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* This work was supported by National Institutes of Health Grant DK37340 (to S. L. F.) and by Bayer A.G. (to S. L. F.).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. ¶ Present address: Service D'Hepatogastroenterologie Groupe Hospitalier, Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France. ‖ Present address: Volcani Agricultural Research Organization Institute, Bet Dagan, Israel 50250. ‡ Present address: Systemix Inc., 3155 Porter Ave., Palo Alto, CA 94304.
We have explored the regulation of transforming growth factor β (TGF-β) activity in tissue repair by examining the interactions of Zf9/core promoter-binding protein, a Kruppel-like zinc finger transcription factor induced early in hepatic stellate cell (HSC) activation, with promoters for TGF-β1 and TGF-β receptors, types I and II. Nuclear extracts from culture-activated HSCs bound avidly by electrophoretic mobility shift assay to two tandem GC boxes within the TGF-β1 promoter but minimally to a single GC box; these results correlated with transactivation by Zf9 of TGF-β1 promoter-reporters. Zf9 transactivated the full-length TGF-β1 promoter in either primary HSCs, HSC-T6 cells (an SV40-immortalized rat HSC line), Hep G2 cells, or Drosophila Schneider (S2) cells. Recombinant Zf9-GST also bound to GC box sequences within the promoters for the types I and II TGF-β receptors. Both type I and type II TGF-β receptor promoters were also transactivated by Zf9 in mammalian cells but not in S2 cells. In contrast, Sp1 significantly transactivated both receptor promoters in S2 cells. These results suggest that (a) Zf9/core promoter-binding protein may enhance TGF-β activity through transactivation of both the TGF-β1 gene and its key signaling receptors, and (b) transactivating potential of Zf9 and Sp1 toward promoters for TGF-β1 and its receptors are not identical and depend on the cellular context.
The abbreviations used are: TGF-β, transforming growth factor β; HSC, hepatic stellate cell; EMSA, electrophoretic mobility shift assay; RI, type I receptor; RII, type II receptor; RIP, type I receptor promoter; RIIP, type II receptor promoter; luc, luciferase; CPBP, core promoter-binding protein.
1The abbreviations used are: TGF-β, transforming growth factor β; HSC, hepatic stellate cell; EMSA, electrophoretic mobility shift assay; RI, type I receptor; RII, type II receptor; RIP, type I receptor promoter; RIIP, type II receptor promoter; luc, luciferase; CPBP, core promoter-binding protein.
is a multifunctional cytokine that plays a key role in the response to injury in a wide variety of tissues (
The liver offers a particularly attractive paradigm in which to explore the role of TGF-β1 in tissue fibrosis. Cell-specific markers have been well characterized for in situ analysis, and methods are established for obtaining pure isolates of both parenchymal and nonparenchymal cell types. Such studies conclusively establish hepatic stellate cells as the primary source of extracellular matrix in liver fibrosis and a major cellular target of TGF-β1 (
) have also established hepatic stellate cells (previously called lipocytes, Ito, or fat-storing cells) as a major source of TGF-β1. Marked up-regulation of TGF-β1 gene expression and activation of latent cytokine are consistent features of both human (
Increased activity of TGF-β1 during hepatic stellate cell activation reflects not only up-regulation and activation of the cytokine but also enhanced responsiveness to TGF-β1 due to increased TGF-β receptor expression. We previously documented induction of TGF-β receptors types I, II, and III during stellate cell activation in vivoand in culture (
). Receptor induction in culture was paralleled by increased TGF-β1 binding and enhanced fibronectin gene expression in response to TGF-β1.
Increased fibrogenesis is only one of many phenotypic responses characterizing hepatic stellate cell activation. To define more broadly the nature of gene induction during this event, we used subtraction cloning to identify mRNAs up-regulated rapidly after acute injury (
). Interestingly, this approach identified the type II TGF-β receptor among the induced mRNAs. Another mRNA encoded a novel zinc finger partial cDNA; full-length cloning identified a Kruppel-like factor that we have termed Zf9 (
). Zf9 contains three carboxyl-terminal C2H2 zinc fingers similar to other Kruppel-like factors and a serine-rich activation domain with homology in the most amino-terminal 47 amino acids to a new member of the Kruppel-like family, UKLF (
). Zf9 mRNA is rapidly induced during stellate cell activation in vivo and in culture, in association with nuclear localization of the protein. Most importantly, the factor transactivates a minimal collagen α1(I) promoter, which contains several GC-rich Sp1 binding sites.
