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Protein Eng Des Sel. 2008 July; 21(7): 463–473.
Published online 2008 May 21. doi:  10.1093/protein/gzn023
PMCID: PMC2575055

Expression, purification and characterization of BGERII: a novel pan-TGFβ inhibitor


Transforming growth factor beta (TGFβ) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFβ isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFβ type II (RII) and betaglycan (BG) have been utilized as TGFβ inhibitors, their antagonistic potency against different TGFβ isoforms varies considerably because RII does not appreciably bind to TGFβ2 whereas BG binds weakly to TGFβ1 and TGFβ3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BGE) and the extracellular domain of RII. The fusion protein (named BGERII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFβ1 and TGFβ3 than a commercially available soluble RII and to TGFβ2 than a commercially available soluble BG. More significantly, whereas BGE or RII alone showed no antagonistic activity towards TGFβ2, BGERII inhibited the signaling of both TGFβ1 and TGFβ2 in cell-based assays including TGFβ-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFβ-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BGERII was found to have greater activity than other potent TGFβ inhibitors in blocking the signaling of TGFβ1 and TGFβ3. Thus, BGERII is a potent pan-TGFβ inhibitor in vitro and has potential for blocking TGFβ-induced pathogenesis in vivo.

Keywords: inhibitors, recombinant proteins, soluble receptors, TGFβ


Transforming growth factor beta (TGFβ) is a multifunctional cytokine mediating many cellular processes (Massague, 1998; Mehra and Wrana, 2002). TGFβ regulates cell proliferation and differentiation as well as the processes for embryonic development, cell adhesion, wound healing and angiogenesis in a tissue-specific manner.

There are three mammalian TGFβ isoforms (TGFβ1, β2 and β3), which are homodimeric polypeptides with a molecular weight of 25 kDa. TGFβ isoforms interact with three membrane receptors that exist as homodimers termed type I, II and III receptors, or RI, RII and RIII. RI and RII are serine/threonine kinases whereas RIII, also known as betaglycan (BG), serves as an accessory ligand-binding receptor (Lopez-Casillas et al., 1993). RII binds with high affinity to TGFβ1 and β3, but with much lower affinity to TGFβ2 (Zuniga et al., 2005). BG binds with a higher affinity to TGFβ2 and with lower affinity to TGFβ1 and β3 (Esparza-Lopez et al., 2001). BG serves to enhance the binding of TGFβ, especially TGFβ2, to RII. In the canonical TGFβ signaling pathway, TGFβ binds to RII, which then heterodimerizes with RI causing its activation through transphosphorylation. RI then initiates intracellular signaling by phosphorylating receptor–Smad proteins, Smad2 and Smad3, which complex with Smad4 and ultimately translocate to the nucleus where they act as transcription factors to regulate the expression of target genes (Massague et al., 2005). Perturbed TGFβ signaling leads to many different disease states in humans such as atherosclerosis, fibrosis, cancer (Blobe et al., 2000; Kim et al., 2005), osteoarthritis (Kraus et al., 2004) and even male pattern baldness (Hibino and Nishiyama, 2004).

Fibrosis is characterized by the excessive and abnormal deposition of extracellular matrix (ECM) components leading to a loss of function in the affected tissue. This can affect various organs including lung (Sheppard, 2006), liver (Breitkopf et al., 2005), kidney (Mizuno et al., 2000) and skin (Lemaire et al., 2006). During fibrosis, fibroblasts are activated for a prolonged period in which scar tissue forms and resolution of the healing process does not occur. Key stimulators of fibrosis are TGFβ and its downstream effector, connective tissue growth factor (Verrecchia and Mauviel, 2007). Normally, TGFβ functions as a central regulator and moderator of tissue repair by controlling immune responsiveness and balancing ECM turnover. TGFβ isoforms are produced in a latent form and are stored at a high concentration in the ECM of most organs. Overactivation of TGFβ, thus, leads to amplification of its function tipping the balance in favor of excessive ECM deposition resulting in fibrosis. Therefore, the ability to control the amount of TGFβ in the ECM is crucial for maintaining its balanced function.

TGFβ signaling has been implicated in both tumor suppression and progression. TGFβ has been shown to inhibit the growth of early carcinomas by arresting cell cycle progression, inducing apoptosis, promoting cellular senescence, maintaining genomic stability and interfering with cellular immortalization. However, in later-stage carcinomas, TGFβ has been shown to promote a more invasive and metastatic tumor phenotype. The loss of TGFβ receptors and an increase in TGFβ ligands are features of many advanced human tumors and are associated with poor prognosis. The stimulation of tumor progression by TGFβ is believed to occur in at least two ways. One is a direct effect on the transformed epithelial cells by promoting invasion and motility. The other is indirectly by generating a more permissive tumor stroma and stimulating angiogenesis. Thus, attenuation of TGFβ signaling in prostate-derived stromal cells was shown to reduce their tumor-promoting activity when co-transplanted with prostate cancer cells in a xenograft animal model (Verona et al., 2007).

Because TGFβ signaling has been shown to promote progression of various human diseases, large- and small-molecule TGFβ inhibitors have been developed and investigated for their efficacy to block the progression of cancer, fibrotic disorders and glaucoma (Yingling et al., 2004; Zhang et al., 2005; Akhurst, 2006; Turley et al., 2007). Several approaches at inhibiting this signaling pathway have been explored including both intracellular and extracellular targets. The antisense oligodeoxynucleotide AP12009, which specifically inhibits TGFβ2 expression, has shown promise in treating several malignant tumors (Schlingensiepen et al., 2006). Several small-molecule kinase inhibitors have been developed, which selectively block the RI kinase, and thus block a key step in the TGFβ signaling pathway (Ge et al., 2004). A drawback to these approaches is that the inhibitor must be internalized to function. Also, although the small molecule RI kinase inhibitors may show high selectivity for the ATP-binding site of RI, there is the possibility of inhibiting other kinases resulting in undesirable side effects.

