Functional Cooperation Between Smad Proteins and Activator Protein-1 Regulates Transforming Growth Factor-β–Mediated Induction of Endothelin-1 Expression
Endothelin-1 (ET-1) is a 21–amino-acid potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells. Expression of the ET-1 gene is subject to complex regulation by numerous factors, among which transforming growth factor-β (TGF-β) is one of the most important. It has been widely documented that TGF-β increases ET-1 mRNA and peptide levels. We have explored the mechanism by which TGF-β upregulates ET-1 expression in endothelial cells. Transcriptional activation of the ET-1 promoter accounted for the TGF-β–induced increase in ET-1 mRNA levels. We have identified within the ET-1 promoter two DNA elements indispensable for TGF-β–mediated induction of ET-1: an activator protein-1 (AP-1) site at −108/−102, known to be important for constitutive and induced expression, and a novel regulatory sequence located at −193/−171, which constitutes a specific binding site for Smad transcription factors. Mutation of both elements abolished TGF-β responsiveness. Binding of Smad3/Smad4 and c-Jun to their corresponding DNA elements was evidenced by electrophoretic mobility shift assays. Furthermore, the coactivator CREB-binding protein (CBP)/p300 was found to play an essential role in the induction of the gene. The simultaneous requirement for two distinct and independent DNA elements suggests that Smads and activator protein-1 functionally cooperate through CBP/p300 to mediate TGF-β–induced transcriptional activation of the ET-1 gene.
Endothelin-1 (ET-1), a potent vasoconstrictor and smooth muscle cell mitogen expressed in endothelial cells of the vascular wall, is synthesized as a precursor molecule of 212 amino acids, the preproendothelin-1 (ppET-1), which is proteolytically cleaved to yield the bioactive form of 21 residues.1–3 Because transcription of the gene is the rate-limiting step of its biosynthesis from endothelial cells, the regulation of the expression occurs mainly at the mRNA level. The molecular analysis of the sequences upstream from exon 1 has revealed that the ET-1 gene has a TATA box–containing promoter.4 Several cis-acting elements have been also implicated in the transcriptional regulation of ET-1 mRNA. An activator protein-1 (AP-1) site at positions −108/−102, which is bound by c-Fos/c-Jun, is required for constitutive ET-1 promoter activity. A GATA motif at positions −136/−131, which is bound by the transcription factor GATA-2 in endothelial cells, is crucial for basal and regulated expression of the ET-1 gene. Cooperation between AP-1 and GATA-2 leads to a synergistic increase in the transactivation of ET-1. Potential binding sites for nuclear factor-1 (NF-1) and hypoxia-inducible factor-1 have also been described.4–7 Nevertheless, in spite of the identification of these cis-regulatory elements, the functional characterization of the promoter is still incomplete. ET-1 mRNA is constitutively expressed but potentially regulated by different biological and pharmacological factors.2
Transforming growth factor-β (TGF-β) is one of the most potent regulators of ET-1 levels. TGF-β strongly increases ET-1 mRNA and protein levels in endothelial cells.8,9 However, the information on the molecular mechanisms of this effect is rather incomplete. In the present study, we identify two DNA elements in the human ET-1 promoter that are indispensable for TGF-β–mediated induction of ET-1: a proximal AP-1 site, known to be important for constitutive and regulated expression, and a novel Smad-binding element (SBE). TGF-β–dependent transcription is mediated by the Smad signaling pathway through a complex mechanism that implies functional cooperation of Smad and AP-1 transcription factors. Furthermore, our data suggest the involvement of the coactivator CREB-binding protein (CBP)/p300 in this cooperation.
Materials and Methods
An expanded version of Materials and Methods can be found in an online data supplement (available at http://www.circresaha.org).
Human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were isolated and maintained in culture using standard procedures. The F9 murine teratocarcinoma cell line was kept in culture by following procedures already described.
RNA Isolation and RNase Protection Analyses
After the corresponding experimental protocol, HUVECs or BAECs were processed for RNA isolation. To detect and quantify human and bovine ET-1 mRNA, RNase protection experiments were performed as described.10 For normalization purposes, human β-actin and 28S rRNA were also determined.
