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Carcinogenesis Advance Access originally published online on February 24, 2008
Carcinogenesis 2008 29(5):905-912; doi:10.1093/carcin/bgn049
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Expression of the type III TGF-β receptor is negatively regulated by TGF-β

Nadine Hempel1,5, Tam How1,{dagger}, Simon J. Cooper2,{dagger}, Tyler R. Green1, Mei Dong1, John A. Copland2, Christopher G. Wood3 and Gerard C. Blobe1,4,*

1 Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
2 Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA
3 Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
5 Present address: Department of Immunology and Microbial Diseases, Albany Medical College, Albany, NY 12208, USA

* To whom correspondence should be addressed. Tel: +1 919 668 1352; Fax: +1 919 681 6906; Email: blobe001{at}mc.duke.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The type III transforming growth factor-beta receptor (TβRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-β) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TβRIII in the pathogenesis of human cancers, with frequent loss of TβRIII expression at the message and protein level. Mechanisms for the loss of TβRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-β1. Here, we define a specific role for TGF-β1 in negatively regulating TβRIII at the message level in breast and ovarian cancer models. TGF-β1 decreased TβRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-β1-mediated TβRIII repression is mediated by the type I TGF-β receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TβRIII expression. Mechanistically, TGF-β1 does not affect TβRIII messenger RNA (mRNA) stability, but instead directly regulates the TβRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-β1 and constitutively active ALK5. Taken together, these studies define TGF-β1-mediated downregulation of TβRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TβRIII expression in human cancers.

Abbreviations: a/s, antisense; mRNA, messenger RNA; PCR, polymerase chain reaction; s, sense; TGF-β, transforming growth factor-beta; TβR, transforming growth factor-beta receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Transforming growth factor-beta (TGF-β) regulates a diverse range of biological functions including differentiation, proliferation, angiogenesis, immunosuppression and motility in a context-dependent manner (1). During the initiation and progression of human cancer, the TGF-β-signaling pathway has a dual role, initially suppressing tumor formation but with elevated levels of TGF-β promoting the growth, progression and metastatic spread of established tumors.

TGF-β elicits its cellular effects via interaction with three cell surface receptors, the type I, II and III transforming growth factor-beta receptors (TβRs). Upon ligand binding, the serine/threonine kinase type II TGF-β receptor (TβRII) associates with and phosphorylates the type I receptor (TβRI or ALK5), activating the TβRI serine/threonine kinase (2). TβRI then recruits, phosphorylates and activates the Smad2/3 transcription factors, which form a complex with the co-Smad and Smad4, and translocate as a complex into the nucleus to regulate transcription of TGF-β-responsive genes (3).

The type III receptor (TβRIII or betaglycan) was originally characterized as a coreceptor for TβRII (4). While TβRIII does not have a functional kinase domain, it binds all the three TGF-β isoforms and inhibin with high affinity and regulates their ability to interact and signal through other TGF-β superfamily signaling receptors (4,5). The importance of TβRIII is evident by the embryonic lethality of TβRIII knockout mice at day 16.6, due to liver and heart developmental defects (6,7), and an essential role in chick heart development (8). In addition, recent studies have broadened the potential roles of TβRIII, including regulating TGF-β receptor levels through interactions with β-arrestin2 and GAIP-interacting protein, C terminus (GIPC) and potentially signaling independently (9,10).

Recently, we have demonstrated that TβRIII expression is significantly downregulated at both the message and protein levels in a broad spectrum of human cancers, including cancers of the breast, lung, ovary, pancreas and prostate (11–15). Further, we demonstrated that TβRIII regulates migration and invasion in these cancers both in vitro and in vivo (11–15).