Blocking TGFβ extracellularly may be a more favorable approach as this alleviates the need for internalization and can block RI-independent TGFβ signaling, which has been implicated in cardiac hypertrophy (Watkins et al., 2006). There are several examples of using the soluble extracellular domains of RII or BG as effective TGFβ antagonists. The soluble RII and an RII fused to the Fc region of human immunoglobulin (Fc-RII) have been shown to antagonize TGFβ1 and β3 (Komesli et al., 1998) and inhibit TGFβ1-induced matrix remodeling (Smith et al., 1999) and liver fibrogenesis (Ueno et al., 2000). The soluble extracellular domain of BG has been shown to inhibit tumor growth, angiogenesis and metastasis in several animal models of human carcinomas (Bandyopadhyay et al., 2002a, 2002b, 2005).

Although these soluble receptors have been shown to inhibit TGFβ-induced pathological processes, there is an apparent disadvantage in using only a single domain as an effective TGFβ inhibitor because they bind different TGFβ isoforms with different affinities (Lyons et al., 1991; De Crescenzo et al., 2006). Thus, their anti-TGFβ activity is likely isoform-dependent. Furthermore, TGFβ isoforms are differentially expressed depending on cell type and context (Levine et al., 1993; Perry et al., 1997; Saed et al., 2002; Rosenthal et al., 2004; Dallas et al., 2005). This differential expression of TGFβ isoforms requires the development of a multivalent TGFβ inhibitor.

This issue was touched upon by the in vitro dimerization of the RII, and BG extracellular domains utilizing a coiled-coil system (De Crescenzo et al., 2004). This method increased receptor binding to TGFβ1 compared with the respective monomers; however, the stability of the dimer complex was limited by the binding affinity of the coiled-coil. The authors suggested the possibility of overcoming this by the introduction of cysteine residues in the coil sequences which would form disulfide bonds upon interaction of the coils.

The aim of the current study focused on the design and synthesis of a TGFβ inhibitor with the ability to inhibit TGFβ signaling extracellularly and to block the activity of all three isoforms. This was accomplished by fusing the BGE and the extracellular domain of RII to generate a chimeric receptor with pan-TGFβ binding ability.

This chimeric receptor, called BGERII, was successfully expressed in bacteria, isolated by immobilized metal ion affinity chromatography (IMAC) and refolded by controlled oxidation. We demonstrate that, as a crude preparation, BGERII binds with higher affinity to all three isoforms of TGFβ and is more effective at blocking TGFβ signaling in cell-based assays when compared with other single-domain soluble receptors. After further purification by gel filtration chromatography, the monomeric species of BGERII was found to have greater antagonistic activity against TGFβ1 and TGFβ3 than two highly potent and well-known TGFβ inhibitors. These results encourage the further development of BGERII as a pan-TGFβ antagonist to block TGFβ-induced pathogenesis in vivo.

Materials and methods

Construction of the chimeric receptor sequence

The nucleotide sequence corresponding to the rat BGE amino acid residues G24-D383 was previously subcloned into a modified pET-32a(+) (EMD Chemicals, Inc., San Diego, CA, USA) plasmid between an engineered KpnI site (GGTATG to GGTACC immediately preceding the S-Tag sequence) and HindIII site. This eliminated the S-Tag, enterokinase site, and the subcloning sites from NcoI to SalI. This rat BGE sequence was previously engineered to mutate Cys278 to a Ser to aid in folding as this cysteine does not participate in native disulfide pairing. This construct also contains three additional mutations. Arg58 to His was introduced to eliminate a thrombin cleavage site and Asn337 to Ala was introduced to eliminate a deamidation site. His116 to Arg was an unintended spurious mutation.

To construct BGERII, the modified rat BGE sequence was amplified using the forward primer 5′-CGGGGTACCGGTCCAGAGCCC-3′, containing a 5′-KpnI site, and the reverse primer 5′-GTGTTGTGGAAGCTTGTCAGGGTCCAGCAGGA-3′, containing a 3′-overhang to overlap the 5′-RII sequence. The human RII sequence corresponding to amino acids P73-D184 was amplified using the forward primer 5′-GACCCTGACAAGCTTCCACAACTGTGTAAATTTTGT-3′, containing a 5′- overhang to overlap the 3′- BGE sequence, and the reverse primer 5′-CCGCTCGAGTCAGTCAGGATTGCTGGTGTTA-3′, containing a 3′-XhoI site preceded by a stop codon. The overhanging sequences of these fragments allowed for in-frame annealing so the BGERII sequence could be amplified by overlapping PCR using the BGE forward primer and the RII reverse primer. The resulting fragment consisted of a 5′-KpnI site, the BGERII sequence followed by a stop codon and a 3′-XhoI site.

The BGERII sequence was initially cloned into the pET-32a(+) vector. It was later transferred into pET-17b vector (EMD Chemicals, Inc.) between the NdeI and XhoI sites. This resulted in the removal of the T7-Tag from pET-17b and the insertion of a construct containing, immediately after the start codon, a 6-histidine tag followed by a thrombin cleavage site, and then the BGERII sequence.

Cell culture and expression

The pET-17b construct was transformed into Origami ™ (DE3)pLysS cells (EMD Chemicals, Inc.). Recombinant protein was expressed by the transformed bacteria cultured in LB broth supplemented with 100 µg/ml ampicillin in an incubator shaker. When the culture was grown to an optical density at 600 nm of ~0.6, it was induced with 1 mM isopropyl-beta-d-thiogalactopyranoside for ~16 h. The cells were then pelleted by centrifugation at 4,000 RPM (4,044 × g). Growth and expression were performed at 37°C and 300 RPM in the incubator shaker.

Protein purification

Cell pellets were resuspended in PBS at 1/50 the original culture volume. Phenylmethanesulfonyl fluoride and lysozyme were added at 0.1 and 2 mg/ml, respectively. The resuspended cells were incubated on ice for 2 h and then lysed by sonication. Cell lysate was then centrifuged at 10 000 RPM (11,300 × g) for 5 min at 4°C. Soluble protein was obtained at this step. The pellet containing the inclusion bodies was washed with PBS followed by centrifugation. This process was repeated several times followed by the solublization of the inclusion bodies in a buffer containing 8 M urea, 50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole and 10 mM 2-mercaptoethanol.