Construction of Reporter Plasmids and Cell Transfection
A luciferase reporter driven by a −650-bp fragment of the human ET-1 promoter (−650-bp ppET-1-prom-luc) was generated as described. 5′- and 3′-deletional constructs were generated by polymerase chain reaction and cloned into pGL3-basic and pGL3-promoter, respectively. Reporter luciferase constructs with specific mutations in the Smad or AP-1 binding sites were generated by polymerase chain reaction–based/site-directed mutagenesis. Single and multiple tandem repeats of the SBEs of the ET-1 promoter cloned into pGL3-promoter were generated by oligonucleotide cloning. Expression vectors used in the present study are explained in detail in the online data supplement. Transient transfection experiments were performed as described.11
Nuclear Extract Preparation and EMSAs
Electrophoretic mobility shift assays (EMSAs) were performed with nuclear extracts or with glutathione S-transferase (GST) fusion proteins (GST-Smad2, GST-Smad3, and GST-Smad4) or purified c-Jun and radiolabeled double-stranded oligonucleotides using standard procedures.
Western Blot Analyses
After the corresponding treatment, cellular proteins from BAECs were isolated, and immunoblotting was performed as described previously.12 Blots were probed with anti-Smad3, anti-Smad4, anti–c-Jun, anti–c-Fos, or anti-p300 antibodies.
Biotinylated Oligonucleotide Pull-Down Experiments
Pull-down experiments using nuclear extracts from BAECs and a biotinylated AP-1 oligonucleotide were performed according to procedures already described. Proteins bound to the probe were analyzed by immunoblotting.
Data were analyzed by unpaired Student t test or using nonparametric tests as appropriate. The probability values obtained are indicated in the text and/or in the figure legends when statistically significant.
Results and Discussion
TGF-β Increases ET-1 Expression Through a Transcriptionally Mediated Mechanism
The effect of TGF-β on ET-1 gene expression was investigated in HUVECs using RNase protection assays to detect and quantify the level of transcripts. TGF-β caused a time- and dose-dependent upregulation of human ET-1 mRNA (data not shown). Although HUVECs constitute a good cell model for investigation on endothelium, they are difficult to transfect, and the cell number for experiments is normally very limited. Therefore, we analyzed the mechanism by which TGF-β increases ET-1 expression in BAECs. TGF-β also time- and dose-dependently upregulated the levels of bovine ET-1 mRNA (Figures 1A through 1C). Induction of ET-1 mRNA by TGF-β did not require de novo protein synthesis as assessed in experiments with cycloheximide (data not shown).
An upstream regulatory sequence (−650/+173) of the human ET-1 gene was cloned in front of a luciferase gene to study whether the effect of TGF-β occurs at the transcriptional level. Transient transfection of BAECs showed that TGF-β induced a potent increase in luciferase activity (12- to 16-fold at 24 hours, data not shown). Therefore, these experiments indicate that ET-1 induction by TGF-β is an early response and that the promoter sequence contains the necessary information to mediate the transcriptional activation of the gene.
TGF-β–Responsive Element in Human ET-1 Promoter Is Responsible for TGF-β–Mediated Induction of Gene Through Smad Signaling Pathway
To identify TGF-β–responsive elements within the −650/+173-bp ET-1 promoter fragment, we generated luciferase constructs under the control of 5′-deletional fragments from −400 to −99 bp and tested their inducibility by TGF-β. Figures 2A and 2B show that the activity of a −650-bp promoter fragment could be induced 12- to 16-fold by TGF-β. Constructs of −400, −329, or −298 bp, which contain an NF-1 site at positions −293/−281, had similar induction rates, but the removal of this putative NF-1 site in the −277-bp construct did not alter the TGF-β effect. The TGF-β response was kept roughly intact until a short sequence between positions −193 and −171 was eliminated. The removal of this DNA fragment caused a dramatic reduction of the TGF-β induction, although significant inducibility was still seen with −171-bp or −117-bp constructs compared with −99-bp or promoterless constructs. Previous assumptions not verified experimentally claimed that regulation of the ET-1 promoter by TGF-β involved the NF-1 site at −293/−281.2,4 This is in clear contrast to our results, which indicate that this site is not involved but that a region between −193 and −171 bp is responsible for the effect.
