Promoter Sequences of the Human Transforming Growth Factor-Bl Gene Responsive to Transforming Growth Factor-/3l Autoinduction”

Two distinct regions of the transforming growth factor (TGF)-Pl promoter are responsive to autoregula- tion. Sequences located between nucleotides -454 to -323 and between the two major transcriptional start sites have positive regulatory activities and are in- duced by TGF-81 in A-549 cells. The chloramphenicol acetyltransferase activity of the upstream human TGF-81 promoter-chloramphenicol acetyltransferase gene is increased 8- to IO-fold by treatment of cells with TGF-81, whereas that of the second promoter is increased approximately 3- to 4-fold. Using an S1 nuclease protection assay of chloramphenicol acetyltransferase mRNA, we found that the steady-state expression of chloramphenicol acetyltransferase mRNA also is markedly increased. Seven distinct factors present in nuclear extracts from A-549 cells in- teract with the sequences between -454 and -323, strongly supporting the involvement of sequence-spe- cific transcription factors in the transcriptional au-toactivation of the human TGF-81 gene.

Two distinct regions of the transforming growth factor (TGF)-Pl promoter are responsive to autoregulation. Sequences located between nucleotides -454 to -323 and between the two major transcriptional start sites have positive regulatory activities and are induced by TGF-81 in A-549 cells. The chloramphenicol acetyltransferase activity of the upstream human TGF-81 promoter-chloramphenicol acetyltransferase gene is increased 8-to IO-fold by treatment of cells with TGF-81, whereas that of the second promoter is increased approximately 3-to 4-fold. Using an S1 nuclease protection assay of chloramphenicol acetyltransferase mRNA, we found that the steady-state expression of chloramphenicol acetyltransferase mRNA also is markedly increased. Seven distinct factors present in nuclear extracts from A-549 cells interact with the sequences between -454 and -323, strongly supporting the involvement of sequence-specific transcription factors in the transcriptional autoactivation of the human TGF-81 gene.
Transforming growth factor-@ (TGF-@)' is a 25,000-dalton homodimeric molecule which belongs to a family of structurally related multifunctional regulatory peptides (for reviews, see Refs. [1][2][3]. The nucleotide sequences and the deduced amino acid sequences of human, murine, porcine, bovine, simian, and chicken (4-9) TGF-Pl mRNAs demonstrate that the mature polypeptide of TGF-P1 is conserved 100% across these species with the exception of a single amino acid substitution in the murine peptide. This extensive amino acid conservation is a characteristic of TGF-p's types 2 and 3 as well, strongly suggesting that the TGF-p's must play a vital role in normal cell physiology.
Several growth factors have been demonstrated to autoregulate expression of their mRNAs, among them PDGF (lo), TGF-a (ll), and TGF-Pl (12). These data imply the existence of an autocrine loop that may serve to amplify response to the growth factor under certain conditions. Such autoregulation may also serve to sustain growth factor action in vivo. As a first step in exploring the molecular basis for the autoregulation of TGF-Pl gene expression, we characterized the promoter region of the human TGF-Pl gene and identified sequences responsible for both promotion and inhibition of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8 To whom correspondence should be addressed.

1) Recipient of National Research Service Award.
The abbreviations used are: TGF, transforming growth factor; PDGF, platelet-derived growth factor; bp, base pair; CAT, choramphenicol acetyltransferase; EGF, epidermal growth factor. transcription by assaying the chloramphenicol acetyltransferase (CAT) activity of TGF-@l promoter-CAT chimeric genes (13). Two promoter regions were identified one located 5' to the most upstream transcriptional start site, and another located between the two major transcriptional initiation sites. In this report, we have localized the specific regions permissive for the optimal autoregulation of TGF-01 gene transcription to both a 130-bp fragment located between -453 and -323 bp upstream of the 5'-most start site and a fragment containing the second promoter.

MATERIALS AND METHODS
Cells and Culture Conditions-Cells were grown in a humidified incubator (5% COa, 95% air) at 37 "C in the presence of antibiotics (50 units/ml penicillin, 50 wg/ml streptomycin). Human lung adenocarcinoma (A-549) cells were grown in Dulbecco's modified Eagle's medium with high glucose supplemented with 5% fetal bovine serum. Other cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All transfections were repeated at least three times.
DNA Constructs-The construction of a series of the human TGF-@1 gene promoter-CAT chimeric genes has been described elsewhere (13). To clone the fragment of the human TGF-01 gene containing sequences between -453 and -323, phTBG104 (13) was first digested with BstEII, followed by treatment with T4 polymerase. The DNA was subsequently digested with HincII, and the 130-bp fragment was isolated and cloned into the HincII site of pGEM 4 (Promega Biotechnology).