Isolation of target protein was achieved by gravity flow over chelating sepharose resin charged with nickel ions; 8 M urea buffer containing 50 mM imidazole was used for washing non-specifically bound protein off the column. The recombinant BGERII was eluted with buffer containing 200 mM imidazole.

Refolding of bacterially-expressed protein

Dithiothreitol (DTT) was added to the IMAC-purified chimeric receptor with slow stirring under a nitrogen overlay to a final concentration of 8 mM. In a separate vessel, the refolding buffer (50 mM Tris, 50 mM glycine, 300 mM NaCl, 5 mM EDTA and 2 mM MgCl2-6H2O at pH 8.0) was sparged with nitrogen with vigorous stirring for ~30 min. The refolding buffer was then added dropwise to the reduced chimeric receptor, while maintaining a constant nitrogen overlay. Upon completion of the 4-fold dilution, l-cystine was added to a concentration of 2 mM, the nitrogen overlay was removed and the mixture was allowed to stir slowly overnight. After ~18 h, CuCl2 was added to 10 µM and stirring was continued for 1 h to catalyze the oxidation of free cysteines. The mixture was then concentrated using an Amicon 8200 stirred cell (Millipore, Billerica, MA, USA) fitted with a Millipore Ultrafiltration Membrane YM filter (NMWL 30000), using nitrogen at 25 psi. The concentrated chimeric receptor was dialyzed against 50 mM Tris, pH 8.0, and then filtered using a 0.2 µm syringe filter. The protein concentration was determined by the absorbance at 280 nm and this material was used for the experiments described in this study. A recombinant rat BGE was bacterially expressed, purified and refolded using the same approach as the BGERII.

SDS–PAGE and western blot analysis

Samples for SDS–PAGE were resuspended in SDS sample buffer containing 2-mercaptoethanol (reduced) or without 2-mercaptoethanol (non-reduced) and boiled for 5 min prior to loading onto a 7.5%, 10% or 12% polyacrylamide gel and a constant voltage was applied. To visualize separated proteins, the gels were stained with Coomassie Brilliant Blue G-250 (Sigma, St Louis, MO, USA) and destained with deionized water. For western blot analysis, SDS–PAGE gels were transferred to a nitrocellulose membrane under constant voltage. The membranes were blocked with 5% non-fat dried milk in TBST (10 mM Tris pH 7.5, 150 mM NaCl and 0.05% (v/v) Tween® 20) followed by washing with TBST. Primary antibodies and secondary antibodies were diluted in TBST and applied with a washing step in between. Proteins were detected using the Amersham ECL western blotting detection kit (GE Healthcare, Piscataway, NJ, USA) and exposure on photographic film. Soluble RII and RIII receptors, expressed in mouse myeloma cells, and the respective antibodies were from R&D systems (Minneapolis, MN, USA). Anti-His Tag was from Upstate (Charlottesville, VA, USA), anti-phosphorylated Smad2 was from Chemicon (Temecula, CA, USA) and anti-phosphorylated Smad3 was a kind gift from Dr Edward Leof and anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Enzyme-linked immunosorbent assay

Two enzyme-linked immunosorbent assay (ELISA) protocols were developed to measure TGFβ ligand binding to receptors. One method utilized the coating of immunoplates (Nunc, Rochester, NY, USA) with the receptors and then binding of ligand, the other method utilized the coating of ligand and binding of receptors. Briefly, in the first method, 10 pmol of each receptor was coated per well in 100 µl carbonate buffer pH 9.7 (25 mM sodium carbonate, 25 mM sodium bicarbonate) overnight at 4°C. The wells were blocked with 300 µl 2% BSA in Dulbecco’s phosphate buffered saline (DPBS) (0.2 g/l KCl, 8.0 g/l NaCl, 0.2 g/l KH2PO4, 1.15 g/l Na2HPO4, 0.1 g/l MgCl2-6H2O and 0.13 g/l CaCl2-2H2O) for 35 min at 37°C then washed once with Tris-buffered saline with Tween 20 [TBST—20 mM Tris–HCl pH 7.6, 150 mM NaCl and 0.05% (v/v) Tween® 20]. Increasing concentrations of TGFβ in 100 µl DPBS per well were allowed to bind for 2 h at room temperature and 600 RPM on a microplate shaker. The wells were washed five times with TBST and then the appropriate primary antibody diluted in 100 µl DPBS per well was applied for 2 h at room temperature with shaking at 600 RPM. The wells were washed five times with TBST and then the appropriate dilution of horse-radish peroxidase-conjugated secondary antibody in 100 µl DPBS per well was applied at room temperature for 2 h with shaking at 600 RPM. The wells were washed five times with TBST and then 100 µl of a 1:1 mixture of TMB substrate and stabilized H2O2 (ImmunoPure® TMB Substrate Kit, Pierce Biotechnology, Rockford, IL, USA) was added to each well and allowed to incubate at room temperature until a deep blue color was developed. The reaction was stopped by the addition of 100 µl 1 N HCl. The absorbance at 450 nm was measured with a plate reader.

The second method was carried out similarly to the first with the following modifications. TGFβ was coated at 0.2 µg per well in 100 µl of carbonate buffer overnight at 4°C. After blocking with BSA, receptors were applied at increasing concentrations and allowed to bind. The subsequent steps were the same as described in the first method.

TGFβ-induced phosphorylation of Smad2 and Smad3

The efficacy of BGERII at inhibiting TGFβ signaling in cells was measured by the level of phosphorylation of Smad2 and Smad3. The non-tumorigenic epithelial cell line, MCF-10A, was originally isolated from a mammary cyst in a 36-year-old Caucasian female ( These cells are highly responsive to TGFβ signaling. The cells were cultured on 60 mm tissue culture dishes in DMEM F12 medium supplemented with 5% horse serum. When cells reached ~90% confluency, they were treated for 30 min with 0.1 ng/ml TGFβ along with various concentrations of receptors. The cells were lysed and normalized for protein concentration. Equal amounts of protein were loaded onto an SDS–PAGE gel followed by transfer to a nitrocellulose membrane. Western blot analysis was performed as described above. Actin was detected as a control for protein loading.

