertical 6% native polyacrylamide gels containing 2% glycerol using 25% TBE buffer (22.3 mM Tris-HCl, 22.3 mM boric acid, 0.25 mM EDTA, pH 8.0). Binding was assessed via autoradiography.
Supershift EMSA-Supershift assays were used to determine which members of the AP-1 family were involved in TGF␤ 1 -stimulation of IL-11 gene transcription. In these studies EMSA were performed as described above except that isotype matched rabbit polyclonal antibodies against AP-1 proteins or control preimmune antiserum were included during the 1-h radiolabeled probe-extract incubation period. All of the antibodies that were used were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). They included antibodies that react with all Jun family members (Pan-Jun) (catalogue number SC-44X), JunB (catalogue number 46X), JunD (catalogue number SC-74X), c-Jun (catalogue number SC822X); all Fos family members (pan-Fos) (catalogue number SC-253X), c-Fos (catalogue number SC-52X), FosB (catalogue number SC-48X), Fra-1 (catalogue number SC-605X), and Fra-2 (catalogue number SC604X).

Respiratory Syncytial Virus (RSV) Preparation and Infection
RSV (A-2 strain) was obtained from the ATCC. Stock virus was prepared in permissive cell lines and titered, and A549 cells were infected with the virus as described previously by this laboratory (17,19).

TGF-␤ 1 and RSV Stimulate IL-11 Gene Transcription-Pre-
vious studies from our laboratory demonstrated that TGF-␤ 1 is a potent stimulator of IL-11 gene transcription in lung fibroblasts (18) and that TGF-␤ 1 and RSV stimulate A549 alveolar epithelial cell IL-11 protein production and mRNA accumulation (17,19). To determine if both stimuli augment IL-11 gene transcription in A549 cells, nuclear run-on assays were performed, and the levels of IL-11 gene transcription were evaluated at base line, after TGF-␤ 1 stimulation and after RSV infection. At base line, the levels of IL-11 gene transcription in A549 cells were near the limits of detection with our assay (Fig.  1). In contrast, both TGF-␤ 1 and RSV caused significant increases in IL-11 gene transcription (Fig. 1).

Characterization of the Transcription Start Site in the IL-11
Promoter-Prior to initiating studies designed to define the stimulation-responsive cis-elements in the IL-11 promoter, primer extension analysis was used to characterize the transcription initiation site in TGF-␤ 1 -stimulated A549 cells. A single transcription initiation site was detected. This start site was 154 bp upstream of the ATG (data not shown) and is within 1-2 bases of the start site previously described in unstimulated PU-34 primate bone marrow fibroblasts by our laboratories (21).
TGF-␤ 1 Stimulates IL-11 Promoter Activity-To begin to characterize the mechanism by which TGF-␤ 1 stimulates IL-11 gene transcription, transient transfection assays were performed with a promoter-luciferase reporter gene construct containing IL-11 promoter elements between nucleotides Ϫ728 and ϩ58 (relative to the transcription start site). The levels of luciferase activity in A549 cells were evaluated at base line and after TGF-␤ 1 stimulation. As can be seen in Fig. 2, only a modest level of luciferase activity was able to be detected in unstimulated A549 cells. In contrast, TGF-␤ 1 was an impressive, dose-dependent stimulator of the promoter activity of this construct (Fig. 2). This demonstrates that the Ϫ728 to ϩ58 fragment of the IL-11 promoter contains TGF-␤ 1 -responsive sequences.
Previous studies from our laboratory demonstrated that RSV also stimulates A549 cell IL-11 production and mRNA accumulation (17,19). The studies noted above demonstrate that this stimulation is, at least in part, transcriptionally mediated. To determine if the cis-elements in the IL-11 promoter that respond to TGF-␤ 1 also respond to RSV, we compared the ability of these two stimuli to stimulate the Ϫ728 to ϩ58 IL-11 promoter-luciferase construct. As noted above, TGF-␤ was a potent stimulator of this construct. In contrast, RSV, an equally potent stimulator of IL-11 transcription in the run-on assays, did not stimulate this construct in an impressive fashion (data not shown). Thus, the response elements between Ϫ728 and ϩ58 in the IL-11 promoter are at least partially stimulus-specific since they respond vigorously to TGF-␤ 1 but not vigorously to RSV.