Careful analysis of the −193/−171-bp DNA segment, very conserved among human, rat, and mouse species, reveals the existence of an SBE, in particular, two inverted repeats of the sequence CAGAC, separated by a GC-rich stretch, an optimal DNA binding sequence for Smad3 and Smad4.13 To assess the contribution of these sites in the mechanism of regulation of the ET-1 promoter by TGF-β, the activity of constructs mutated in the Smad binding sites (SBE1 and SBE2) and in the GC-rich linker was analyzed by luminometry (Figure 2C). Mutation of either SBE1 or SBE2 resulted in a significant inhibition of TGF-β induction. Combination of mutations in SBE1 and SBE2 drastically reduced TGF-β induction. Disruption of the GC-rich segment also caused an inhibitory effect. The TGF-β response was almost abolished when mutations of both SBE1/SBE2 and the GC-rich linker were combined. The introduction of any of these mutations did not significantly modify the basal promoter activity (data not shown).
TGF-β regulates cellular processes by binding to a heteromeric complex of type I and type II kinase receptors. The type I receptor acts downstream from the type II receptor and propagates the signal to the nucleus by phosphorylating specific members of the Smad family, receptor-regulated (R)-Smads. Phosphorylated R-Smads form complexes with the common partner (Co)-Smad (Smad4), which accumulate in the nucleus, where they participate in transcriptional regulation of target genes. Members of a third class of Smad proteins (I-Smads: inhibitory Smad6 and Smad7) are capable of inhibiting TGF-β signaling.14 To investigate the role of Smad family members in the mechanism of ET-1 upregulation by TGF-β, the R-Smads (Smad2 and Smad3), Co-Smad4, and I-Smad7 were overexpressed in BAECs, and their effect on basal and TGF-β–induced promoter activity was analyzed. Figure 3 shows that Smad2 overexpression produces a small but significant increase in TGF-β–induced ET-1-promoter activity. A stronger potentiation of both basal and TGF-β–induced activity was observed with Smad3 and Smad4 expression vectors. Combination of Smad3 and Smad4 resulted in an even greater amplification of ET-1 promoter activity. By contrast, overexpression of the inhibitory Smad7 significantly reduced TGF-β–induced promoter activity.
Western blotting analyses of total and nuclear extracts from BAECs showed a constitutive expression of Smad3 and Smad4, and the amount of both forms increases in the nuclei from cells treated with TGF-β, which correlates with their translocation after TGF-β activation (data not shown). We also checked their expression in Smad3- and Smad4-transfected cells and found a significant fraction in the nuclei from cells incubated in the absence of TGF-β (data not shown). This may explain the fact that ET-1 promoter activity was increased in Smad3- and Smad4-transfected cells under basal conditions. This apparent activation of the pathway in the absence of TGF-β may be explained by the fact that R-Smad overexpression overcomes the limiting levels of endogenous Smad-anchor for receptor activation (SARA), a protein adaptor for TGF-β signaling that locks Smad3 in its inactive monomeric conformation in the absence of a proper receptor signal.15
In conclusion, we were able to locate and identify within the human ET-1 promoter a TGF-β–responsive element that behaves as a potential binding site for Smad transcription factors. Overexpression of Smad3/Smad4 members strongly potentiated the effect of TGF-β on ET-1 promoter activity.