Our cloning and characterization of the promoters for both TGF-β1 (
) TGF-β receptors has positioned us to explore the potential regulation of these genes. In particular, all three promoters contain GC-rich Sp1 binding sites. Such consensus sequences raise the possibility that one or more of these genes might be regulated by Zf9. In this study, we indeed demonstrate that Zf9 transactivates all three genes following transient transfection in mammalian cells, and we compare these interactions with those of Sp1, the prototype GC box-binding protein.
Cells and Cell Lines
Primary hepatic stellate cells were isolated from normal Sprague-Dawley rats by in situperfusion and density gradient centrifugation as described previously (
). Cells were maintained on uncoated plastic in medium 199 with 20% serum (1:1, horse:calf). The immortalized rat hepatic stellate cells line HSC-T6 was generated by transfection of primary cells at day 15 after plating using LipofectAMINE containing an expression plasmid encoding the SV40 large T antigen. The resulting clone HSC-T6 retains all features of activated stellate cells, including expression of desmin, α smooth muscle actin, and glial acidic fibrillary protein, and it can esterify retinol into retinyl esters. The cells maintain a stable phenotype for at least 40 passages (more detailed characterization of this line will be reported elsewhere). These cells, as well as HepG2 cells, were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Hyclone).Drosophila Schneider (S2) cells were maintained in S2 medium (Life Technologies, Inc.) with 10% fetal bovine serum.
Cellular Fibronectin mRNA Quantitation
Expression of the EIIIA isoform of cellular fibronectin mRNA by HSC-T6 cells was analyzed by RNase protection, exactly as described previously (
Expression plasmids containing rat Zf9 (pCIneo-Zf9) and human Sp1 (pCIneo-Sp1) for mammalian and Drosophila cells (pPAC-Zf9; pPAC-Sp1) have been described previously (rat Zf9 GenBankTM accession numberU73759) (
). These included phTG6, −323 to +11; phTG7, −175 to +11; and phTG7–4, −60 to +11. These promoter regions were subcloned into a luciferase reporter from their corresponding CAT vectors by digestion with HindIII, fill-in to create blunt ends, digestion with KpnI, and then ligation into theSmaI/KpnI sites of pGL2-basic(Promega).
The TGF-β receptor I reporter constructs pTβRIP-luc(−867 to −169) and pTβRIP-luc (−867 to −228) were constructed by digesting the RI promoter with either NaeI and KpnI or SmaI and KpnI, respectively, and subcloning into KpnI/BglII site of the pGL2-basic after BglII site was filled. Type I receptor constructs pTβIP-luc (−495 to −65),pTβRIP-luc (−425 to −65), and pTβRIP-luc(−283 to −65) were generated by digesting the RII promoter withBglII, HinfI, and PvuI, respectively, filling in the ends, digesting these fragments again withXhoI, and subcloning into SmaI/XhoI site of the pGL2-basic.
Production of Recombinant GST-Fusion Proteins
Recombinant full-length rat Zf9- fusion protein was expressed in JM109 bacteria and purified by affinity chromatography as described previously (
). Purified Sp1 (Promega)-DNA or Zf9-GST fusion protein-DNA complexes were formed by incubating at room temperature for 20 min with 10,000 cpm of32P-labeled probe, 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, and 300 μg/ml bovine serum albumin in 20 μl of binding mixture. Competition reactions were performed by adding an unlabeled double-stranded oligonucleotide to the reaction mixture. Reactions were electrophoresed on a 6% NOVEX precasted nondenaturing polyacrylamide gel at 100 V for 1 h in a 100 mm Tris borate-EDTA buffer. Gels were vacuum-dried and analyzed by autoradiography.