Definition of the TGF-␤ 1 Response Element in the IL-11 Promoter-To define further the cis-element(s) in the IL-11 promoter that responds to TGF-␤ 1 , intrinsic restriction sites and Bal-31 digestion were employed to obtain IL-11 promoter fragments that contain progressively larger 5Ј deletions. These promoter constructs were then cloned into our luciferase reporter construct, and their responsiveness to TGF-␤ 1 was as- sessed. Constructs whose 5Ј end extended from Ϫ728 to Ϫ81 were generated and tested. The parent (Ϫ728 to ϩ58) and all of the 5Ј deletion constructs did not express significant levels of luciferase activity in unstimulated A549 cells. TGF-␤ 1 , however, was a potent stimulator of IL-11 promoter-driven luciferase activity in the parent construct (Fig. 3). Interestingly, 5Ј deletions extending from Ϫ728 to Ϫ100 did not significantly alter TGF-␤ 1 responsiveness (Fig. 3). In contrast, deletions past Ϫ100 markedly diminished TGF-␤ 1 responsiveness. When stimulated with TGF-␤ 1 , these constructs had Յ5% of the TGF-␤ 1 inducibility of the wild type Ϫ728 to ϩ58 parent construct (Fig. 3). These studies demonstrate that elements that are essential for TGF-␤ 1 induction exist proximal to nucleotide Ϫ100 in the IL-11 promoter.
Importance of AP-1 Sites in TGF-␤-mediated Transcriptional Activation-Inspection of the sequence immediately 3Ј of Ϫ100 in the IL-11 promoter demonstrated two AP-1-like elements separated by 3 base pairs (Fig. 4). To define the role that these elements play in conferring TGF-␤ 1 responsiveness, constructs were prepared that contained point mutations at these sites, individually and in combination. Neither mutation caused a significant increase in the basal levels of IL-11 promoter activity (Fig. 5). Individual mutation in the 5Ј (distal) or 3Ј (proximal) sites caused an approximately 75% decrease in TGF-␤ 1 responsiveness (Fig. 5). Interestingly, the simultaneous mutation of both the 5Ј and the 3Ј AP-1-like sites abrogated TGF-␤ 1 responsiveness in this system. These constructs had Յ5% of the TGF-␤ 1 responsiveness of the wild type promoter-luciferase construct (Fig. 5). These studies demonstrate that both of these AP-1 sites play important roles in TGF-␤ 1 -induced IL-11 activation.
Effect of TGF-␤ 1 on AP-1 Protein-DNA Binding-To gain additional insight into the trans-acting factors that bind to the AP-1 sites in the IL-11 promoter, electrophoretic mobility shift assays (EMSA) were performed using labeled oligonucleotides identical to the 5Ј and 3Ј AP-1-like sequences in the IL-11 promoter. At base line, protein-DNA binding was detected at the 5Ј site but not the 3Ј site (Fig. 6). TGF-␤ 1 stimulation caused an impressive increase in binding to the 5Ј site. This stimulation was noted 2-6 h and peaked approximately 12-24 h after the addition of TGF-␤ 1 to the A549 cell cultures (Fig. 6).
TGF-␤ 1 stimulation also induced protein-DNA binding at the 3Ј AP-1 site in the IL-11 promoter. This induction had a slower kinetic than the induction at the 5Ј site. It was, however, readily detected after 12-24 h of TGF-␤ 1 -cell incubation (Fig.  6). In both cases, the protein-DNA binding was eliminated by the addition of excess unlabeled oligonucleotides encoding the 5Ј AP-1 site, 3Ј AP-1 site, or a consensus AP-1 sequence but not by oligonucleotides encoding NF-B, AP-2, or SP-1 sequences (data not shown). When viewed in combination, these studies demonstrate that TGF-␤ 1 enhances AP-1 family protein binding to both the 5Ј and 3Ј AP-1 sites in the IL-11 promoter.