While multiple mechanisms potentially account for the loss of TβRIII expression in these human cancers, including loss of heterozygocity of the TGFBR3 gene and epigenetic regulation (11–13), we had also identified TGF-β1-mediated repression of TβRIII message levels in a breast cancer model (11). Here, we investigate the mechanisms by which TGF-β negatively regulates TβRIII messenger RNA (mRNA) expression at the transcriptional level in both breast and ovarian cancer model systems.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture and reagents
Cell lines were cultured in 5% CO2 at 37°C. Human ovarian cancer Ovca420 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum. Human breast cancer MDA-MB-231 cells were cultured in Modified Eagle's Medium, supplemented with non-essential amino acids, sodium pyruvate and 10% fetal bovine serum. Cells were serum starved for 3 h unless otherwise indicated and treated with TGF-β1 (R&D Systems, Minneapolis, MN) with indicated concentrations. Actinomycin D and SB431542 were purchased from Sigma (St Louis, MO). Cells were infected with the ALK5QD-expressing adenovirus 100 plaque forming units, generously provided by Dr Carlos Arteaga, using a GFP-expressing adenovirus (100 plaque forming units) as a control.

RNA isolation, complementary DNA synthesis and semiquantitative real-time reverse transcription–polymerase chain reaction
Cells were lysed using QiaShredder columns (Qiagen, Valencia, CA) and RNA is isolated using the RNeasy extraction kit (Qiagen). RNA (500 ng) was reverse transcribed with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers according to the manufacturer’s instructions. Real-time semiquantitative reverse transcription–polymerase chain reaction (PCR) was carried out on a MyiQ thermal cycler (Bio-Rad, Hercules, CA) using SYBR green reagent (Bio-Rad), 2 µl of template and 0.25 µM of each of the following primer pairs: human type I TGF-β receptor (TβRI): sense (s) 5'-acggcgttacagtgttctg-3', antisense (a/s) 5'-ggtgtggcagatatagacc-3'; human TβRII: s 5'-gcaggtgggaactgcaagat-3', a/s 5'-gaaggactcaacattctccaaattc-3'; human TβRIII: s 5'-ctgttcacccgacctgaaat-3', a/s 5'-cgtcaggaggcacacactta-3'. An annealing temperature of 58°C was used for all primer pairs. After amplification, specificity of the reaction was confirmed by melt curve analysis. Data were analyzed using the comparative CT method with values normalized to glyceraldehyde-3-phosphate dehydrogenase (s 5'-gagtcaacggatttggtcgt-3' and a/s 5'-ttgattttggagggatctcg-3') and expressed relative to untreated controls.

125I-TGF-β binding and cross-linking
TβRIII protein was visualized using binding and cross-linking of the receptor to 125I-labeled TGF-β, following immunoprecipitation with TβRIII-specific antibody, as described previously (16).

TβRIII promoter constructs
A 700 bp region of the proximal TβRIII promoter was isolated by PCR from human female genomic DNA (Promega, Madison, WI) with Platinum Taq polymerase (Invitrogen) using s 5'-cgcccaccatcagagcgtga-3' and a/s 5'-ggaggaaagcggcggcaagt-3' primers. The PCR product was cloned into TOPO TA pCR2.1 vector (Invitrogen) followed by subcloning into pGL3Basic using SacI and XhoI restricition site. Promoter truncations were created using PCR with the following primers: –165 s, 5'-ggggtaccggcggaggagcgcccttc-3'; –75 s, 5'-ggggtaccgggtcggcctgatgg-3'; –13 s, 5'-ggggtacccagcgagtgaaggag-3'; common a/s, 5'-ggaatcgcgcagggaaagtgg-3'.

The following primers were used to amplify the distal promoter: 3.3 kb s, 5'-tcactcctagatgtggaac-3' and 3.3 kb a/s, 5'-cctcaatgttgccagagctctgc-3'; 2.3 kb s, 5'-gggacgcgtgcttaggatgcagaacatatgag-3' and 2.3 kb a/s, 5'-cccgctcgaggctcatgcggttctttcagt-3'; 1 kb s, 5'-aaggtacatagctctgggtgcac-3' and 1 kb a/s, 5'-cctcaatgttgccagagctctgc-3'. These were cloned into pCR2.1 vector, subloned into pGL3Basic and used as a template to create truncation mutants by PCR with the following primers: –314 s, 5'-gggacgcgtgagacggagtctcaccctgtc-3'; –190 s, 5'-gggacgcgtactacacccggccaattttt-3'; –139 s, 5'-gggacgcgttagtagagacggggtttcaccatg-3'; –30 s, 5'-gggacgcgtcgcccgtccagaattatctt-3'; common a/s, 5'-cccgctcgaggctcatgcggttctttcagt-3'.