TGFβ-responsive promoter-luciferase assay

The efficacy of BGERII at blocking TGFβ signaling in cells was compared with other receptors using transformed mink lung cells (TMLC) which are mink lung epithelial cells stably transfected with a luciferase reporter under the control of the TGFβ-responsive plasminogen activator inhibitor-1 (PAI-1) promoter. This assay was described previously (Abe et al., 1994). Briefly, TMLCs were plated at 1500 cells per well onto 96-well tissue culture plates in 100 µl DMEM supplemented with 10% fetal bovine serum. The cells were grown at 37°C in a 5% CO2 incubator for 3 days. The cells were then treated with medium containing 0.5 ng/ml TGFβ and the appropriate concentrations of TGFβ inhibitor for ~18 h. The cells were lysed in buffer containing 10 mM K2HPO4, 1% Triton X-100 and 1 mM DTT and incubated at room temperature for 20 min. Reaction buffer (160 mM K2HPO4, 24 mM MgSO4 and 8 mM ATP) was added to each sample followed by d-luciferin immediately before measuring light emission in a luminometer.

Alternatively, TMLCs were plated at 5 × 104 cells per well in DMEM (4.5 g/l glucose) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate and 250 µg/ml G-418 sulfate (Geneticin) and allowed to attach for 3–5 h. The medium was aspirated and then replaced with the same medium containing 0.1% BSA and 1.0 or 2.0 ng/ml TGFβ with the appropriated concentration of inhibitor. After ~18 h, the cells were lysed and d-luciferin was added to each sample immediately before measuring light emission in a luminometer. Fc-RII (molecular weight according to the vendor 82 kDa) and pan-specific anti-TGFβ antibody were from R&D systems. RI kinase inhibitors (HTS466284 and the 1,5-Naphthyridine derivative) were synthesized in-house according to published methods (Sawyer et al., 2003; Singh et al., 2003; Gellibert et al., 2004).

Gel filtration chromatography

Twelve microgram of the crude preparation of BGERII was loaded in 1 ml onto a HiLoad 16/60 Superdex 75 prep grade column (Amersham Biosciences, GE Healthcare Bio-Sciences Corp.). The column was connected to a Pharmacia 500-series FPLC unit (GE Healthcare) set to run at 1.0 ml/min for 1 column volume and 1.0 ml fractions were collected. Fractions were loaded in the order of elution and at equal volume onto non-reducing SDS–PAGE gels and visualized by Coomassie staining. Fractions corresponding to the four major species were pooled and concentrated to a final volume of 0.7 ml using centriprep ultracel YM-10 centrifugal filtration devices (Millipore).


Design of a BGERII expression construct in the pET-17b vector

To obtain the chimeric BGERII fusion protein, the cDNA encoding the rat BGE-domain (amino acids G24-D383) and the human RII receptor (amino acids P73-D184) were joined in-frame by an overlapping PCR (Javed et al., 2008). The final construct contains an N-terminal 6-histidine tag followed by a thrombin cleavage site and a lysine–leucine linker between the C-terminus of BGE and the N-terminus of RII (Fig. 1A). The construct was subcloned into the pET-17b vector between the NdeI and XhoI sites, thus removing the T7-Tag of this vector. The construct was sequenced to make sure that no mutation was introduced during PCR amplification. Protein properties were estimated by entering the predicted amino acid sequence into the ProtParam Tool on the ExPASy Proteomics Server of the Swiss Institute of Bioinformatics ( The expressed BGERII has 492 amino acid residues, a molecular weight of 55 372.0 Da, a theoretical pI of 6.0 and an extinction coefficient of 40 910/M cm at 280 nm assuming no cysteines appear as half cystines.

Fig. 1
Active BGERII was purified from a bacterial culture, isolated by IMAC and refolded by controlled oxidation. (A) The cDNA encoding rat BG E-domain (amino acids G24-D383) and human RII receptor (amino acids P73-D184) were joined in-frame by an overlapping ...

Expression of BGERII in a bacterial culture and purification from inclusion bodies by IMAC under denaturing and reducing conditions

Inclusion bodies containing BGERII were washed several times with PBS to remove soluble background proteins. The inclusion bodies were then solubilized in a urea-containing buffer and samples at each step were visualized by reduced SDS–PAGE and Coomassie staining (Fig. 1B). BGERII was effectively separated from the bulk of background proteins by IMAC at a yield of ~100 mg/l of bacterial culture (Fig. 1C).

Preparation of active BGERII by refolding using air oxidation

Previous reviews on protein folding (Rudolph and Lilie 1996; Clark, 1998; Lilie et al., 1998) were consulted for the design of a feasible scheme for refolding BGERII. After concentration, dialysis and filtration, the final yield was ~10 mg/l of bacterial culture. This represents a loss of ~90% due mostly to precipitation. The purity of refolded BGERII was determined by Coomassie staining and western blot analysis using antibodies to the RIII receptor and to the 6-histidine tag (Fig. 1D). This revealed that although our preparation appears to be of high purity by Coomassie staining, there are lower molecular weight proteins that appear to be C-terminal truncations of the mature protein. This is evidenced by lower molecular weight proteins containing a recognizable RIII sequence and even lower molecular weight proteins containing a recognizable His-Tag sequence. The relative purity and concentration of the soluble TGFβ receptors used in this study were visualized by the western blot analysis (Fig. 1E). Soluble human RII and RIII receptors (R&D Systems) were expressed in mouse myeloma cells and, therefore, glycosylated. This is visualized by the smearing in the migration pattern on the representative western blots which is absent in the BGE and BGERII samples which were expressed in bacteria. The western blot confirms immunoreactive sequences of both RII and RIII in the purified BGERII sample.

