Vol. 10, No. 4
MOLECULAR AND LELLULAR lI1OLOGY, Apr. 1990, p. 1492-149/
0270-7306/90/041492-06$02.00/0 Copyright © 1990, American Society for Microbiology
Autoinduction of Transforming Growth Factor the AP-1 Complex
Is Mediated by
KAZUE HATTORI,' KYUNG YOUNG KIM,' MICHAEL B. SPORN,' MICHAEL KARIN,' AND ANITA B. ROBERTS' Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892,1 and Department of
KIM,'* PETER ANGEL,2 ROBERT LAFYATIS,1
Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 920932 Received 7 September 1989/Accepted 9 December 1989
The multifunctional actions of transforming growth factor ,1 (TGF-131) indicate that it has a pivotal control function in many physiological and pathological processes. An important property of TGF-11 is its ability to activate its own mRNA expression and thereby increase its own secretion. Two distinct regions of the promoter of the TGF-,1 gene are responsive to autoregulation: one 5' to the upstream transcriptional start site and another located between the two major start sites. In both promoter regions, autoinduction is mediated by binding of the AP-1 (Jun-Fos) complex. An important contribution to this positive regulation is the autoactivation of c-jun transcription by AP-1. Cotransfection of antisense c-jun or antisense c-fos expression vectors prevents TGF-131 autoinduction. These results demonstrate that both components of the AP-1 complex are required for TGF-11 autoinduction. Induction of jun expression by TGF-131, as well asjun autoinduction, may amplify the action of TGF-01 during normal development and oncogenesis.
BG104 (18) was adapted for cloning by filling in 5' protruding ends with Klenow fragment and was inserted upstream of the promoter in a herpes simplex virus thymidine kinasechloramphenicol acetyltransferase (CAT) recombinant. For construction of the sense and antisensefos constructs under the control of the metallothionein promoter, a mouse mammary tumor virus promoter fragment from pHfosAS or pHfoss (15) was replaced with a 1.6-kilobase EcoRI-BglII fragment of the mouse metallothionein I promoter. The antisensejun construct was obtained from R. Chiu, University of California, San Diego. The other CAT plasmids used here have been described elsewhere (17, 18). CAT assays. All cell lines were transfected by the calcium phosphate coprecipitation method (21), with 10 jig of the appropriate plasmids purified by banding in CsCl. In some experiments, transfection frequencies were monitored by cotransfection with 3 ,ug of pCH110 (Pharmacia LKB Biotechnology Inc.), a ,B-galactosidase expression vector. To analyze the inducibility by TGF-,1l, we transfected 10 ,ug of the appropriate TGF-131 plasmid DNA into A-549 cells. After 24 h in the absence or presence of TGF-1l (5 ng/ml), cells were harvested and CAT activity was assayed. To measure the transactivation by Jun/AP1, we transfected 10 ,ug of the various CAT constructs into F9MTcfos cells, together with 1 ,ug of the Rous sarcoma virus (RSV)-c-jun construct. At 24 h after transfection, the cells were either left untreated or treated with 5 ,uM Cd2 to induce the expression of Fos by the hMT-IIA c-fos construct stably integrated in these cells (8). Six hours later, the cells were harvested and CAT activity was determined. All transfections were repeated at least three times. DNase I footprinting. DNase I footprinting reactions were performed as described previously (2) with either a HeLa whole-cell extract partially purified on a heparin-agarose column or AP-1 protein highly purified by three cycles of DNA sequence-specific affinity chromatography (3). In addition, extracts of Escherichia coli expressing c-Jun as TrpE fusion proteins were used as described by Angel et al. (1). Analysis ofjun and TGF-0I1 expression. Total cellular RNA was isolated by the method of Chirgwin et al. (7). The levels