Full Induction of ET-1 Gene by TGF-β Requires Cooperation Between Smad and AP-1 Transcription Factors
Although SBEs are critical for TGF-β–dependent transcriptional activity, target gene selection by TGF-β cannot be explained solely by the presence of SBEs in their promoters. The affinity of Smads for DNA is relatively low, and they have been found to require other sequence-specific binding factors to bind efficiently to TGF-β–regulated promoters.14 Therefore, we wondered whether the induction of ET-1 gene expression by TGF-β requires the functional cooperation between the Smad3/Smad4 complex and other transcription factors relevant for ET-1 expression. For that purpose, we tried to locate and identify additional DNA regions also necessary for TGF-β induction by studying the capacity of 3′-deletional fragments of the human ET-1 promoter to enhance luciferase expression driven by a constitutive simian virus 40 (SV40) promoter. Figure 4A shows that TGF-β did not induce the expression of this promoter. When an ET-1 promoter fragment composed of nucleotides −621 to −223, containing the NF-1 site at −293/−281, was inserted in front of the SV40 promoter, TGF-β was unable to induce it. The inclusion of the sequence up to position −147, downstream from the SBEs, resulted surprisingly in lack of induction. Thus, the isolated presence of SBEs in this construct is not sufficient to confer TGF-β inducibility. Studies using a more proximal construct, up to position −124, including a GATA-2 site at −136/−131, revealed that it was also not responsive to TGF-β. Only when a DNA sequence containing an AP-1 site at positions −108/−102 was included did we find significant induction by TGF-β. These results indicate that induction of the ET-1 gene by TGF-β requires not only the SBEs but also a DNA fragment with an AP-1 binding site 100 bp apart. Site-directed mutagenesis studies (Figure 4B) showed that the mutation of the two SBEs caused a significant reduction in the TGF-β–induced promoter activity. Point mutation of the AP-1 element resulted in a decrease in the basal activity and in a strong inhibition in the TGF-β inducibility. Again, the isolated presence of intact SBEs in this mutant construct was not sufficient to promote a maximal response to TGF-β. Finally, the small induction observed with the mutated AP-1 construct was significantly reduced when compared with the double SBE/AP-1 mutant, which was almost not responsive to TGF-β. In conclusion, full induction of the ET-1 gene by TGF-β requires cooperation between Smad and AP-1 transcription factors.
By overexpressing Smad3 and Smad4 together, we investigated whether the SBEs and the AP-1 site could function independently for TGF-β induction. As shown previously, Smad3/Smad4 overexpression resulted in a strong potentiation of the induction driven by a wild type −193-bp ET-1 promoter (Figure 5A). Although to a lesser extent, Smad3/Smad4 overexpression almost equally potentiated the induction of the promoter constructs with mutated SBEs and with mutated AP-1. Smad3/Smad4 overexpression caused no significant effect on the activity of the double-mutant construct. These results indicate that although both SBEs and AP-1 elements may operate independently, they are capable of mediating a vigorous transcriptional response when they function in a concerted fashion, an observation that suggests the existence of a synergistic relationship. c-Jun and c-Fos components of AP-1 are highly expressed in BAECs, and their overexpression did not modify TGF-β induction of ET-1 expression (data not shown). Therefore, to confirm the existence of such a synergistic effect, we chose the c-Jun–deficient F9 cell model. In these cells, TGF-β had no effect on ET-1 promoter activity (data not shown). Figure 5B shows that the expression in F9 cells of c-Jun produced a 5-fold increase in ET-1 promoter activity. Smad3/Smad4 overexpression caused a similar 5-fold effect, which confirms that their action can take place in the absence of AP-1. The overexpression of both c-Jun and Smad3/Smad4 produced a synergistic effect on ET-1 promoter activity (15-fold increase). This effect was more evident when c-Jun and c-Fos were simultaneously overexpressed (7.8-fold for c-Jun/c-Fos versus a 26-fold increase for c-Jun/c-Fos/Smad3/Smad4, P<0.01).