Transient Transfection and Luciferase Assays
For transient expression assays, cells were transfected with the indicated plasmids using Lipofectin (for HepG2 cells) or LipofectAMINE (for stellate cells) (Life Technologies, Inc.). Following transfection with either reagent, cells were incubated for 48 h. Luciferase activity was normalized to co-transfected β-galactosidase activity to correct for transfection efficiency. Drosophila Schneider cells were transfected by the calcium phosphate coprecipitation method, using 10 μg of the appropriate reporter plasmids with 100 ng of either a control plasmid, pPAC; the Sp1 expression plasmid, pPAC-Sp1; or the Zf9 expression plasmid, pPAC-Zf9. Cells were harvested 48 h after addition of the DNA, and extracts were assayed for luciferase activity. All transfection were repeated at least three times.
Hepatic Stellate Cells Respond to TGF-β1, and Their Nuclear Extracts Bind to the TGF-β1 Promoter
) have previously documented that TGF-β1 increases matrix gene expression in primary stellate cells. This finding was confirmed in the immortalized HSC-T6 cells in order to establish the relevance of this line to studies of TGF-β regulation in hepatic fibrosis. As previously reported in activated primary stellate cells (
) a high level of basal cellular fibronectin mRNA was detected in HSC-T6 cells, which was increased an additional 42% in the presence of 1 ng/ml recombinant TGF-β1 (data not shown). We also examined the interaction of nuclear extracts from both primary and immortalized stellate cells with a GC-rich region located between −209 and −239 of the human TGF-β1 promoter (
). Nuclear extracts from both primary and HSC-T6 cells bound to a labeled oligonucleotide containing this sequence, and the interaction was abolished by competition with excess cold oligonucleotide (Fig. 1). Binding was more intense in the highly activated immortalized stellate cells than in the less activated primary cells, consistent with the observation that stellate cell activation is associated with increased TGF-β1 expression (
Recombinant Zf9 Binds to GC-rich Regions in the TGF-β1 Promoter
The TGFβ1 promoter contains three GC-rich regions, which were designated TGFβ1/1, TGFβ1/2, and TGFβ1/3 (Fig.2A). Oligonucleotides TGFβ1/1 and TGFβ1/2 contain tandem Sp1 sites, whereas TGFβ1/3 contains only a single site. Oligonucleotides representing each of these motifs were used to assess interactions with recombinant Zf9-GST by EMSA. As shown in Fig. 2B, Zf9-GST bound in a specific manner to all three regions, but with much greater affinity to oligonucleotides TGFβ1/1 and TGFβ1/2 than TGFβ1/3. Addition of anti-Zf9 antiserum to nuclear extracts abolished protein-DNA interaction with TGFβ1/1 and TGFβ1/2 regions (not shown). This is similar to our previous result examining the interaction of Zf9-GST with an Sp1 consensus motif by EMSA (
Zf9 Transactivates the TGF-β1 Promoter in Stellate Cells and Other Cellular Contexts
Transient cotransfections were performed to determine whether Zf9 transactivated TGF-β1 in stellate cells, as well as in other cellular contexts. As shown in Fig.3, Zf9 transactivated the TGF-β1 promoter in stellate cells and Hep G2 cells, although to a greater extent in Hep G2. Transactivation also was examined inDrosophila Schneider cells, which provide an important cellular context devoid of Sp1 and Zf9. Zf9 retained transactivating activity toward the TGF-β1 promoter inDrosophila Schneider cells (Fig. 3A), which contrasts with its inability to transactivate collagen α1(I) in this cell type (
). Transactivation by Zf9 in DrosophilaSchneider cells was proportionate to the amount of transfected Zf9 expression plasmid (Fig. 3B).
TGF-β1 promoter-luciferase constructs with progressive 5′ deletions were used to map the regions required for Zf9 responsiveness in stellate cells and Hep G2 (Fig. 4). Identical patterns of transactivation were observed between both cell types. Transactivation by Zf9 required at least the region from −175 to +11 in both cell types, whereas a reporter construct containing from −60 to +11 was unresponsive (Fig. 4). These functional data corresponded to the relative affinities of Zf9-GST for the three GC-rich regions (Fig. 2).