Comparison of the binding activity of BGERII and other soluble receptors to TGFβ1, β2 and β3

ELISA analysis revealed that refolded BGERII is active and not only binds to TGFβ, but binds with higher affinity to TGFβ1 and β2 than equimolar concentrations of either the full-length RIII receptor or its E-domain, BGE (Fig. 2A and B). Furthermore, BGERII binds TGFβ1, β2 and β3 with higher affinity than equimolar concentrations of the soluble RII receptor (Fig. 2C–E). This demonstrates that fusing both domains into one chimeric receptor resulted in a single protein with the ability to bind to all three isoforms of TGFβ with higher affinity than equimolar amounts of either soluble RII or RIII.

Fig. 2
BGERII binds to TGFβ isoforms with higher affinity than a full-length soluble RIII, RII or the E-domain of RIII, BGE. An ELISA was developed to compare the binding of BGERII with other TGFβ receptors to different TGFβ isoforms. ...

Comparison of the level of inhibition of TGFβ-induced phosphorylation of Smad2 and Smad3 by BGERII to other soluble receptors

Phosphorylation of the C-terminal serine residues of Smad2 and Smad3 is indicative of intracellular signaling stimulated by TGFβ binding to its receptors. The results of this study show that BGERII was more effective at inhibiting the phosphorylation of Smad2 and Smad3 induced by 0.1 ng/ml TGFβ1 compared with equimolar concentrations of other receptors (Fig. 3A). Whereas soluble RIII or BGE was not effective inhibitors of TGFβ1 in this cell-based assay, the soluble RII was effective at a concentration of 100 nM. However, BGERII was more effective at inhibiting Smad phosphorylation even at a 5-fold lower concentration of 20 nM. More significantly, although neither the soluble type II receptor nor the single domain, BGE, was an effective inhibitor of TGFβ2 in this cell-based assay, their combination as a fusion protein (BGERII) yielded an effective TGFβ2 antagonist (Fig. 3B). In fact, BGERII appeared slightly more effective at inhibiting the phosphorylation of Smad2 and noticeably more effective at inhibiting the phosphorylation of Smad3 than the soluble RIII at 100 nM (Fig. 3B).

Fig. 3
BGERII blocks TGFβ-induced phosphorylation of Smad2/3 and transcription of a TGFβ-responsive promoter more effectively than other receptors. Human mammary epithelial MCF-10A cells were treated with various concentrations of various TGFβ ...

Comparison of the level of inhibition of TGFβ-induced transcription by BGERII to other soluble receptors

Receptor-phosphorylated Smad2 and Smad3 are known to act as transcription enhancers at promoters of TGFβ target genes containing Smad-binding elements. Therefore, we also compared the efficacy of the soluble receptors in blocking TGFβ-induced luciferase activity driven by the PAI-1 promoter, which contains Smad-binding elements and was stably transfected into TMLC (Abe et al., 1994). BGERII showed a stronger dose-dependent inhibition of TGFβ1-induced luciferase activity compared with equimolar concentrations of the other soluble receptors tested (Fig. 3C). At the lowest and middle concentrations of receptors tested, the soluble RII and RIII actually stimulated TGFβ1 signaling, whereas BGERII showed inhibitory activity at all concentrations tested. The single domain, BGE, was not an effective inhibitor of TGFβ1 signaling at any concentration tested. Neither soluble RII nor BGE displayed the ability to inhibit TGFβ2-induced luciferase activity at any of the concentrations tested in this cell-based assay (Fig. 3D). Both soluble RIII and BGERII showed dose-dependent inhibition of TGFβ2-induced luciferase activity; however, at equimolar concentrations, BGERII was a more effective inhibitor.

Comparison of the level of inhibition of TGFβ-induced transcription by BGERII to several potent TGFβ inhibitors

We next compared the TGFβ inhibitory activity of BGERII with other more potent TGFβ inhibitors including Fc-RII, a pan-specific anti-TGFβ antibody and two RI kinase inhibitors. At a concentration of 10 nM, the Fc-RII was clearly the most potent inhibitor of TGFβ1, but BGERII was more active than either of the RI kinase inhibitors and the pan-specific anti-TGFβ antibody (Fig. 4A). At a concentration of 50 nM, the Fc-RII and pan-specific antibody showed the highest activity, whereas the BGERII showed similar activity to the two RI kinase inhibitors. At 250 nM, all compounds inhibited 1.0 ng/ml TGFβ1-induced signaling to baseline or below. Fc-RII did not inhibit 1.0 ng/ml TGFβ2-induced transcriptional activity, whereas BGERII showed a dose-dependent inhibition, though not as effective as the pan-specific antibody or the RI kinase inhibitors (Fig. 4B). The Fc-RII was apparently the most effective inhibitor of 1.0 ng/ml TGFβ3-induced transcriptional activity (Fig. 4C). The pan-specific antibody was more effective than BGERII at the higher concentrations tested; however, BGERII showed similar activity at 10 nM. The HTS466284 kinase inhibitor and BGERII showed similar activity at all concentrations and the naphthyridine-derived kinase inhibitor showed the lowest activity in this comparison.

Fig. 4
Activity of BGERII compared to Fc-RII, pan-specific anti-TGFβ antibody and two RI-kinase inhibitors in transformed mink lung cells (TMLC). TMLCs were plated in a 96-well plate and treated with various concentrations of Fc-RII, a pan-specific anti-TGFβ ...

Isolation of highly active monomeric BGERII by gel filtration chromatography

Comparing reduced and non-reduced samples of BGERII by SDS–PAGE showed that the crude preparation is a heterogeneous mixture of monomer and various disulfide-linked multimers (Fig. 5A). Further purification by gel filtration chromatography resulted in the separation of four major protein species (Fig. 5B). Fractions corresponding to each of the species were pooled, concentrated and an equal amount of each was visualized by non-reducing SDS–PAGE (Fig. 5C). The species labeled number 1 corresponds to high molecular weight multimers. Number 2 corresponds approximately to the molecular weight of the dimer of BGERII. Number 3 corresponds to the correct molecular weight of monomeric BGERII and number 4 appears to consist of a C-terminal truncation of BGERII. The activities of all four species were compared with that of the load (L, BGERII crude preparation). At the concentration of 250 nM, the species corresponding to the monomeric BGERII is clearly the active species present in the load capable of inhibiting 2 ng/ml of TGFβ1-, β2- and β3-induced transcription in TMLCs (Fig. 5D–F). The inhibitory activity against TGFβ1 and β3 of the fractions corresponding to the multimer, dimer and truncated species is likely due to some monomeric BGERII that co-eluted with each of them.