Effects of TGF-␤ 1 on AP-1 Subunit Composition-To understand further the trans-acting factors binding to the AP-1 sites in the IL-11 promoter, supershift EMSAs were performed using antibodies to a variety of AP-1 family proteins. At both the 5Ј and 3Ј sites the importance of AP-1 family members was confirmed since anti-pan Jun and anti-pan Fos antibodies (antibodies against all Jun proteins and all Fos family proteins, respectively) caused impressive supershifts in this assay ( Fig. 7 and data not shown). In addition, characteristic patterns of AP-1 subunit usage were noted when selective antibodies were employed. At base line, the binding to the 5Ј IL-11 AP-1 site was composed almost entirely of JunD AP-1 moieties (Fig. 7). Interestingly, TGF-␤ 1 increased the contribution of c-Jun, Fra-1, and Fra-2 proteins while simultaneously decreasing the contribution of JunD proteins to the 5Ј AP-1-DNA binding. Significant alterations in c-Fos, FosB, and JunB were not noted (Fig. 7). Similar alterations in AP-1 subunit binding to the 3Ј site were noted (data not shown). These studies demonstrate that TGF-␤ 1 stimulation of IL-11 gene transcription is associated with impressive and selective alterations in the composition of the AP-1 moieties that bind to the IL-11 promoter. These changes are characterized by an enhanced contribution from c-Jun, Fra-1, and Fra-2 at the 5Ј and 3Ј sites and a simultaneous decrease in the base line binding of JunD to the 5Ј site.
TGF-␤ 1 Response Elements Confer Responsiveness on a Minimal Promoter Construct-To understand further the TGF-␤ 1 response elements in the IL-11 promoter, studies were undertaken to determine if they could confer TGF-␤ 1 responsiveness on a minimal promoter-reporter gene construct. This was done by generating constructs containing IL-11 promoter fragments between Ϫ103 and Ϫ77, in the sense and antisense direction, in series with the herpes simplex virus minimal promoter and luciferase reporter gene. The activity of these constructs in the presence and absence of TGF-␤ 1 was compared with the activity of the minimal promoter-luciferase construct under the same conditions. The parent minimal promoter-luciferase construct did not demonstrate significant levels of activity in the presence or absence of TGF-␤ 1 (Fig. 8). In contrast, TGF-␤ 1 was a potent stimulator of the sense and antisense IL-11 promoterminimal promoter-Luc constructs (Fig. 8). These studies demonstrate that the sequences between Ϫ103 and Ϫ77 in the IL-11 promoter act as an enhancer and confer TGF-␤ 1 responsiveness on heterologous promoter elements. DISCUSSION Studies from a number of laboratories have demonstrated that TGF-␤ 1 is a potent stimulator of IL-11 production. To understand further the biology of IL-11, the mechanism of this stimulation was characterized. Nuclear run-on studies demonstrated that this stimulatory effect is, at least in part, transcriptionally mediated, and transient transfection assays demonstrated that TGF-␤ 1 is a potent stimulator of IL-11 promoterreporter gene constructs. Two AP-1 motifs located between nucleotides Ϫ100 and Ϫ82 in the IL-11 promoter were noted to be necessary for this induction. In addition, a 25-base pair nucleotide sequence that contains these AP-1 sites was able to FIG. 2. TGF-␤ 1 stimulation of IL-11 promoter-luciferase activity. A549 cells were transfected with wild type IL-11 promoter-luciferase constructs that contained IL-11 sequences extending from nucleotides Ϫ728 to ϩ58 relative to the transcription start site (pXP2-IL-11-728). The cells were then incubated for 24 h in the presence and/or absence of the noted concentrations of TGF-␤ 1 ; cell lysates were prepared, and luciferase activity was quantitated as described under "Experimental Procedures." The luciferase activities in the lysates from cells incubated with concentrations of TGF-␤ 1 Ͻ10 ng/ml are expressed as a percent of the activity in lysates from cells incubated with 10 ng/ml TGF-␤ 1 . The noted values represent the mean Ϯ S.E. of three separate determinations.