Crucial information about the mechanism of action of TGF-β on ET-1 expression can be obtained by analyzing the nature of potential proteins interacting with transcription factor binding sites within the ET-1 promoter. To this end, we have performed EMSA with a radiolabeled DNA probe containing the SBE (1× SBE) using nuclear extracts from BAECs treated or not with TGF-β for 6 hours. The 1× SBE probe was not shifted by protein complexes either in the absence or the presence of TGF-β (Figure 6A), a result that clearly confirms the low-affinity DNA-binding capacity of Smads in their interaction with isolated CAGA boxes and thus the absolute requirement of cooperative binding. The contact with tandem copies of potential DNA elements can stabilize binding and is therefore a useful approach to reveal weak DNA-protein interactions. As also shown in Figure 6A, we were able to detect specific protein binding to a radiolabeled probe composed of three copies in tandem of the SBEs (3× SBEs), and the intensity of the bands increased in nuclear extracts from TGF-β–treated cells. This binding could be competed out efficiently by an excess of unlabeled 3× SBEs but poorly with the 1× SBEs and was not modified with a mutant SBE. This increased binding capacity has important consequences in the transactivation properties of these DNA elements. As shown in Figure 6B, 1× SBE was unable to enhance TGF-β–induced luciferase expression driven by a constitutive SV40 promoter. However, three copies in tandem significantly enhanced TGF-β inducibility, an effect more potent with an 8× SBE construct (28.5-fold for 8× SBEs and 3.9-fold for 3× SBEs versus 1.1-fold for the empty vector; P<0,01). Figure 6C also shows EMSA experiments performed with the radiolabeled AP-1 probe. Under basal conditions, we observed a specific protein complex whose intensity increased with longer times of TGF-β incubation. Similar results were observed when we used a 93-bp probe containing both the SBE and the AP-1 binding site (−193/−101) (Figure 6D). Interestingly, the binding to this complex was competed out by an excess of unlabeled AP-1 but not by the SBE oligonucleotide. Taken together, EMSA experiments indicate the presence within the ET-1 promoter of two specific binding sites for nuclear proteins responsive to TGF-β: a strong and dominant AP-1 site and a low-affinity binding element for Smad proteins. To confirm these results, we performed EMSA with purified recombinant GST-Smads and c-Jun. It has been shown that the GST component exhibits a strong tendency to dimerize, which may enhance significantly the affinity of GST-Smads for SBEs compared with the endogenous Smads.16 Under these more favorable binding conditions, we were able to detect, using the 1× SBE probe, DNA-protein interactions with GST-Smad3 and especially with GST-Smad4 (Figure 7A). The combination of Smad3 and relatively lower amounts of Smad4 increased the intensity of the bands obtained separately, suggesting the existence of mutual interactions between Smad3 and Smad4. We also studied the contribution to the binding of the CAGAC repeats and of the GC-rich linker within this SBE in the presence of increasing concentrations of unlabeled wild-type, mutated GC-rich, mutated SBE1/2 and triple-mutant oligonucleotides. As shown in Figure 7B, wild-type oligonucleotide effectively competed the Smad4 binding, whereas mutated GC-rich or SBE1/2 did it with significant lower affinity. The triple mutant oligonucleotide did not modify it.
We have also studied the binding of purified recombinant c-Jun to the ET-1 promoter AP-1 site. Figure 7C shows that c-Jun can interact with the radioactive AP-1 probe and that this band can be competed out by a 100-fold excess of unlabeled wild-type AP-1 but not of mutated AP-1 or of a SBE probe.
In conclusion, these results indicate that an SBE and an AP-1 site within the ET-1 promoter constitute specific elements for the binding of their corresponding transcription factors.