Growth Factors and Reagents-TGF-@l and -82 from porcine platelets were obtained from R & D Systems (Minneapolis, MN); human PDGF was a gift of Dr. Thomas Deuel (Washington University, St. Louis, MO); basic fibroblast growth factor was obtained from Sigma; and epidermal growth factor (EGF) was purified to homogeneity from mouse submaxillary glands.
RNA Extraction and SI Nuclease Analysis of CAT mRNA-Following experimental treatment of cells transfected with TGF-pl gene promoter-CAT chimeric plasmids, RNA was isolated according to the guanidine isothiocyanate/cesium chloride procedure by the method of Chirgwin et al. (14). RNA (50 Kg) was probed for CAT mRNA using a single-stranded DNA probe uniformly labeled with [32P]dCTP (3000 Ci/mmol) during strand synthesis from a single-stranded M13 phage, M13 CAT, constructed by insertion of a BglII-EcoRI fragment from pAlOCAT2 (15). After synthesis of the [32P]dCTP-labeled probe, a 458-nucleotide single-stranded segment containing 256 bp of CAT coding sequences was liberated by HincII digestion and purified on a 6% polyacrylamide-urea gel. S1 nuclease digestion was performed (13) and the products were analyzed by electrophoresis in a 5% polyacrylamide sequencing gel containing 8 M urea, followed by autoradiography.
Nuclear Extracts and Gel Mobility Shift Assay-Nuclear extracts were made from A-549 cells essentially as described by Dignam et al. (16); final protein concentrations were typically 1-2 mg/ml. Gel mobility shift assays were performed as previously described (17). Specifically, plasmid DNA (phTBG104), linearized with BstEII and end-labeled by T4 polynucleotide kinase, was cleaved with HincII, and the appropriate fragments were isolated by electroelution after electrophoresis through a 6% polyacrylamide gel. The DNA-protein binding reaction was performed in 25-pl incubations: nuclear extracts 7041 (1-3 ~1 ) were incubated with 1 Fg of poly(d1:dC) (Pharmacia LKB Biotechnology Inc.) in binding buffer (10 mM Tris-HCI, pH 7.5, 50 mM NaCI, 5% glycerol, 5.0 mM MgCI,, 1.0 mM dithiothreitol, and 1.0 mM EDTA) for 10 min on ice before addition of approximately 0.1 ng of 32P-labeled probe (40,000 cpm) after which the incubation was continued for an additional 15 min. Competitor DNAs were added at thepreincubation step. Protein-DNA complexes were electrophoresed in 6% polyacrylamide gels in 0.25 X TBE (1 X TBE = 90 mM Tris-HC1, 90 mM boric acid, 1 mM EDTA, pH 8.3). The gels were dried and radioactivity was detected by exposure to Kodak XAR-5 film with an intensifying screen at -70 "C.

RESULTS
Localization of Regions for TGF-PI Autoregulation-A restriction endonuclease map of the 5' end of the human TGF-61 gene and the structures of the TGF-P1 promoter-CAT chimeric plasmids used in this study are shown in Fig. 1; the arrow denotes the first major transcriptional start site. The recombinant plasmids were transfected into A-549 cells and the cells were then incubated in the absence or presence of TGF-Pl(5 ng/ml). All transfections were transient expression experiments in which TGF-81 was added 24 h after transfection. After a predetermined interval, the cells were harvested and CAT activity was measured. These cells showed increased levels of CAT activity after TGF-Pl treatment ( Fig. 2 and Table 1). The mean results in A-549 cells of several experiments with each plasmid are summarized on the right of  Table I) (13). This level of CAT activity was stimulated approximately 8to 10fold when cells transfected with phTG5 were incubated with TGF-P1, indicating that the sequences permissive for TGF-Pl optimal autoregulation are located in that region. The activity of plasmids phTG1, 2, 3, 4, 6, and 7 was stimulated approximately 1-to 4-fold by TGF-61 treatment, but the levels of CAT activity were only 5-10% of that seen with phTG5. These results with phTG1, 2, 3, and 4, as well as previously published results (131, suggest that sequences upstream from -453 contain a negative regulatory region which suppresses the activity of sequences in phTG5. Recent experiments have demonstrated that TGF-PI can directly enhance the transcription level of the mouse LY (2) I collagen gene and that this effect is mediated by NF-1 binding sites (18). Since the human TGF-01 gene contains a sequence similar to the consensus sequence of NF-1 at nucleotides -260 to -240, it has been suggested that NF-1 may also be involved in autoregulation of TGF-81 gene transcription (12). However, plasmid phTG6, which contains the sequences related to NF-1 showed only a low level of expression, even after a 3.5-fold induction by TGF-01.