confer TGF-␤ 1 responsiveness on a heterologous minimal promoter. Supershift EMSA demonstrated that JunD is the major AP-1 protein binding promoter DNA in unstimulated cells. TGF-␤ 1 stimulation was associated with enhanced protein-DNA binding to both AP-1 motifs with an augmented contribution by c-Jun, Fra-1, and Fra-2 proteins and a decreased contribution by JunD proteins. These are the first studies to investigate, in depth, the mechanisms that stimulate IL-11 gene transcription. All in all, they demonstrate the importance in this inductive response of complex alterations in AP-1 transcription factors and a cis-enhancer element that contains two closely approximated AP-1 motifs.
TGF-␤ 1 is a pleiotropic cytokine with far reaching effects on tissue homeostasis. Prominent in this regard are its ability to stimulate tissue fibrosis, regulate matrix molecule elaboration, and inhibit tissue inflammation. In keeping with its biologic importance, a significant amount of effort has been directed at characterizing the mechanism(s) by which TGF-␤ mediates its biologic effects. Studies of TGF-␤ 1 regulation of cytokine production (33), cell proliferation (34), and retinoic acid receptor expression (35) have demonstrated that AP-1 activation can play a major role in these processes. Our studies add to this body of data by demonstrating that TGF-␤ 1 stimulation of IL-11 elaboration is also mediated via an AP-1-dependent pathway.
AP-1 was initially identified as a DNA binding activity in HeLa cell extracts that bound to cis-elements within the promoter and enhancer sequences of the human metallothionein IIA gene and simian virus 40 (36). It is now a term that is used to refer to dimeric proteins produced by the complex immediate-early gene family that couple a variety of extracellular stimuli from the cell surface to the nucleus to initiate alterations in gene expression and cell phenotype (37,38). Investi-

FIG. 5. Effect of AP-1 mutations on TGF-␤ 1 responsiveness.
Luciferase reporter gene constructs were prepared that contained IL-11 promoter sequences extending from Ϫ324 to ϩ58 relative to the transcription start site (pXP2-IL-11-324). They were then transfected into A549 cells, and luciferase activities were assessed in the presence (solid bars) and absence (open bars) of TGF-␤ 1 (10 ng/ml) treatment. Comparisons were made of wild type constructs and constructs with mutations in the 5Ј AP-1 site, 3Ј AP-1 site, and both AP-1 sites (double mutant). Luciferase activities are expressed as a percent of the activity in lysates from cells transfected with the wild type construct and stimulated with TGF-␤ 1 . The noted values represent the mean Ϯ S.E. of at least three separate determinations.
gations of these moieties have revealed a consensus binding site for these dimers, the palindromic sequence, 5Ј-TGA(G/ C)TCA-3Ј (36). Our studies demonstrated that the sequence between Ϫ100 and Ϫ82 plays a crucial role in the transcriptional activation of the IL-11 promoter. Inspection of this region revealed a classic AP-1 binding site (the distal (5Ј) AP-1 sequence) and a binding site that differs at a single nucleotide (the proximal (3Ј) AP-1 sequence). Both sites were shown to bind AP-1 moieties. In addition, mutation of each AP-1 site significantly decreased and the simultaneous mutation of both AP-1 sites totally abrogated TGF-␤ 1 -induced IL-11 promoter activation. Furthermore, oligonucleotides made up largely of these 2 AP-1 sites conferred TGF-␤ 1 responsiveness upon a minimal heterologous promoter. These studies demonstrate that both AP-1 sites play important roles in IL-11 transcriptional activation and the enhancer function of this crucial promoter region. This is in keeping with studies of other genes such as involucrin (39) and tissue factor (40) whose promoters contain multiple AP-1 sites that play important roles in gene activation.