Transactivation of the TGF-β RI Gene by Zf9 Can Be Mediated through the Multiple GC Regions
We examined whether Zf9 might also contribute to up-regulation of the type I TGF-β receptor. Hep G2 cells were used in these and subsequent transfections because their transfection efficiency and relative transactivation were much higher than stellate cells, facilitating more reproducible functional mapping of the promoter. In part, this was due to a much higher basal transactivation of the reporter in stellate cells (not shown), similar to that seen for collagen α1(I) (
). Transiently transfected Zf9 markedly transactivated a full-length TGF-β RI promoter-reporter in Hep G2. To identify the sequences responsible for regulation by Zf9, we tested a series of deletion constructs of the human TGF-β RI promoter fused to a luciferase reporter. Transfection of Zf9 cDNA induced the expression of the TGF-β RI-luciferase reporter genes containing 5′ sequences out to position −867 (pTβRIP-luc−867/−65) by 9-fold compared with transfection of the empty vector pCI-neo (Fig.5). The induction was reduced when GC-rich sequences between −65 to −228 and sequences between −867 to −284 were deleted (Fig. 5). These regions contain multiple GC-rich sequences that may be target sequences for Zf9. Because transcription starts at −232, the results suggest that GC-rich sequences located at downstream of the transcription start site play an important role in the basal and inducible expression of the TGF-β RI gene.
The glutathione-Sepharose affinity-purified GST-Zf9 fusion protein and human recombinant Sp1 were used in EMSAs (Fig.6). Two series of radiolabeled probes and competitor oligonucleotides were used, which represented the sequences between −360 and −349 and between −179 and −151 in the TGF-β RI promoter containing the GC-rich sequences. The GC-rich sequences in the TGF-β RI promoter were found to bind the purified Zf9-GST in the EMSA (Fig. 6). This binding is specific, as shown by competition studies using wild type unlabeled excess competitor oligonucleotides. Sp1 also formed a specific complex with both sites.
The effect of either Sp1 or Zf9 on transactivation of the TGF-β RI promoter was examined in Drosophila Schneider cells in order to determine whether Zf9 could activate in the absence of Sp1. The TGF-β RI-luciferase chimeric constructs were cotransfected with a Drosophila actin promoter-Sp1 or −Zf9 expression plasmid (Fig. 7). The activity of pTβRIP-luc-867/-65 was induced 40-fold by Sp1, whereas the activities of constructspTβRIP-luc-867/-169 and pTβRIP-luc-867/-228were not induced. Moreover, constructs that deleted sequences between −283 and −867 were still responsive to Sp1, suggesting that the region between −65 and −169 is the target sequence for Sp1. In contrast to Sp1, Zf9 was unable to induce TGF-β RI gene expression in Drosophila Schneider cells (Fig. 7).
Transcriptional Activation of the TGF-β RII Promoter by Zf9
Zf9 transactivation of the type II TGF-β receptor was also examined (Fig. 8). Zf9 was a potent transcriptional activator of the TGF-β RII promoter-luciferase reporter in HepG2. As seen in Fig. 8,pTβRIIP-luc−1670/+36 construct was induced 7-fold by Zf9, and the construct pTβRIIP-luc−500/+36 was induced almost 10-fold. Transactivating activity of Zf9 was maintained with deletions to −219 to +36 (pTβRIIP-luc−219/+36) but dropped to the basal level when the deletion reached −70 (pTβRIIP-luc−70/+36). These results suggest that sequences between −219 and −70, including the multiple GC-rich sequences, are important for positively regulating TGF-β RII expression in the presence of high levels of the Zf9 protein.
EMSA was used to confirm the interactions between Zf9 or Sp1 and the TGF-β RII promoter (Fig. 9). To demonstrate that Zf9 and Sp1 can bind directly to the sequences between −219 and −70 in the TGF-β RII promoter, bacterially purified GST-Zf9 protein was used in EMSA using the three series of radiolabeled oligonucleotides and competitors, which represented sequences between −152 and −127, between −118 and −85, and between −39 and −10. Both GST-Zf9 and Sp1 bound to the −152/−127 and −118/−85 oligonucleotide probes with high affinity, whereas the −39/−10 oligonucleotide probe formed a very low affinity complex with these proteins, consistent with the reduced transactivation of a reporter construct containing this region (Fig. 8).