Fig. 5
BGERII monomer separated by gel filtration contains the highest TGFβ inhibitory activity. (A) Reduced (R) and non-reduced (NR) BGERII (5 µg) was electrophoresed on a 7.5% SDS–PAGE gel and visualized by Coomassie staining. This ...

Comparison of the level of inhibition of TGFβ-induced transcription by monomeric BGERII to Fc-RII and a pan-specific anti-TGFβ antibody

Because the monomeric BGERII was more potent than the crude preparation, we again compared its antagonistic activity against TGFβ isoforms at a concentration of 2.0 ng/ml with that of the pan-specific anti-TGFβ antibody and Fc-RII. Monomeric BGERII inhibited TGFβ1- and β3-induced transcription in TMLCs more effectively than the pan-specific antibody (Fig. 6A and C). However, the pan-specific antibody was a more effective inhibitor of TGFβ2 (Fig. 6B). Monomeric BGERII was also a more effective inhibitor of TGFβ1 and β3 when compared with Fc-RII (Fig. 6D and E). Comparing reduced with non-reduced samples of monomeric BGERII, Fc-RII and the pan-specific antibody by SDS–PAGE revealed that the Fc-RII and the pan-specific antibody exist as disulfide-linked multimers (Fig. 6F).

Fig. 6
Activity of monomeric BGERII compared with the pan-specific anti-TGFβ antibody and Fc-RII. TMLCs were plated in a 96-well plate and treated with various concentrations of monomeric BGERII or Pan-Ab (AC) or Fc-RII (D and E) in the presence ...


TGFβ signaling is a therapeutic target for many pathological conditions such as fibrosis, cancer and ocular disorders (Nakamura et al., 2004; Breitkopf et al., 2005; Saunier and Akhurst, 2006). There are several approaches to TGFβ inhibition including antisense oligonucleotides, small molecule kinase inhibitors, neutralizing antibodies and soluble receptors. Representatives of each type of inhibitor are at various stages of development and each type of inhibitor has its own advantages and disadvantages. The rationale for TGFβ inhibition and the design and efficacy of the various TGFβ inhibitors has been reviewed (Yingling et al., 2004; Akhurst, 2006).

Several obstacles have been encountered in the development of effective TGFβ inhibitors such as delivery to the target location and specificity for the target. In addition, any or all of the three TGFβ isoforms may be expressed by a given cell in a specific context. Although small molecule kinase inhibitors have the advantage of high-throughput screening, their synthesis could be costly. Another disadvantage of kinase inhibitors is the possible side effects and toxicity, in part, due to their non-specific inhibition of other kinases (Foringer et al., 2005; Park et al., 2006). TGFβ RI (ALK5) kinase inhibitors typically inhibit ALK4 and ALK7 with high affinity as well. TGFβ RI and ALK4 are identical at the ATP binding site (; Drug Discovery, 2006—September 2006, Use of ALK4 as a Surrogate Kinase for TGFβ RI).

Since excessive TGFβ signaling can lead to pathological conditions, but some TGFβ signaling is required for normal development and homeostasis, a better strategy may be to inhibit excess TGFβ extracellularly. Evidence for this approach has been shown in animal models of human carcinomas and fibrotic disease by utilizing a soluble RIII to block TGFβ-induced cell migration, invasion, angiogenesis, metastasis and firbrosis (Bandyopadhyay et al., 1999, 2002a, 2002b, 2005; Liu et al., 2002; Dong et al., 2007). Furthermore, in a mouse model of metastatic breast cancer, transgenic mice expressing the Fc-RII showed suppressed metastasis with no apparent side effects after lifetime exposure to this TGFβ antagonist (Yang et al., 2002). This suggests that soluble TGFβ antagonists can reduce excessive TGFβ extracellularly without affecting normal TGFβ function required to maintain healthy tissue homeostasis.

Fc-RII is one of the most potent receptor-based inhibitors of TGFβ1 and β3; however, it does not inhibit TGFβ2 (Komesli et al., 1998). Some cancers are known to specifically overexpress TGFβ2 such as malignant gliomas (Schlingensiepen et al., 2006) and prostate cancer cells (Dallas et al., 2005), whereas other types of cancer are known to overexpress all three TGFβ isoforms including breast and gastric cancer (Reiss, 1999). All three TGFβ isoforms were also shown to mediate renal fibrogenesis, and it was suggested that blockade of all three TGFβ isoforms may yield the best therapeutic results (Yu et al., 2003). Therefore, in addition to effectively inhibiting excess TGFβ extracellularly, it is also necessary to effectively inhibit all three isoforms in certain diseases where all three isoforms are implicated in disease progression.

Receptor-based inhibitors are relatively easy and cost-efficient to produce with the added possibility of scale-up. It was previously found that the antagonistic potency of soluble receptors is inversely related to the rate of dissociation from TGFβ (De Crescenzo et al., 2001). Dimerization of the Fc portion of an antibody fused to the RII extracellular domain (Fc-RII) dramatically increased antagonistic potency. Homo and heterodimerization of TGFβ receptor extracellular domains through the introduction of coiled-coils was shown to enhance antagonistic potency (De Crescenzo et al., 2004). Using this system, an RII extracellular domain homodimer was generated along with an RII extracellular domain and RIII membrane proximal domain heterodimer. It was found that the dimers had better antagonistic potency for TGFβ1 than the corresponding monomers.