Luciferase assay
Cells were transfected with promoter constructs using Lipofectamine 2000 transfection reagent (Invitrogen). The TβRIII promoter luciferase reporter constructs or the empty pGL3Basic vector (200 ng per well) was transfected together with control Renilla luciferase reporter vector pRL-SV40 (10 ng per well) in 24-well plates. Media was changed 24 h posttransfection and cells were treated with indicated amounts of TGF-β for an additional 48 h. In cotransfection experiments, 200 ng per well of ALK5QD or empty vector were co-transfected with promoter constructs. Luciferase activities of the lysed cells were measured using the Dual Luciferase Kit (Promega), according to the manufacturer’s instructions. Each result represents mean ± SEM of three transfections and assays were repeated 2–3 times to confirm reproducibility.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
TGF-β1 downregulates TβRIII expression
We have demonstrated previously that TβRIII mRNA levels are decreased in response to TGF-β1 treatment in MDA-MB-231 breast cancer cells (11). To further characterize this regulatory mechanism, we treated MDA-MB-231 cells and an ovarian cancer cell line, Ovca420, with TGF-β1 and assessed the levels of TβRIII mRNA levels using semiquantitative real-time reverse transcription–PCR. Both cell lines normally express detectable levels of endogenous TβRIII (11,12). Treatment with 100 pM TGF-β1 resulted in a 60–80% decrease in TβRIII levels in both cell lines, with maximal downregulation observed at 6–9 h (Figure 1A and B). TGF-β1 was quite potent in mediating this effect, with low doses (10–30 pM) resulting in near maximal repression of TβRIII message levels and higher levels (100 pM) only slightly increasing the effect (Figure 1C and D). These decreases in TβRIII message levels were sufficient to result in decreases in steady-state cell surface TβRIII expression in MDA-MB-231 cells (Figure 1E) and Ovca420 cells (data not shown) and soluble TβRIII protein levels in both Ovca420 and MDA-MB-231 cells (Figure 1E).


Figure 1
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  		Fig. 1. TGF-β1 downregulates TβRIII expression. Ovca420 (A) and MDA-MB-231 (B) cells were treated 100 pM TGF-β1 for the indicated times. For dose-response experiments Ovca420 (C) and MDA-MB-231 (D) cells were treated for 6 hours with the indicated amounts of TGF-β1. RNA was isolated, reverse transcribed and Real time semi-quantitative PCR carried out with primers against human TβRIII. Data was normalized against GAPDH levels and expressed relative to fold reduction in TβRIII levels compared to non-treated controls (n = 3, mean +/– SEM, *P < 0.05, **P < 0.01, ***P < 0.001, student's T-test). E. Ovca420 and MDA-MB-231 cells were treated for 24 hours with the indicated amounts of TGF-β1 (non-treated controls: NT), and TβRIII protein levels were detected by binding and crosslinking of 125I-TGF-β1 to the receptor. TβRIII was immunoprecipitated from lysed cells (cell surface) or media (soluble) using TβRIII specific antibodies, followed by protein electrophoresis and visualization using autoradiography. The arrow points to the position of TβRIII on the gel and the position of the 165 kDa molecular weight marker is indicated.

 
To assess whether the effects of TGF-β1 on TβRIII were specific, we analyzed the role of TGF-β1 on regulating TβRI and TβRII levels in Ovca420 and MDA-MB-231 cells. In both cell lines, no significant changes in TβRII levels were observed upon TGF-β1 treatment of various doses and treatment times (Figure 2). While we observed an increase in TβRI expression in Ovca420 cells, this was not consistently, statistically significant (Figure 2A and C). In addition, TβRI mRNA levels were not increased following the TGF-β1 treatment of MDA-MB-231 cells (Figure 2B and D). These results indicate that of the TGF-β receptors, TβRIII is the only receptor downregulated by TGF-β1 in ovarian and breast cancer cells.