Several growth factors including platelet-derived growth factor (22), transforming growth factor ox (TGF-a) (10), and TGF-pl (28) autoregulate the expression of their mRNAs, resulting in increased secretion of the respective peptides. Such autoinduction can amplify responses to these growth factors during development or in disease process such as carcinogenesis (16). Since the ultimate response of a target cell to activation by a growth factor is a change in its pattern of gene transcription, it is likely that nuclear transcription factors are part of the signaling pathway whereby growth factors regulate cell function. The phorbol ester-induced transcription factor AP-1 (3, 20), a complex of the Jun and Fos nuclear oncoproteins (1, 5, 8, 12, 24, 29), has been implicated as playing a pivotal role during cell growth, differentiation, and development (6; M. Karin, in Proceedings of the Bristol-Meyers Symposium on Cancer Research, in press). Moreover, like the growth factor genes, the c-jun gene is also positively autoregulated by its gene product, the Jun/AP-1 protein (2). Therefore, it is conceivable that autoinduction of growth factors is mediated through transcription factors with autoregulatory properties similar to those of Jun/AP-1. To investigate the mechanism of autoinduction of TGF13, we have characterized the promoter regions of the TGF-,1l gene responsive to autoinduction (17-19). In this paper, we present data demonstrating that autoinduction is mediated by binding of the AP-1 complex in both promoter regions. MATERIALS AND METHODS Cells. Human lung adenocarcinoma (A-549) cells were grown in high-glucose Dulbecco modified Eagle medium supplemented with 5% fetal bovine serum. F9MTcfos embryonal carcinoma cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Plasmid construction. For construction of the plasmid phTG-H/B, a HincII-BstEII restriction fragment from phT *
Corresponding author. 1492
AUTOINDUCTION OF TGF-pl
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cA,r FIG. 1. Activation of plasmids phTG-H/B, phTG16, and 1 x TRE-tk CAT by Jun in F9MTcfos cells. Each of the plasmids was transfected into F9MTcfos cells either with or without 1 ,ug of the RSV-c-juin construct. At 24 h after transfection, the cells were either left untreated or treated with 5 p.M Cd'2 to induce the expression of Fos by the hMT-IIA c-fos construct stably integrated in these cells (8). Six hours later, the cells were harvested and CAT activity was determined. 1 xTRE-tkCAT, a construct containing a single copy of the collagenase TRE inserted in front of the tkCAT gene, was used as a control for the Jun transactivation.
of specific transcripts were analyzed by the RNase protection technique, as described previously (3). The derivation of the probes used for detection ofjun-specific transcripts was described by Hattori et al. (14). For Northern (RNA) blot analyses, equal amounts of RNA (10 ,.g) were electrophoresed on 1% agarose gels containing 0.66 M formaldehyde and transferred to nitrocellulose membranes. Blots were hybridized with 32P-labeled probes by the method of Church and Gilbert (9). Labeling of the 218-base-pair singlestranded TGF-41 probe, complementary to the mature coding region of human TGF-pl mRNA, has been described previously (27).
Ap1 ni I
Identification of sequences responsible for both TGF-41 .autoinduction and transactivation by Jun. Plasmids containing sequences located 5' to the upstream transcriptional start site (the first promoter, phTG-H/B) and between the two major transcriptional initiation sites of the TGF-,1l gene (the second promoter) linked to the bacterial CAT gene (13) (Fig. 1 and 2) were transfected into A-549 cells (human lung adenocarcinoma cells) to analyze their activity in the absence or presence of TGF-p1. Since each of these promoter constructs contained sequences that appeared to be potential AP-1 sites, also known as TREs (3, 20), they were also cotransfected into F9MTcfos embryonal carcinoma cells with RSV-c-jin or RSV-mut-jun vectors (8), which specify the production of either the wild-type Jun/AP-1 protein or a mutant protein that can no longer bind DNA. CAT activities of the first promoter, CAT (phTG-H/B), and the second promoter, CAT (phTG16), as well as of lxTRE-tkCAT (3), were transactivated by c-jun in F9MTcfos cells (Fig. 1). The results show that the expression of phTG-H/B is induced 10-fold by TGF-1l in A-549 cells and 29-fold in response to transactivation by Jun in F9MTcfos cells treated with Cd'+ to induce the expression of Fos protein from the inducible hMT-IIA c-fos construct (Fig. 2). The second promoter construct, phTG16, was also induced 4-fold by TGF-,1 in A-549 cells and 18-fold in response to transactivation. Both the induction by TGF-1l and the transactivation by Jun dropped almost to the basal level when the sequences between +145 and +173 were deleted (phTG22). However, the level of activity resulting from either transactivation or TGF-pl induction was increased again when the deletion reached + 228, suggesting that sequences between +173 and +228 contain a negative regulatory region. The downstream sequences from +289 do not contain the sequence element responsible for the TGF-P autoinduction. The importance of the TRE-like elements in autoinduction of TGF-,1 is further supported by reduction of the autoinduction to background levels when this site is deleted from first promoter (19). The results on inducibility of TGF-pl obtained by measuring CAT activity were confirmed by RNA analysis (Fig. 3). The TGF-11-CAT chimeric genes were cotransfected t°s&