Our study describes the design, production and characterization of a fusion protein containing both the RII receptor extracellular domain and the RIII receptor endoglin domain. Incorporating both domains into one protein has the advantages of blocking the activity of all three isoforms of TGFβ with high affinity and for equal delivery of both domains to the same physiological or pathological location. The production of the chimeric receptor is relatively simple and cost-effective. TGFβ ligands are differentially expressed and bind heterologously to RII and RIII. In cell-based affinity labeling experiments, RII was previously shown to bind with similar affinity to both TGFβ1 and β3 (Kd ~40 pM), but with much lower affinity to TGFβ2 (Kd ~500 pM) (Cheifetz et al., 1990). While TGFβ1, β2 and β3 share 70–80% sequence identity, the lower affinity of RII for TGFβ2 was found to lie in three key residues on TGFβ2 (De Crescenzo et al., 2006). When these residues were substituted to match the residues present in TGFβ1 and β3, binding affinity of RII to this variant of TGFβ2 was restored to that seen with TGFβ1 and β3. Although the type III receptor serves limited signaling function in the canonical TGFβ pathway, it has been shown to enhance the binding of TGFβ, particularly TGFβ2, to RII (Ohta et al., 1987; Cheifetz et al., 1990; Lopez-Casillas et al., 1993). Its soluble extracellular domain binds to all three isoforms of TGFβ, but with highest affinity to TGFβ2, followed by TGFβ3 and then TGFβ1 (Vilchis-Landeros et al., 2001). The extracellular domain of RIII is composed of two distinct domains. The membrane proximal domain shows sequence similarity to uromodulin (U-domain) and the membrane distal domain shows sequence similarity to endoglin (E-domain). Mutagenesis analysis has shown that both domains can bind independently to TGFβ (Fukushima et al., 1993; Lopez-Casillas et al., 1994; Pepin et al., 1994, 1995). Competition assays have shown that, similar to wild-type RIII, both subdomains bind with higher affinity to TGFβ2 than TGFβ1 (Esparza-Lopez et al., 2001). The same study showed that while only the E-domain enhanced TGFβ binding to RII, both domains enhanced intracellular signaling mediated by TGFβ2. Therefore, while RII preferentially binds to TGFβ1 and β3, RIII binds preferentially to TGFβ2. Theoretically, joining the two proteins together should generate a single chimeric protein that could bind all three isoforms of TGFβ with high affinity. RIII has also been shown to bind inhibin A. This would be an undesirable characteristic as the main goal is to bind only TGFβ. Although RIII contains two separate TGFβ-binding domains, there is no apparent difference in the ability of either domain to bind TGFβ. However, the inhibin A binding ability has been shown to reside exclusively in the U-domain (Esparza-Lopez et al., 2001; Wiater et al., 2006). Thus, utilization of the E-domain would be more favorable than the U-domain or wild-type RIII for a chimeric receptor. Therefore, we designed a fusion protein consisting of an N-terminal BGE and C-terminal soluble RII.

The expression and purification methods described in this study were used to rapidly produce sufficient quantities of BGERII to determine its biological activity and feasibility as a pan-TGFβ inhibitor. The purification and refolding steps, which may be further optimized, proved to be adequate for the production and characterization of this novel protein. Our study demonstrates that this novel fusion protein can be expressed to a moderate level in a prokaryotic expression system and that active protein can be purified and recovered by dilution into non-denaturing buffer and through air oxidation. BGERII binds with high affinity to all three isoforms of TGFβ and is a more effective inhibitor of TGFβ signaling than equimolar concentrations of soluble RII or RIII. BGERII binds with higher affinity to TGFβ1 and β2 compared with RIII alone and with higher affinity to TGFβ1, β2 and β3 compared with RII. One might conclude that this is the result of adding an extra binding domain in the receptor which would effectively double the relative concentration. However, our results suggest that is not the case. Because RII does not bind significantly to TGFβ2, one would expect that BGERII should have the same binding affinity to TGFβ2 as RIII or even more closely to BGE. But our data show that BGERII binds TGFβ2 with higher affinity than RIII or BGE. Furthermore, BGERII is a potent TGFβ2 antagonist whereas BGE and soluble RII show little or no antagonistic activity against TGFβ2. Therefore, this is not just an effect of adding another mole of receptor. At least one possible explanation for this is an avidity effect, wherein BGERII simultaneously contacts TGFβ at two separate sites and that the binding energy provided by such simultaneous contacts is additive.

BGERII contains 16 cysteines which participate in the formation of eight intramolecular disulfide bonds in the native conformation. This fact severely hampers a critical step in the purification of any recombinant protein overexpressed in a bacterial system: refolding to the native conformation. We found that after refolding BGERII exists as a heterogenous mixture of monomer and soluble disulfide-linked multimers and that only the monomer contains high activity. Monomeric BGERII was shown to have higher TGFβ1 and β3 inhibitory activity compared with the two most potent TGFβ inhibitors, Fc-RII and the pan-specific anti-TGFβ antibody, with the additional advantage over Fc-RII of being able to inhibit TGFβ2, albeit to a lesser degree than the pan-specific antibody.

BGERII has some advantages when compared with Fc-RII and the pan-specific antibody. These two large-molecule inhibitors exist as disulfide-linked complexes in their native form. The multimeric conformation of these two inhibitors is likely the basis for their very high activity, but also makes them quite large. Being a smaller molecule with high activity may give BGERII a therapeutic advantage with respect to its accessibility to target tissues. Another advantage of BGERII is our ability to produce it in a bacterial expression system which is much faster, has the potential for scale-up and would be relatively more cost-effective compared with the mammalian cell systems used to produce the other two inhibitors. Additionally, BGERII can be engineered using native host sequences, thus reducing a potential immune response. On the other hand, further studies are needed to determine the stability, bioavailability and clearance of BGERII in vivo and whether it will induce more unwanted side effects for being a more potent TGFβ inhibitor in comparison with other TGFβ inhibitors. Recently, RIII has been shown to also bind bone morphogenetic proteins (BMPs) and to enhance BMP activity (Kirkbride et al., 2008). Because TβRII does not bind BMP and BGE alone does not antagonize TGFβ activity, it is not expected that BGERII will inhibit BMP signaling. Nonetheless, further investigation is needed to address this issue.