Figure 2
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  		Fig. 2. TGF-β1 does not significantly regulate TβRI and TβRII expression. Ovca420 (A) and MDA-MB-231 (B) cells were treated 100 pM TGF-β1 for indicated times. For dose-response experiments Ovca420 (C) and MDA-MB-231 (D) cells were treated for 6 hours with indicated amounts of TGF-β1. RNA was isolated, reverse transcribed and Real time semi-quantitative PCR carried out with primers against human TβRI and TβRII. Data was normalized against GAPDH levels and expressed relative to fold changes in TβRI or TβRII levels compared to non-treated controls (n = 3, mean +/– SEM, *P < 0.05, **P < 0.01, student's T-test).

 
Downregulation of TβRIII expression is TβRI/ALK5 dependent
To determine whether TGF-β1-mediated downregulation of TβRIII was elicited via the serine/threonine kinase receptor complex of TβRI and TβRII, we assessed the effects of the TβRI/ALK5 inhibitor SB431542 on TβRIII levels in the presence and absence of exogenous TGF-β1. SB431542 treatment induced TβRIII expression in the absence of exogenous TGF-β1 in both cell lines (Figure 3A), suggesting that baseline levels of TβRIII may be suppressed by autocrine TGF-β1. Consistent with this, SB431542 treatment effectively abrogated TGF-β1-mediated Smad2/3 phosphorylation (data not shown) and TGF-β1-mediated downregulation of TβRIII in both cell types (Figure 3A). To further investigate a role of ALK5, we infected MDA-MB-231 cells with an adenovirus expressing constitutively active ALK5, ALK5QD. Expression of ALK5QD was sufficient to stimulate Smad2/3 phosphorylation (data not shown) and decrease TβRIII message levels to an extent similar to that observed upon TGF-β1 treatment (Figure 3B). These results suggest that the ALK5 pathway mediates the effects of TGF-β1 in downregulating TβRIII expression.


Figure 3
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  		Fig. 3. TGF-β acts via TβRI (ALK5) to down-regulate TβRIII expression. A. Ovca420 and MDA-MB-231 cells were pretreated with the ALK5 inhibitor SB431542 (20 µM) for 30 min, following by 6 hours of 100 pM TGF-β1 treatment. RNA was isolated, reverse transcribed and real time semi-quantitative RT-PCR carried out with TbRIII specific primers. Values were normalized against GAPDH levels and expressed relative to fold reduction in TβRIII levels (n = 3, mean +/– SEM, **P < 0.01, ***P < 0.001, student's T-test). B. MDA-MB-231 cells were infected with adenoviral construct expressing the constitutively active form of ALK5 (ALK5QD) or control adenovirus expressing GFP. 48 hours post infection RNA was isolated, reverse transcribed and real time semi-quantitative RT-PCR carried out with TβRIII specific primers. Values were normalized against GAPDH levels and expressed relative to fold reduction in TβRIII levels (n = 2, mean +/– SEM, *P < 0.05, student's T-test).

 
TGF-β1 does not affect TβRIII message stability
As downregulation of TβRIII was observed at the message level, we investigated the mechanisms by which TGF-β1 could regulate the message level. TGF-β1 has been demonstrated to regulate message stability of a variety of genes, including both increasing and decreasing RNA stability (17). To assess the role of RNA stability in the regulation of TβRIII expression by TGF-β1, the half-life of TβRIII message was determined by blocking transcription with actinomycin D and examining the levels of TβRIII message in the presence or absence of TGF-β1. While the half-life of TβRIII message was somewhat variable between the Ovca420 cell line (t1/2 = 2 h) and the MDA-MB-231 (t1/2 = 4 h), the rate of TβRIII message degradation was not significantly different in the presence or absence of TGF-β1 in either cell line (Figure 4). These studies demonstrate that the effects of TGF-β1 in downregulating TβRIII message level are not due to the effects on message stability.