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TGF-f1 INDUCIBILITY 10.3 8.5 2.5 4.2
phTG H/B 28.9 ph TG5 12.7 ] +729 phTGl5 16.6 +102 1 +432 18.2 phTG16 +102 1 +289 phTG17 4.7 19.0 +145 l J +289 4.5 phTG18 16.8 i +289 +173 1.1 phTG22 1.5 +228 EJ +289 2.7 phTG25 8.9 +247 0J +289 2.9 phTG26 13.2 FIG. 2. Autoinducibility of TGF-P2-CAT chimeric genes by TGF-,1l parallels their transactivation by Jun/AP-1. Deletional analysis of TGF-pl promoter constructs for TGF-pl and Jun activation is shown. At the top is an extended map of the two active promoter regions of the human TGF-pl gene, indicating the two major transcription initiation sites (P1 and P2), the locations of various protein-binding sites, and the positions of several restriction enzyme sites. The inducibility by TGF-1l refers to the ratio of CAT activity in A-549 cells treated with TGF-,1l to that in untreated cells; each represents the average of five different experiments. The numbers in the transactivation column indicate the ratio of CAT activity in F9MTcfos cells transfected with RSV-c-jun to that in cells transfected with RSV-mut-jln (8) and are the average of three independent experiments. -453
MOL. CELL. BIOL.
KIM ET AL. F9MTcfos P-1
prTG H B phTG16 ^s,A
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FIG. 3. RNase protection analysis of TGF-pl-CAT gene expression. TGF-31-CAT constructs (10 ,ug) plus RSV-c-jun (+) or RSV-mut-jun (-) expression vectors (1 ,ug) were cotransfected into F9MTcfos cells, and the cells were treated with 5 ,uM Cd2+ to induce expression of Fos. Six hours later the cells were analyzed by RNase protection for the level of CAT transcript (21). The CAT riboprobe was 480 nucleotides long, whereas the fragment protected from RNase digestion was 256 nucleotides long.
into inducible F9MTcfos cells with RSV-c-juin or RSVmut-jun expression vectors, and CAT mRNA was analyzed by the RNase protection assay with a CAT-specific riboprobe. Cotransfection with the wild-type Jun/AP-1 expression vector led to increased CAT mRNA transcription by the transfected plasmids (phTG-H/B and phTG16) (Fig. 3). The vector expressing the mutant Jun protein (RSV-mut-jun) had no effect on induction of CAT expression (data not shown). We also demonstrated correct initiation of mRNA transcripts from phTG16 in A-549 cells treated with TGF-1l (data not shown). Binding of the AP-1 complex to the TGF-j1 promoter. To
confirm that the sites which are responsible for the TGF-1l autoinduction and transactivation by jun are AP-1-binding sites, we used DNase I footprinting to locate binding sites for sequence-specific DNA-binding proteins. Using a HeLa cell extract partially purified by heparin-agarose chromatography, we detected various protected sites in the two TGF-1l promoter regions (data not shown). One of these protected regions is located between positions -365 and -371. This site is also protected by either affinity-purified HeLa cell AP-1 or bacterially expressed c-Jun fusion protein, indicating that it functions as a high-affinity AP-1-binding site (Fig. 4). Under the conditions of the footprinting assay, no pro-
FIG. 4. Binding of proteins to the TGF-1l promoter regions. For the first promoter of the TGF-pl gene, a 280-base-pair DNA fragment from positions -167 (ThaI) to -453 (HincII) was subcloned into the SmaI site of pUC13. The plasmid was linearized with EcoRI or BamHI and labeled with Klenow fragment to generate sense and antisense probes. The probes were incubated with 25 ,ug of bovine serum albumin (lane BSA) or affinity-purified AP-1 (approximately 2 to 4 ng, together with 25 ,ug of bovine serum albumin) and subjected to DNase I footprinting reactions as described elsewhere (2). These probes were also incubated with 1 jig of renatured TrpE-c-Jun fusion protein, and DNase I footprinting was performed. The site protected by AP-1 is shown on the left.