TGF-␤ 1 is one of a number of stimuli that induce epithelial cell IL-11 production in vitro. Previous studies from our laboratory demonstrated that a variety of respiratory tropic viruses also stimulate A549 cell production of IL-11 protein and the accumulation of IL-11 mRNA (17,19). The studies in this manuscript demonstrate that RSV stimulates A549 cell IL-11 production via a mechanism that is, at least in part, transcriptionally mediated. They also demonstrate that the magnitude of induction of IL-11 induced by RSV is comparable to that seen with TGF-␤ 1 . At the start of these studies, we hypothesized that RSV and TGF-␤ 1 would activate the IL-11 promoter via similar mechanisms. This did not, however, prove to be true. Although RSV was as potent as TGF-␤ 1 in the induction of IL-11 protein production, mRNA accumulation, and gene transcription, it did not efficiently stimulate the IL-11 promoter-reporter gene constructs used in these studies. This demonstrates that TGF-␤ 1 and RSV activate the IL-11 promoter via different mechanisms. It also provides insight into the stimulus specificity of the pathways used to activate IL-11 gene transcription.
AP-1 proteins are made up of homo-and heterodimers composed of Fos, Jun, and activating transcription factor subunits (37). At least 24 different combinations have been described (36). This complexity is the result of important tissue-specific, stimulus-specific, and temporally regulated differences in AP-1 subunit expression (36,40,41). This results in AP-1 moieties that differ in their DNA binding capacities, transactivation capacities, and biologic effects (42)(43)(44). To gain insight into the AP-1-mediated events involved in TGF-␤ 1 stimulation of IL-11, we used supershift gel mobility shift assays to define the Fos and Jun proteins involved in this inductive response. These studies demonstrate that, in the absence of stimulation, JunD is the major AP-1 moiety that can be detected. This is in keeping with studies from a number of different laboratories demonstrating that JunD is constitutively expressed in a variety of cells and tissues (45) and studies from our laboratories that demonstrate that JunD plays an important role in the constitutive elaboration of IL-11 by PU34 cells (21). Our studies also demonstrate that TGF-␤ 1 stimulation is associated with an increase in c-Jun, Fra-1, and Fra-2 and a decrease in FIG. 6. Electrophoretic mobility shift assay (EMSA) characterizing the protein binding to the 5 and 3 AP-1 sites in the IL-11 promoter. A549 cells were incubated in medium alone or in the presence of TGF-␤ 1 for the noted periods. Nuclear lysates were prepared and EMSA were performed using oligonucleotides encoding the 5Ј and 3Ј AP-1 sites in the IL-11 promoter as described under "Experimental Procedures." The arrows highlight the shifted bands.

FIG. 7. Characterization of the TGF-␤ 1 -induced alterations in AP-1 subunit binding to the 5 (distal) AP-1 site in the IL-11
promoter. A549 cells were incubated in medium alone or with TGF-␤ 1 (10 ng/ml) for the noted periods. Nuclear lysates were then prepared, and supershift EMSA were performed as described under "Experimental Procedures." A, comparisons are made of the protein-DNA binding seen with preimmune antiserum (Pre-immune), antibody that binds all Jun moieties (Pan-Jun Ab), antibody to JunB proteins (JunB Ab), antibody to c-Jun proteins (c-Jun Ab), and antibody to JunD proteins (JunD Ab). B, comparisons are made of the effects of antibodies that bind to all Fos moieties (Pan-Fos Ab), antibody that binds c-Fos proteins (c-Fos Ab), antibody that binds FosB proteins (FosB Ab), antibody that binds Fra-1 proteins (Fra-1 Ab), and antibody that binds Fra-2 proteins (Fra-2 Ab). In this figure the nonspecific bands are highlighted by the arrows labeled A; the TGF-␤ 1 -induced AP-1 binding reactions are highlighted by the arrows labeled B, and the supershifted bands are highlighted by the arrows labeled C.