Figure 4
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  		Fig. 4. TGF-β1 does not alter TβRIII mRNA stability. Ovca420 (A) and MDA-MB-231 (B) cells were treated with 10 µg/ml Actinomycin D and with or without 100pM TGF-β1 for the indicated times. RNA was isolated, reverse transcribed and real time semi-quantitative RT-PCR carried out with TβRIII specific primers. Values were normalized against GAPDH levels and expressed relative to fold reduction in TβRIII levels (n = 3, mean +/– SEM).

 
Characterization of the human TGFBR3 gene promoter
While the rat and mouse TGFBR3 gene promoters have been isolated and described (18,19), the human TGFBR3 promoter has not been characterized. The TGFBR3 gene is located on chromosome 1, position 1p33–p32, and contains three non-coding exons in its 5' region, according to expressed sequence tag (EST) (www.ensembl.org, OTTHUMG00000010097; Figure 5A, untranslated exons A, B and C). Published mRNA species have been identified to contain either 5' untranslated region (UTR) A (accession no. NM_003243 [GenBank] ) or both B and C (L07594 [GenBank] ). The potential transcriptional start site was marked as –1 on each promoter, based on the 5' end of the most common complementary DNA species identified. The presence of different 5' UTRs indicate that the TβRIII message is either alternatively spliced posttranscriptionally, or that the TGFBR3 gene contains alternative promoters.


Figure 5
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  		Fig. 5. Activity of the proximal and distal promoters of the TGFBR3 gene. A. Schematic of the 5' region of the human TGFBR3 gene. Grey boxes represent the location of the alternate 5'UTRs of TβRIII cDNA species identified in the literature and black box the first coding exon of TβRIII. The 5'UTR transcriptional start sites of the proximal and distal promoters are labeled as +1. The location of the promoter constructs and potential transcription factor binding sites are indicated B. Promoter activities of sequences flanking the alternate 5'UTRs of TβRIII in Ovca420 and MDA-MB-231 cells. All promoters were cloned into the pGL3Basic vector, transfected into cells and lysed cells assayed for luciferase activity as stated in Methods. Results are corrected for Renilla luciferase activity of the pRLSV40 transfection standard and represent the mean +/– SEM, n = 3. Results are expressed as fold increases in luciferase activity relative to the empty pGL3Basic vector.

 
Regions flanking the 5' UTR A and C of the TGFBR3 gene were cloned and classified as proximal and distal TGFBR3 promoters, respectively. The proximal and distal promoters are located ~25 and 45 kb from the translational ATG start codon of TβRIII, respectively. Cloning of these promoters into a pGL3Basic luciferase reporter vector and transfection into Ovca420 and MDA-MB-231 cells revealed that both promoters displayed high basal activity when compared with empty vector (Figure 5B). To identify the basal transcriptional unit of each promoter, we created a series of deletion mutants. Transfection of these into cells revealed that the proximal promoter unit is located between base –165 and –75 from the proximal transcriptional start site (Figure 5B). This region contains an Sp1 site that is conserved among the rat, mouse and human TGFBR3 promoters. This, together with an upstream GC-rich region containing an additional Sp1 site, has been shown as a crucial component driving the rat TGFBR3 promoter via activator protein 1 (19). Other potential transcription factor response elements identified using AliBaba, Match and Transfac database programs (http://www.gene-regulation.com) are indicated (Figure 5A). Activities of deletion constructs of the distal promoter demonstrated that a crucial response element exists between bases –500 and –314 from the distal transcriptional start site. Additionally, a potential repressor site between –314 and –199 may exist on this promoter, as deletion of this region resulted in marked increase in promoter activity in Ovca420 cells and to a lesser extent in MDA-MB-231 cells (Figure 5B).