tection of the other two potential AP-1-binding sites, located between +155 and +170 (5'-CTGAGACGAG-3') and between +247 and +289 (5'-TTGAGACTT-3'), was detected. However, previous mobility shift experiments had implicated these sequences as recognition sites for proteins whose binding is inhibited by oligonucleotides having an AP-1 consensus sequence (17). Therefore, it appears that these sites are low-affinity AP-1-binding sites which are occupied only at high protein concentrations. Two Splbinding sites (11), between -211 and -219 (data not shown) and between +172 and +185 (18), have also been confirmed by DNase I footprinting with partially purified Spl. Antisense jun or antisense fos blocks the TGF-0I1 autoinduction. To investigate whether AP-1 is also responsible for autoactivation of the TGF-fi1 promoter in vivo, we cotransfected into A-549 cells the TGF-13-CAT chimeric genes and plasmids bearing either a fragment of human c-jun in an antisense orientation under the control of an RSV promoter (26) or a fragment of human c-fos in an antisense or sense orientation (17) under the control of a metal-inducible metallothionein promoter. In A-549 cells cotransfected with antisense c-jun or antisense c-fos expression vectors, the
AUTOINDUCTION OF TGF-1l
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0 0.5 1 3 5 8h
--_---4 FIG. 5. Inhibition of TGF-pl autoinduction by antisense c-jiulz or antisense c-fos. Plasmids phTG-H/B, and phTG16 were transfected into A-549 cells with 1 ,jg of either RSV antisense c-jun or antisense or sense c-fos under the control of the metal-inducible hMT-IIA promoter. At 12 h after the transfection, the cells were either treated with TGF-Pl (5 ng/ml) or left untreated. The cells cotransfected with the fos constructs were treated with 5 pLM Cd-2 for 24 h to induce the expression of antisense or sense c-fos. At 24 h after the TGF-l1 treatment, the cells were harvested and CAT enzyme activity was determined (13). Sense-fos
induction of CAT activity of both promoter constructs (phTG-H/B or phTG16) by TGF-131 was completely abolished (Fig. 5). Interestingly, antisense c-jin lowered even the basal level of expression. These results strongly suggest that the AP-1-binding sites mediate the autoregulation of TGF-,1l and that expression of both the Jun/AP-1 and c-Fos components of the AP-1 complex is required. Regulation of jun, fos, or TGF-0i1 expression by TGF-0i1. TGF-f31 has been shown to stimulate the expression of the transcription factor genes jinB and c-jun in various cell lines (23). For this reason, we also examined the expression of c-jun in A-549 cells in the absence and presence of TGF-,B1. Incubation of A-549 cells under serum-free conditions with TGF-pl resulted in prolonged induction of jun mRNA, lasting at least 24 h (Fig. 6A). In contrast, TGF-1l treatment elicited a rapid and transient induction of c-fos mRNA (Fig. 6C) (23). A-549 cells contained detectable levels of c-fos mRNA before treatment with TGF-,11; the levels of 2.2kilobase c-fos mRNA were elevated 1 h after stimulation with TGF-1l and then returned to normal. Under similar conditions, TGF-,11 treatment also increased the steadystate level of its own mRNA (Fig. 6B). Previously, we showed that this rise in the level of TGF-,B1 mRNA is accompanied by a parallel increase in secretion of TGF-,11 protein into the culture medium of treated cells (4). The enhanced mRNA levels of both TGF-1I (Fig. 6B) and c-juIn (Fig. 6A) persisted as long as TGF-41 was present in the culture medium. These data suggest that the prolonged induction of c-jun and TGF-41 mRNAs by TGF-,1I may be due, in part, to the positive feedback loop of c-juntl autoinduction (2), which could amplify the TGF-31 response. In contrast, transient elevation of c-fos expression may result from the ability of c-fos to down regulate its expression (25). Induction of jun-CAT chimeric genes by TGF-,1. Recent findings show that Jun/AP-1 stimulates the transcription of
FIG. 6. Induction of juin, fos, and TGF-31 mRNAs by TGF-p1. (A) Analysis of the effect of TGF-pl on c-jun mRNA expression. Confluent A-549 cells were incubated with TGF-pl (5 nglml) for 0, 2, 8. or 24 h, after which total cellular RNA was extracted. RNA from these cells was hybridized to radiolabeled jun and analyzed by RNase protection. (B) Time course of TGF-1l effects on TGF-1l expression in A-549 cells. Total RNA was isolated from cells following 0. 2. 8, or 24 h of treatment in serum-free medium with 5 ng/ml TGF-13. RNA was hybridized to radiolabeled single-stranded TGF-1l probe (27). (C) Analysis of the effect of TGF-pl on c-fos expression. Total RNA was extracted from the A-549 cells after 0, 0.5. 1. 3, 5. or 8 h of incubation with TGF-pl (5 nglml) and hybridized to radiolabeled c-fos probe.