A time course experiment showed that the increase in CAT activity of phTG5 is slow during the early time points but then increases steadily beginning at about 18 h after the onset of TGF-Bl treatment (Fig. 3). The precise interpretation of this time course is difficult, since we do not know how much time is needed for the transfected DNA to reach the nucleus and become actively transcribed.
Effects of Other Growth Factors on the Activity of the Human TGF-PI Promoter-A previous report (12) showed that treatment of normal rat kidney cells with EGF and PDGF also increased the TGF-81 mRNA levels, and that the combined effects of EGF and TGF-P1 on TGF-Pl mRNA expression were greater than those of either effector alone. To examine whether these other growth factors also regulate TGF-P1 promoter activity, A-549 cells transfected with the plasmid phTG5 were treated with various growth factors. As shown in Fig. 4, EGF, PDGF, and basic fibroblast growth factor were unable to stimulate CAT gene expression, but TGF-82 elicited a 4-to 6-fold stimulation of CAT gene expression in these cells. The combined effects of these growth factors with TGF-01 showed little additional effect on CAT activity. Previously, it had been observed that TGF-81 mRNA levels in normal rat kidney cells treated for 24 h with EGF are unaffected by the addition of cycloheximide, whereas the levels in cells treated with TGF-P1 are severely reduced (12). Our data support this observation and suggest that the effect of TGF-61 on the regulation of its gene expression might occur at a transcriptional level through nuclear factors, while EGF or other growth factors might contribute to stabilization of mRNA.
Autostimulation of Transcripts from the First Promoter as Well as the Second Promoter of the TGF-81 Gene-Previous investigations identified a second promoter region in the TGF-P1 gene located between the two transcriptional start sites at +I and +271 (13). To determine whether this second promoter region was sensitive to autoregulation, A-549 cells were transfected with another plasmid, phTG15, containing the second promoter region (positions +95 to +727); they were then incubated in the presence or absence of TGF-PI.
Extracts examined at 48 h contained a high basal level of CAT activity (Fig. 5A) in the absence of TGF-PI. In the presence of TGF-Pl, the activity of phTG15 was increased 3to 4-fold. To establish that the increase in CAT activity after   treatment of TGF-01 was due to elevated levels of CAT mRNA, we measured steady-state CAT mRNA by quantitative S1 nuclease analysis (Fig. 5B). The probe used in the S1 analysis was synthesized from a single-stranded M13 recombinant plasmid as described under "Materials and Methods." The size of the probe was 458 nucleotides, whereas the size of a fragment protected from S1 nuclease digestion would be 256 nucleotides. A SI protected DNA fragment of 256 bases was detected in cultures transfected with either phTG5 or phTG15 in the presence (Fig. 5B, lunes 4 and 9) or absence (Fig. 5B,  nuclease is shown in Fig. 5B, lanes 1 and 6. As a positive control, RNA extracted from the cells transfected with a HTLV-I LTR construct (19) was used (Fig.  5B, lune 5). Following treatment with TGF-P1, the CAT mRNA level was elevated 5-to 6-fold in cells transfected with either phTG5 or phTG15, even though the basal level of CAT mRNA in cells transfected with phTG5 was 4 times lower than in cells transfected with phTG15.  (phTG5 and phTG15). A, regulation of phTG5 and phTG15 by TGF-01 and $2 in A-549 cells. Plasmids were transfected into A-549 cells and incubated in the presence or absence of TGF-01 or TGF-P2 (5 pglml). The CAT activities generated by these constructs were measured. ACM, chloramphenicol acetylated; CM, chloramphenicol. B, S1 nuclease analyses of CAT mRNA synthesized in A-549 cells in the presence or absence of TGF-Pl after transfection with phTG5 or phTG15. The upper panel shows the schematic diagram of both plasmids and the single strand probe. Samples (60 pg) of total RNA were subjected to S1 nuclease protection analysis for CAT mRNA as described under "Materials and Methods." Lanes: I and 6, yeast tRNA; 2 and 7, total RNA from A-549 cells before transfection; 3 and 4, total RNA from A-549 cells transfected with phTG15; 8 and 9, total RNA from A-549 cells transfected with phTG5; 3 and 8, no TGF-131 treatment; 4 and 9, TGF-Dl treated after transfection; 5, as a positive control, CAT mRNA of HTLV I-LTR CAT (19); M, molecular weight markers. The size of CAT-specific fragment protected from S1 nuclease digestion is 256 nucleotides.