TGFBR3 proximal promoter activity is inhibited by TGF-β1
Our data indicated that TGF-β1 acts to downregulate TβRIII expression directly at the transcriptional level. To directly explore this, the proximal and distal TGFBR3 promoter constructs were transfected into Ovca420 cells and their activity assessed following TGF-β1 exposure. The activity of the TGF-β-responsive promoter, p3TP, was induced 4-fold upon TGF-β1 treatment, serving as a positive control for TGF-β responsiveness (Figure 6A). In contrast, the luciferase activity of the proximal TGFBR3 promoter was significantly reduced (~40%) by TGF-β1 treatment (Figure 6A), whereas the TGFBR3 distal promoter or the empty pGL3Basic vector was not significantly affected (Figure 6A). Further, the TGFBR3 proximal promoter was repressed by TGF-β1 in a dose-dependent fashion, whereas the TGFBR3 distal promoter was not (Figure 6B). To establish whether the effects of TGF-β1 on the TGFBR3 proximal promoter were via ALK5, we infected Ovca420 cells with adenovirus-expressing ALK5QD. Similar to TGF-β1 stimulation, ALK5QD stimulated the 3TP promoter and repressed the proximal promoter, while not affecting the distal promoter (Figure 6C). These data indicate that downregulation of TβRIII message levels by TGF-β1 occurs via direct inhibition of the proximal promoter of the TGFBR3 gene.


Figure 6
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  		Fig. 6. The proximal TβRIII promoter is negatively regulated by TGF-β1. A. The empty pGL3Basic vector, the TGF-β responsive p3TP promoter, the proximal (–559/+60) and the distal (–2330/+80) TβRIII promoter constructs were transfected into Ovca420 cells and treated for 48 hours with 100pM TGF-β1. Cells were lysed and assayed for luciferase activity as stated in Methods. Results are corrected for Renilla luciferase activity of the pRLSV40 transfection standard and represent the mean +/– SEM, n = 3. Results are expressed as fold changes in luciferase activity relative to each untreated control (NT, **P<0.01, ***P<0.001, student's T-test). B. TGF-β down-regulates TβRIII proximal promoter activity in a dose-dependent manner. Ovca420 cells were transfected with proximal and distal promoter as stated above and treated with indicated amounts of TGF-β1 for 48 hours. Results are expressed as fold changes in luciferase activity relative to each untreated control (0 pM; mean +/– SEM, n = 3, *P<0.05, **P<0.01, student's T-test). C. TβRIII proximal promoter downregulation is ALK5-dependent. Constitutive active ALK5QD was cotransfected with either the p3TP promoter construct, the TβRIII proximal or distal promoter into Ovca420 cells. 48 hours post-transfection luciferase activity was measured as stated above and treated with indicated amounts of TGF-β1 for 48 hours. Results are expressed as fold changes in luciferase activity relative to each untransfected control (NT; mean +/– SEM, n = 3, **P<0.01, student's T-test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Here, we demonstrate that TβRIII, an essential component of TGF-β superfamily signaling pathways, is downregulated by TGF-β1 and that this downregulation occurs at the transcriptional level. Further, for the first time, we characterize the human TGFBR3 promoter, defining alternative promoters for the TGFBR3 gene. Finally, we establish that the effects of TGF-β1 are through the ALK5 pathway and via direct effects on the proximal promoter.