JunD-IL-11 promoter binding. These observations are in accord with the known inducibility of these AP-1 subunits and the well documented stimulus specificity of AP-1 subunit induction (36,40,43,44,46,47). It is impossible to determine, from these studies, the degree to which the TGF-␤ 1 -induced alterations in c-Jun, Fra-1, Fra-2, and/or JunD individually contribute to the induction of IL-11 studied in the present analysis. It is possible, however, to hypothesize that all may play an important role. JunD is a potent transactivator but, in contrast to many other AP-1 subunit moieties, is not induced in a major fashion by extracellular stimuli (21,45). The JunD homodimers that would form in unstimulated cells under conditions of JunD excess would therefore play an important role in the regulation of basal IL-11 production but be unable to meet the enhanced transcriptional demands after TGF-␤ 1 stimulation. In contrast, TGF-␤ 1 stimulation enhances the contribution of c-Jun, Fra-1, and Fra-2 resulting in AP-1 dimers that are responsive to the conditions of stimulation. The entry of c-Jun, Fra-1, and Fra-2 into the AP-1⅐DNA binding complex can thus be speculated to be a key event underlying TGF-␤ 1 -stimulated transcription of the IL-11 gene in A549 cells. A similar transcriptional activation paradigm has been proposed to explain the contribution of AP-1 in the stimulation of tissue factor by serum in mouse fibroblasts (40).
Our studies demonstrate that two closely approximated AP-1 sites in the IL-11 promoter play important roles in the stimulation of IL-11 gene transcription by TGF-␤ 1 . It is important to keep in mind, however, that transcriptional activation is frequently a multi-factorial process that involves the concerted and coordinated interaction of a number of different transcription factors. This is well documented for the AP-1 moieties that are known to interact with a variety of other transcription factors including nuclear factor-B (NF-B), CREB, nuclear factor IL-6 (NF-IL-6), liver regeneration factor-1 (LRF-1), and polyoma virus enhancer A binding protein-3 (PEA 3 ) (36, 48). The transcriptional activities of AP-1 moieties are also regulated by a variety of other proteins including the Jun dimeriz-ing protein (37) and the Jun activation domain binding protein 1 (JAB1) (37). In accord with this information, it is important to point out that the present studies, while implicating AP-1 in the regulation of TGF-␤ 1 -stimulated IL-11 transcription, do not address the importance of each of these other moieties. It is likely that additional investigations will demonstrate that other cis-elements and/or trans-activating factors are involved in the coordinated production of IL-11 under a variety of circumstances.
In summary, the present studies demonstrate that TGF-␤ 1 stimulates IL-11 gene transcription in A549 cells and that this stimulation is mediated via a complex AP-1-dependent activation pathway. They also highlight two closely approximated AP-1 sites in the IL-11 promoter that are essential for this activation and demonstrate that DNA sequences that contain these two sites can confer TGF-␤ 1 responsiveness on a heterologous minimal promoter. Lastly, these studies demonstrate that, in the absence of TGF-␤ 1 stimulation, JunD is the major AP-1 subunit involved in IL-11 promoter-protein binding and that TGF-␤ 1 stimulation is associated with increased c-Jun, Fra-1, and Fra-2 and decreased JunD-DNA binding. FIG. 8. Demonstration that the double AP-1 sequence in the IL-11 promoter can confer TGF-␤ 1 responsiveness on a heterologous promoter. Luciferase reporter constructs were prepared that contained the herpes simplex virus tk minimal promoter and the sequence in the IL-11 promoter from nucleotide Ϫ103 to Ϫ77 (a stretch that contains both AP-1 sites) inserted in a sense (pIL11(ϩ)tk) and antisense (pIL11(Ϫ)tk) orientation. These constructs were transfected into A549 cells, and the luciferase activities in these cells were assessed after incubation for 24 h in medium alone or with TGF-␤ 1 (10 ng/ml). In these experiments the responses of the IL-11 minimal promoter-luciferase constructs were compared with the responses of the parent tkminimal promoter-luciferase construct (ptkXP2). The luciferase activities in these experiments are expressed as a percent of the luciferase activities in cells transfected with pIL-11(ϩ)tk and stimulated with TGF-␤ 1 . The noted values represent the mean Ϯ S.E. of a minimum of three separate experiments.