Responsible for the Autoregulation-A gel electrophoresis mobility shift assay was utilized to assess binding of factors in nuclear extracts from A-549 cells to the human TGF-01 promoter. The fragment that extends from -453 to -323 was used because this fragment contains the sequences critical for expression and autoregulation; two 70-bp synthetic duplex probes representing the sequences from -453 to -383 (Cl) and -393 to -323 (C2) (Fig. 7 , upper panel) were constructed to span this region. The radiolabeled probes were incubated with a nuclear extract from A-549 cells as described under "Materials and Methods," and protein-DNA complexes were separated from free DNA by electrophoresis on low ionic strength polyacrylamide gels (Fig. 7). These oligomers bound specifically to nuclear factors as demonstrated by the retardation in mobility relative to the free oligomer at the bottom of the gel. The sequence specificity of this binding was investigated by forming complexes in the presence of specific and nonspecific competitor DNA fragments. Six DNA-protein complexes (bl-b6) were seen with the C1 oligomer and two (b7 and b8) with the C2 oligomer (Fig. 7, lanes 1 and 3 ) . Addition of 1 pg (100-fold molar excess) of unlabeled homologous oligomer reduced almost completely the binding of labeled probe in bands 1-5 for C1, and bands 7 and 8 for C2 (Fig. 7, lanes 2 and 4 ) ; band 6 was not competed under these conditions, suggesting that the band 6 complex is nonspecific. These results suggest that bands 1-5, 7, and 8 represent specific complexes formed by cellular transcription factors with the promoter fragment spanning the region -453 to -323.

DISCUSSION
In this report, we have identified two distinct regions of the TGF-01 promoter that are particularly responsive to autoinduction. Previously, we reported that sequences located between nucleotides -454 to -323 have positive regulatory activity; the transcriptional capacity of the upstream TGF-01 promoter was completely abolished by removal of this region (13). We had also demonstrated that sequences located between the two major transcriptional start sites have even greater relative promoter activity than the upstream regula- Lanes: 1 and 2, binding reaction of C1 probe with A-549 nuclear extracts; 2 and 4 , binding reaction of C2 probe with A-549 nuclear extracts; 2 and 4 , competitions with the unlabeled C1 and C2, respectively. tory sequences (13). In this report, we have characterized more extensively the positive regulatory sequences located between nucleotides -454 to -323 because these sequences are more strongly induced by TGF-Pl; treatment of A-549 cells with TGF-pl increased the level of CAT activity of the upstream and downstream human TGF-01 promoter-CAT chimeric genes 8-to 10-fold and 3-to 4-fold, respectively.
Seven sequence-specific DNA .protein complexes. were formed upon incubation of nuclear extracts from A-549 cells with the upstream region of the TGF-Pl promoter responsible for autoregulation, suggesting the involvement of sequencespecific transcription factors in the transcriptional autoactivation of the human TGF-Pl gene. These major and minor band complexes did not appear to be cell-type specific because similar complexes were also detected following incubation with HeLa cell nuclear extracts (data not shown). This sug-gests that the proteins responsible for formation of the specific complexes might be common to several different cell types.
In previous experiments demonstrating autoinduction of TGF-01 mRNA (12), it had been suggested that the autoinductive effects might be mediated by an NF-1 binding site, as had been demonstrated for induction by TGF-P1 of mouse a(2)I collagen gene expression (18). Although sequences similar to the NF-1 consensus sequence have been identified at positions -260 to -240 of the TGF-Dl promoter, the CAT activity of the plasmid containing these sequences (phTG6) showed only a low level of expression, even after induction by TGF-61, compared to the CAT activity for plasmid phTG5, which contained an additional 130 bp. This 130-nucleotide fragment, as mentioned above, is essential to elicit the maximum expression of the hybrid gene. These results suggest that the mechanisms involved in the autoinduction of TGF-D l expression and the induction of the mouse a(2)I collagen gene expression are different. Since TGF-Pl has been shown to increase expression of a number of genes for structural and matrix-associated proteins (20-22) and protease inhibitors (23)(24)(25), it remains to be seen whether there will be common or gene-specific mechanisms mediating the response to TGF-Dl. Moreover, both with respect to its autoinduction and to its induction of other genes, it will be important to determine if the effects of TGF-P1 are mediated through the modification of pre-existing transcription factors, or through the induction of specific transcription factors.