How does TGF-β1 repress TβRIII expression? Transcriptional profiling studies have demonstrated that TGF-β regulates approximately 3–5% of genes in the genome, with about two-thirds of those being upregulated and one-third of them being downregulated. While mechanisms for promoter activation through the Smad transcriptions factors interacting with Smad-binding elements on responsive promoters have been well established, mechanisms by which TGF-β represses target genes are less well understood. Mechanisms for negative transcriptional regulation by TGF-β have been demonstrated primarily in the context of genes regulating the cell cycle, including downregulation of the growth-promoting transcription factors including c-Myc, ID1 and ID2. TGF-β-mediated ID1 downregulation occurs through the interaction of activating transcription factor 3 with Smad3/4 (20), whereas TGF-β-mediated c-Myc downregulation occurs via interaction of Smad3/4 with the cofactor E2F4/5 and the transcriptional repressor p107 (21). In turn, loss of c-Myc expression negatively regulates ID2 expression as ID2 transcription is normally induced by binding of c-Myc at E-boxes on the Id2 gene promoter (22). Alternatively, TGF-β can repress ID2 transcription by inducing Mad expression, which forms a complex with the c-Myc cofactor Max and displaces c-Myc/Max from the Id2 gene promoter (23). In terms of TGF-β-mediated TβRIII downregulation, the proximal promoter on which TGF-β functions contains a potential E-box sequence, –382 bp from the potential transcriptional start site. In addition, while c-Myc is generally believed to be upregulated in cancers, mRNA levels of c-Myc were significantly decreased in ovarian and breast cancer specimens relative to normal tissue levels [http://www.oncomine.org; (24–27)]. Similarly, TβRIII mRNA levels were also decreased in all of the aforementioned studies. The importance of c-Myc and the E-box sequence in the negative regulation of the TGFBR3 promoter by TGF-β remains to be elucidated.

In addition to interactions with transcriptional activators and corepressors, the Smad3/4 complex has been demonstrated to act as a repressive factor itself. For example, Smad3/4 interacts with CCAAT/enhancer binding protein (C/EBP)β and {delta} to prevent C/EBP transcriptional activity on peroxisome proliferator-activated receptor {gamma} and leptin promoters (28). As the TGFBR3 proximal promoter contains two conserved C/EBP-binding sequences, inhibition of C/EBP binding to these regions also represents a possible regulatory mechanism for TGF-β downregulation.

Whether TβRIII repression results directly from association of Smads with transcriptional repressors on the TGFBR3 promoter or via non-canonical, Smad-independent pathways remains to be defined. Smads bind the canonical CAGA element, but have been shown to have affinity for GC-rich promoters. There are two CAGA sites within –490 and –559 bp form the transcriptional start site and the proximal promoter has a high GC rich content. The role of Smad proteins in the downregulation of TβRIII promoter activity is currently under investigation.

The presence of two functional promoters of the TGFBR3 gene accounts for the two TβRIII mRNA species that have been isolated as ESTs (NM_003243 [GenBank] and L07594 [GenBank] ). From the relative frequency of isolation of these mRNA species, it appears that the most abundant transcript arises from transcription at the proximal promoter. A BLAST of human ESTs with the first coding exon of TβRIII revealed the presence of 80 ESTs, 75 of which contained sequences homologous to the proximal 5' UTR (A). The other five ESTs had no additional 5' UTRs beyond the first coding exons. We failed to identify an EST that contained 5' UTRs B or C in this search. A BLAST with the sequence of 5' UTR B or C pulled out two ESTs, however, these did not contain recognizable sequences of the remaining TβRIII-coding region. In addition, only the proximal promoter has sequence identity with the previously described rat and mouse TGFBR3 promoters (supplementary Figure 1, available at Carcinogenesis Online) (18,19). High sequence homology was observed within 450 bp upstream of the transcriptional start site. An alignment of this region revealed that the human promoter shares 73 and 74% sequence identity with the mouse and rat promoters, respectively. This raises the question of whether and when the distal promoter is required to drive TβRIII transcription. Perhaps, the two promoters function dependent on the transcription factor milieu in a given cell type, thereby regulating TβRIII transcription in a cell context-dependent manner. Interestingly, the distal promoter contains numerous nuclear receptor sites including retinoic acid receptor (RAR{alpha} and RXRβ) and the glucocorticoid receptor, which may induce TβRIII expression in circumstances of high steroid levels. Indeed, the mouse TGFBR3 promoter can be positively regulated by retinoic acid and the muscle differentiation factor MyoD (19). A MyoD-binding element was also found on the human distal TGFBR3 promoter. Further, the rat TGFBR3 promoter is regulated by dexamethasone (18). While these elements exist in the mouse and rat promoters that share homology with the proximal TGFBR3 promoter, the response elements are found on the distal promoter of the human gene. Whether these response elements are functional in regulating the human distal promoter remains to be elucidated.