Next, we examined the effect of either Sp1 or Zf9 on TGF-β RII promoter in Drosophila Schneider cells (Fig.10). The activities of constructs ofpTβRIIP-luc−1883/+36 andpTβRIIP-luc−219/+36 were induced 8- and 6-fold, respectively, by Sp1, whereas the activity of constructpTβRIIP-luc−70/+36 was not induced. This finding suggests that one or more GC-rich sequences located between −219 and −70 in the TGF-β RII promoter are responsible for the Sp1-mediated transcription. Zf9 was unable to induce TGF-β RII gene expression in Drosophila Schneider cells, similar to its lack of effect on the type I receptor promoter in this cell type (Fig.10).
Our results identify a potential role for the Kruppel-like factor Zf9/CPBP in the increases in TGF-β1 and its receptors which are characteristic of liver injury. A number of studies have identified TGF-β1 mRNA induction in liver injury (
Both nuclear extracts from activated stellate cells and recombinant Zf9-GST interact with GC-rich regions within the TGF-β1 promoter. The potential importance of GC-rich regions in this promoter has been emphasized in our earlier publication (
). Here, we have documented strong interaction of Zf9-GST with the tandem Sp1 binding sites between −239 and −209, less interaction with a fragment from −124 to −98, and minimal interaction with a single Sp1 site from −82 to −63. This EMSA pattern corresponds very closely with the promoter requirements for Zf9 transactivation in both stellate cells and Hep G2. Moreover, the binding affinity of nuclear extracts from stellate cell to the tandem Sp1 consensus oligo from −124 to −98 (TGFβ1/2) is proportional to the state of cellular activation, with far more nuclear binding in highly activated, immortalized cells than in primary cells. Transactivation of TGF-β1 by Zf9 occurs both in mammalian cells (stellate cells and Hep G2) andDrosophila cells, which lack Sp1.
We have also documented transactivation of the key signaling receptors for TGF-β1 by Zf9. Within mammalian (Hep G2) cells, transactivation is functionally linked to the presence of key GC-rich regions from −360 to −349 and from −179 to −151 and correlates with interaction between recombinant Zf9-GST and each of these regions. Similarly, transactivation of the TGF-β1 type II receptor promoter requires the presence of GC-rich regions from −152 to −127 and from −118 to −85. Zf9-GST did not bind to a third Sp1 site from −39 to −10, which correlated with a lack of transactivation of a reporter-luciferase construct containing a region from −70 to +36.
In contrast to the TGF-β1 promoter, however, transactivation of TGF-β types I and II receptor genes by Zf9 occurred only in a mammalian cellular environment (Hep G2) and not inDrosophila. This finding underscores the unique requirements for each promoter in driving gene expression and raises the possibility that Sp1 is an essential component required for Zf9-mediated transactivation of the TGF-β types I and II receptors. This conclusion is based on several lines of evidence: 1) Sp1 is absent fromDrosophila Schneider cells; 2) both this study and previous analyses by Ji et al. (
) emphasize the importance of Sp1 binding sites for basal and stimulated expression of the type I receptor promoter; and 3) Zf9 and Sp1 can functionally synergize with one another in several promoter contexts (
). Whereas the cognate sequences recognized by Zf9 are similar to Sp1, the cellular factors required for transactivation by each protein are different, as evidenced by differing activities in Drosophila Schneider cells. In preliminary studies, we have confirmed direct interaction between these two proteins (data not shown). Interestingly, in this earlier study examining Sp1 binding in activated stellate cells (
), a significant amount of nuclear protein that bound to an Sp1 consensus sequence did not supershift with anti-Sp1 antibody. This suggests that other GC box-binding proteins may be present in activated stellate cells, including not only Zf9, but possibly BKLF (
These data provide a potentially important link in stellate cells between induction of a transcriptional activator, Zf9, and increased expression of TGF-β1, a key fibrogenic mediator. TGF-β1 induces a large number of matrix genes in stellate cells (
), as in other tissues. The findings further suggest that Zf9 may enhance by the biologic effect of TGF-β1 in vivo not only by transcriptional induction of the gene but also by up-regulation of type I and II TGF-β receptors. This finding may contribute to the increased expression of these receptors during stellate cell activationin vivo and in culture (
). Whereas transcriptional induction of TGF-β receptors may explain a key mechanism underlying enhanced fibrogenesis, intracellular signaling events downstream of TGF-β1 signaling are only beginning to be understood (