Smads and AP-1 Form a Nucleoprotein Complex Within the ET-1 Promoter: Role of Coactivator CBP/p300
Transactivation and EMSA results suggest the existence of a mechanism of ET-1 induction by TGF-β in which Smads may interact both with DNA (via SBE) and also with the AP-1 transcription factor, activating in this way an AP-1–dependent promoter activity. In this regard, it has been already described that Smad3 interacts in vitro with c-Jun, and the residues involved in this interaction have been mapped.17 Therefore, we analyzed whether Smads and AP-1 associate within the ET-1 promoter. In terms of affinity, EMSA experiments showed that the protein complex binding to the ET-1 promoter is mainly governed by the AP-1 site. Therefore, using a biotinylated AP-1 oligonucleotide probe, we have pulled down this complex and analyzed by Western blotting the identity of the proteins involved. The specificity of this experimental approach was evidenced by the fact that the absence of the biotinylated AP-1 oligonucleotide did not pull down potential DNA-interacting proteins (Figure 8A, left column). Using this experimental approach, we show in Figure 8A (right column) that c-Jun and c-Fos can be detected in the complex from cells incubated under basal conditions. Small amounts of Smad3 and Smad4 can also be visualized in these experiments. Incubation of cells with TGF-β permits higher amounts of Smad3 and Smad4 to be pulled down together with c-Jun/c-Fos. As the TGF-β incubation time increases, the relative presence of c-Jun and c-Fos is more patent, a result similar to that obtained by EMSA with an AP-1 probe. Induction of ET-1 by TGF-β was found to be an early response (maximum at 2 to 4 hours) that does not require protein synthesis to be initiated. Hence, increases in AP-1 activity (and expression as assessed by Western blotting, data not shown) observed at longer times of TGF-β incubation should not be involved in the activation of the mechanism but rather should help to keep ET-1 expression elevated at late stages.
The results obtained with the biotinylated oligonucleotide pull-down experiments indicate that Smads and AP-1 associate with each other, but they do not demonstrate a direct interaction between the factors. To elucidate this issue, the ability of c-Jun to modify Smad3/Smad4 binding to SBE was analyzed by EMSA with recombinant purified proteins. c-Jun inhibited Smad3 binding without significantly affecting Smad4 or Smad3/Smad4 binding. On the other hand, Smad4 and especially Smad3 strongly inhibited c-Jun binding to the AP-1 site (data not shown). Although the behavior of recombinant purified proteins may not represent that of the endogenous proteins, these results suggest that if there is any, the interaction between Smads and AP-1 would result in repression of transcriptional activity rather than activation, as recently described.18 Therefore, we hypothesized that the interaction between Smads and AP-1 can be indirect and mediated by other protein factors. In this regard, the coactivator CBP/p300 has been described to specifically interact with both Smad3 and c-Jun.19,20 CBP/p300 has also been shown to play important roles as a bridge for different transcription factors (GATA-2, AP-1, and hypoxia-inducible factor-1) within the ET-1 promoter.7 Therefore, it is plausible that CBP/p300 mediates the association of the Smad/AP-1 complex. To evaluate its role in the mechanism of TGF-β action, we analyzed the effect of overexpressing p300 on ET-1 promoter activity. Figure 8B shows that the overexpression of p300 markedly amplified both basal and TGF-β–induced ET-1 promoter activity. CBP/p300 modulates gene transcription either by allowing multiprotein complex formation or by acetylation of histones or specific transcription factors. We analyzed whether an intact acetylase activity is required for amplification of the TGF-β effect. Figure 8B also shows that the overexpression of an acetylase-deficient point mutant of p300 (DY mutant) resulted also in an even stronger potentiation of the TGF-β–induced signal, a result suggesting that acetylase activity of p300 is not required for the effect. The adenoviral E1A oncoprotein can physically associate with CBP/p300 and interfere with its ability to induce transcription of target genes. Therefore, E1A is a useful experimental tool to examine the requirement for CBP/p300. E1A overexpression drastically reduced TGF-β–induced promoter activity without affecting basal activity. As a control, a deletion mutant of E1A (E1AΔ2-56) incapable of interacting with CBP/p300 did not alter basal or TGF-β–induced ET-1 promoter activity. In addition, as shown in Figure 8A (right lower panel), CBP/p300 was identified by biotinylated oligonucleotide pull-down experiments as part of the protein complex interacting with the AP-1 site of the ET-1 promoter. Similar to c-Jun/c-Fos, detection of CBP/p300 increased at longer times of incubation with TGF-β. These results suggest that CBP/p300 is involved in the mechanism of TGF-β action on ET-1 expression.