While investigating methylation targets of TGFBR3 during carcinogenesis, we established that the distal TGFBR3 promoter is highly methylated in a variety of cell lines, including in breast and ovarian cancer cell lines (N.H. and G.C.B., unpublished observations). However, this methylation profile did not correlate with the expression profile of TβRIII in these cells, suggesting that methylation of the distal promoter was not the target of transcriptional inactivation by hypermethylation during carcinogenesis. Instead, constitutive methylation of the distal promoter suggests that the distal promoter represents a dormant promoter, at least in these cancers, that may be epigenetically regulated in other tissues or during development. The relevance of epigenetic silencing of the distal promoter remains to be elucidated.

The negative regulation of TβRIII by TGF-β1 has implications both for the role of elevated TGF-β1 and the role for repressed TβRIII levels in human cancers. Elevated levels of TGF-β1 have been documented in a broad spectrum of human cancers. These elevated levels often correlate with more invasive and metastatic disease and a poorer prognosis for these patients (11–15). However, the mechanisms by which elevated TGF-β1 levels exert their tumor-promoting effects have not been elucidated. As we have defined the loss of TβRIII expression as a common event in the same human cancers with elevated TGF-β1 levels, with the loss of TβRIII expression correlating with disease progression and poorer prognosis, the current studies support the loss of TβRIII expression as one mechanism for the tumor-promoting function of TGF-β1.

In a reciprocal manner, the ability of TGF-β1 to specifically downregulate TβRIII levels, while TβRII and TβRI levels remain relatively unaffected, provides an important mechanism for the widespread decrease in TβRIII expression in human cancers. While loss of heterozygosity and epigenetic regulation of the TGFBR3 gene may also have a role, studies have revealed that the TGFBR3 gene locus is not mutated in human cancers (11–13). Given the frequency with which TGF-β1 levels are elevated and TβRIII levels are repressed, the transcriptional regulation mechanism described here may be a dominant mechanism for regulating TβRIII expression during cancer progression. As the levels of TGF-β1 necessary to repress TβRIII expression (~10 pM) are physiologically relevant, the control of TβRIII levels by TGF-β1 may be an important negative feedback for the TGF-β-signaling pathway. Consistent with this hypothesis, treatment with the ALK5 inhibitor, SB431542, was sufficient to induce TβRIII levels (Figure 3). We had described previously the regulation of TβRIII cell surface levels, through interactions with the scaffolding proteins GIPC and β-arrestin2 (9,29). In these studies, stabilization of TβRIII through interaction with GIPC was sufficient to increase TGF-β signaling (29), whereas internalization and downregulation of TβRIII through interaction with β-arrestin2 was sufficient to decrease TGF-β signaling (9). Taken together, these studies suggest that regulation of TβRIII expression is tightly controlled at both the message and protein level, with this expression allowing the regulation of TGF-β signaling. This supports an essential and rate-limiting role for TβRIII in the TGF-β-signaling pathway.

In conclusion, we have identified that human TβRIII mRNA is negatively regulated by TGF-β1 in ovarian and breast cancer cells. This regulation is dependent on TβRI/ALK5 and occurs at the transcriptional level on the TGFBR3 gene. Further, we have identified the presence of alternative promoters for human TGFBR3, of which the proximal promoter confers the negative regulation by TGF-β1. These results provide the first step toward elucidating the transcriptional regulation of human TβRIII, an important regulator of TGF-β1 signaling during carcinogenesis.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Susan G. Komen Breast Cancer Foundation to N.H. and M.D.; National Institutes of Health/National Cancer Institute (R01-CA106307 [GenBank] ) to G.C.B.; National Institutes of Health/National Cancer Institute (R01-CA104505 [GenBank] ) to J.A.C. and C.G.W.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Carlos Arteaga for generously providing the ALK5QD adenoviral construct.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received November 5, 2007; revised January 22, 2008; accepted February 9, 2008.


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