its own gene through the AP-1-binding site in its promoter (2). To test whether the induction of c-jun mRNA by TGF-I1 might also be mediated by this site, the CAT activity of juin-CAT chimeric genes containing segments of the jun 5'-flanking region was assayed in A-549 cells. TGF-,1l stimulates the expression of constructs containing c-jun sequences from -132 to +170 15-fold (Fig. 7); this sequence contains an AP-1-binding site as well as binding sites for Spl and CTF. The importance of the AP-1-binding site is underscored by deleting the binding sites for Spl and CTF. The expression of this construct (-79/+170) was induced 20-fold by TGF-41. In contrast, TGF-p1 failed to stimulate the activity of -79/+170 Ajuin (2), which contains point mutations that interfere with the binding of AP-1 (Fig. 7). DISCUSSION In this study, we have identified three distinct regions of the human TGF-,1 promoter that are responsive to autoinduction. These regions contain the sequences homologous to the phorbol ester responsive element (TRE), demonstrated to act as an inducible transcriptional enhancer activated by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) (3). Although the sequences of these sites deviate somewhat from the previously derived AP-1 consensus [TCA(C/G)TCAG], they nonetheless bind AP-1. The binding site for AP-1 between -365 and -371 has high affinity for both AP-1 and the bacterially expressed c-Jun protein. Antisense c-jun and antisense c-fos each completely blocks the TGF-p1 autoinduction of CAT expression from both first and second promoter constructs. Moreover, cotransfection of a c-jun expression vector into F9MTcfos cells together with TGF-,B1 promoter-CAT chimeric genes
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**E X:LnCAT. ,
LITERATURE CITED 1. Angel, P., E. A. Allegretto, S. T. Okino, K. Hattori, W. J. Boyle, T. Hunter, and M. Karin. 1988. Oncogene jun encodes a sequence-specific trians-activator similar to AP-1. Nature (Lon-
2. Angel, P., K. Hattori, T. Smeal, and M. Karin. 1988. The junll
FIG. 7. Activation of jin-CAT chimeric genes by TGF-,1l in A-549 cells. Supercoiled plasmids (-132/+170jun-CAT. -79/+170 jlun-CAT, and -79/+170 Ajuin-CAT) (2) were introduced into A-549 cells by the calcium phosphate coprecipitation technique. The cells were then incubated for 24 h in the presence or absence of TGF-l31 (5 ng/ml) and assayed for CAT activity.
results in a significant transactivation of the CAT activities. These two sets of experiments both suggest that binding of the AP-1 complex to TRE-like elements in the TGF-,11 promoters is important in TGF-1l autoinduction. TGF-41 treatment rapidly induces the expression of junl mRNA in A-549 cells (23) and also increases the level of its own mRNA; we have shown that each of these, as well as juin autoinduction, is mediated by binding of AP-1 complex. In contrast, induction of c-fos mRNA by TGF-f1 is rapid and transient. Moreover, the level of induction of TGF-,B1 of jlun mRNA far exceeds that of c-fos mRNA. Our data suggest that the basal levels of c-fos expression may be sufficient to contribute to the TGF-31 autoinduction; antisense c-fos has been shown to reduce c-fos protein synthesis by 80 to 90% (J. Holt, personal communication). Taken together, these data demonstrating the interactive nature of TGF-11 and c-jun autoinduction suggest that the ability of growth factors, such as TGF-41, and nuclear proto-oncogenes/transcription factors, such as c-jlan1, to control cellular growth, differentiation, and development may depend in part on their ability to regulate each other's expression. The interrelated nature of these two autocrine loops could also be important in oncogenesis, since it suggests that aberrant expression of either TGF-3 or Jun would lead to amplification of both. Finally, in addition to transcriptional control, posttranslational modifications of transcription factors by growth factors may be important. Possible effects of TGF-431 on posttranslational modifications of Fos and Jun remain to be determined and may further extend our knowledge of the mechanistic interplay of these two sets of proteins. ACKNOWLEDGMENTS
We are indebted to J. Holt and R. Chiu for the gift of fios constructs and antisense c-jimn, respectively: T. K. Jeang, W. J. Boyle, and A. Glick for helpful suggestions; and Dawid for a critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (M.K.), the Department of Energy (M.K.). the Deutscher Akademischer Austauschdienst (P.A.). and Johnson & Johnson Baby Products Co. (K.Y.K.).
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