In conclusion, our results demonstrate that TGF-β–induced ET-1 expression is mediated by a functional cooperation between Smads and AP-1. Previous studies have demonstrated the importance of AP-1 for signal-regulated and constitutive transcription of the ET-1 gene.5–7 The present work reinforces its pivotal role in the control of ET-1 expression, inasmuch as we demonstrate the absolute requirement of AP-1 for a complete response to TGF-β. Other genes of vascular relevance, such as plasminogen activator inhibitor-1, the α2 chain of type I collagen, and interstitial collagenase, are also examples of AP-1–dependent TGF-β responsiveness.21–23 Our results are consistent with a mechanistic model in which Smads can establish both protein-DNA and protein-protein interactions. Through association with AP-1, Smads increase AP-1–dependent ET-1 promoter activity. A full induction is observed when the complex of Smads/AP-1 (containing also CBP/p300) is further stabilized by direct interaction of Smads with the SBE. Further experiments will be required to confirm this hypothetical model as well as to evaluate its physiological relevance.
This study was supported by grants SAF2000-0149 from Plan Nacional de I+D+I and Fundación Ramón Areces. Dr Rodríguez-Pascual has a postdoctoral position supported by the I3P Program of the C.S.I.C. We are very grateful to Angel Corbí, Luisa Botella, Tilman Sanchez-Elsner, Javier Rey-Campos (CIB, Madrid, Spain), and Ana Aranda (IIB, Madrid, Spain) for their valuable discussions and assistance with experiments.
Original received October 31, 2002; resubmission received March 21, 2003; revised resubmission received May 13, 2003; accepted May 13, 2003.
Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A. 1989; 86: 2863–2867.
Inoue A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T. The human preproendothelin-1 gene: complete nucleotide sequence and regulation of expression. J Biol Chem. 1989; 264: 14954–14959.
Kawana M, Lee ME, Quertermous EE, Quertermous T. Cooperative interaction of GATA-2 and AP1 regulates transcription of the endothelin-1 gene. Mol Cell Biol. 1995; 15: 4225–4231.
Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshizumi M, Quertermous T. Regulation of endothelin-1 gene expression by Fos and Jun. J Biol Chem. 1991; 266: 19034–19039.
Yamashita K, Discher DJ, Hu J, Bishopric NH, Webster KA. Molecular regulation of the endothelin-1 gene by hypoxia: contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP. J Biol Chem. 2001; 276: 12645–12653.
Rodriguez-Pascual F, Hausding M, Ihrig-Biedert I, Furneaux H, Levy AP, Forstermann U, Kleinert H. Complex contribution of the 3′-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene: involvement of the RNA-binding protein HuR. J Biol Chem. 2000; 275: 26040–26049.
Hernandez-Perera O, Perez-Sala D, Soria E, Lamas S. Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells. Circ Res. 2000; 87: 616–622.
Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, Sanchez-Pascuala R, Hernandez G, Diaz C, Lamas S. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest. 1998; 101: 2711–2719.
Moustakas A, Heldin CH. From mono- to oligo-Smads: the heart of the matter in TGF-β signal transduction. Genes Dev. 2002; 16: 1867–1871.
Liberati NT, Datto MB, Frederick JP, Shen X, Wong C, Rougier-Chapman EM, Wang XF. Smads bind directly to the Jun family of AP-1 transcription factors. Proc Natl Acad Sci U S A. 1999; 96: 4844–4849.
Topper JN, DiChiara MR, Brown JD, Williams AJ, Falb D, Collins T, Gimbrone MA Jr. CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells. Proc Natl Acad Sci U S A. 1998; 95: 9506–9511.
Chung KY, Agarwal A, Uitto J, Mauviel A. An AP-1 binding sequence is essential for regulation of the human α2(I) collagen (COL1A2) promoter activity by transforming growth factor-β. J Biol Chem. 1996; 271: 3272–3278.
Keeton MR, Curriden SA, van Zonneveld AJ, Loskutoff DJ. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor β. J Biol Chem. 1991; 266: 23048–23052.
Mauviel A, Chung KY, Agarwal A, Tamai K, Uitto J. Cell-specific induction of distinct oncogenes of the Jun family is responsible for differential regulation of collagenase gene expression by transforming growth factor-β in fibroblasts and keratinocytes. J Biol Chem. 1996; 271: 10917–10923.