This report is meant to introduce BGERII as a novel and potent pan-TGFβ inhibitor that was successfully produced by bacterial expression and has the potential to be developed further. Ongoing work is focusing on improving purification and refolding conditions to increase the yield of monomeric BGERII. Additional experiments are being designed to evaluate the efficacy of BGERII as a TGFβ inhibitor as well as its tolerability in vivo.


National Institutes of Health (R01CA75253, Subcontract from P01CA40035) to L-Z.S.; Department of Defense Prostate Cancer Research Project (DAMD17-03-1-0133) to L-Z.S; San Antonio Cancer Institute.


We thank Dr Olga Pakhomova for providing the rat BGE sequence in the modified pET-32a(+) vector.


Edited by Feng Ni


  • Abe M., Harpel J.G., Metz C.N., Nunes I., Loskutoff D.J., Rifkin D.B. Anal. Biochem. 1994;216:276–284. [PubMed]
  • Akhurst R.J. Curr. Opin. Investig. Drugs. 2006;7:513–521. [PubMed]
  • Bandyopadhyay A., Zhu Y., Cibull M.L., Bao L., Chen C., Sun L. Cancer Res. 1999;59:5041–5046. [PubMed]
  • Bandyopadhyay A., Lopez-Casillas F., Malik S.N., Montiel J.L., Mendoza V., Yang J., Sun L.Z. Cancer Res. 2002;a 62:4690–4695. [PubMed]
  • Bandyopadhyay A., Zhu Y., Malik S.N., Kreisberg J., Brattain M.G., Sprague E.A., Luo J., Lopez-Casillas F., Sun L.Z. Oncogene. 2002;b 21:3541–3551. [PubMed]
  • Bandyopadhyay A., Wang L., Lopez-Casillas F., Mendoza V., Yeh I.T., Sun L. Prostate. 2005;63:81–90. [PubMed]
  • Blobe G.C., Schiemann W.P., Lodish H.F. N. Engl. J. Med. 2000;342:1350–1358. [PubMed]
  • Breitkopf K., Haas S., Wiercinska E., Singer M.V., Dooley S. Alcohol Clin. Exp. Res. 2005;29:121S–131S. [PubMed]
  • Cheifetz S., Hernandez H., Laiho M., ten Dijke P., Iwata K.K., Massague J. J. Biol. Chem. 1990;265:20533–20538. [PubMed]
  • Clark E.D.B. Curr. Opin. Biotechnol. 1998;9:157–163. [PubMed]
  • Dallas S.L., Zhao S., Cramer S.D., Chen Z., Peehl D.M., Bonewald L.F. J. Cell Physiol. 2005;202:361–370. [PubMed]
  • De Crescenzo G., Grothe S., Zwaagstra J., Tsang M., O’Connor-McCourt M.D. J. Biol. Chem. 2001;276:29632–29643. [PubMed]
  • De Crescenzo G., Pham P.L., Durocher Y., Chao H., O’Connor-McCourt M.D. J. Biol. Chem. 2004;279:26013–26018. [PubMed]
  • De Crescenzo G., et al. J. Mol. Biol. 2006;355:47–62. [PubMed]
  • Dong M., How T., Kirkbride K.C., Gordon K.J., Lee J.D., Hempel N., Kelly P., Moeller B.J., Marks J.R., Blobe G.C. J. Clin. Invest. 2007;117:206–217. [PMC free article] [PubMed]
  • Esparza-Lopez J., Montiel J.L., Vilchis-Landeros M.M., Okadome T., Miyazono K., Lopez-Casillas F. J. Biol. Chem. 2001;276:14588–14596. [PubMed]
  • Foringer J.R., Verani R.R., Tjia V.M., Finkel K.W., Samuels J.A., Guntupalli J.S. Ann. Pharmacother. 2005;39:2136–2138. [PubMed]
  • Fukushima D., Butzow R., Hildebrand A., Ruoslahti E. J. Biol. Chem. 1993;268:22710–22715. [PubMed]
  • Ge R., et al. Biochem. Pharmacol. 2004;68:41–50. [PubMed]
  • Gellibert F., et al. J. Med. Chem. 2004;47:4494–4506. [PubMed]
  • Hibino T., Nishiyama T. J. Dermatol. Sci. 2004;35:9–18. [PubMed]
  • Javed A., et al. J. Biol. Chem. 2008;283:8412–8422. [PMC free article] [PubMed]
  • Kim I.Y., Kim M.M., Kim S.J. J. Biochem. Mol. Biol. 2005;38:1–8. [PubMed]
  • Kirkbride K.C., Townsend T.A., Bruinsma M.W., Barnett J.V., Blobe G.C. J. Biol. Chem. 2008;283:7628–7637. [PubMed]
  • Komesli S., Vivien D., Dutartre P. Eur. J. Biochem. 1998;254:505–513. [PubMed]
  • Kraus V.B., Huebner J.L., Stabler T., Flahiff C.M., Setton L.A., Fink C., Vilim V., Clark A.G. Arthritis Rheum. 2004;50:1822–1831. [PubMed]
  • Lemaire R., Bayle J., Lafyatis R. Curr. Opin. Rheumatol. 2006;18:582–587. [PubMed]
  • Levine J.H., Moses H.L., Gold L.I., Nanney L.B. Am. J. Pathol. 1993;143:368–380. [PubMed]
  • Lilie H., Schwarz E., Rudolph R. Curr. Opin. Biotechnol. 1998;9:497–501. [PubMed]
  • Liu M., Suga M., Maclean A.A., St George J.A., Souza D.W., Keshavjee S. Am. J. Respir. Crit. Care Med. 2002;165:419–423. [PubMed]
  • Lopez-Casillas F., Wrana J.L., Massague J. Cell. 1993;73:1435–1444. [PubMed]
  • Lopez-Casillas F., Payne H.M., Andres J.L., Massague J. J. Cell Biol. 1994;124:557–568. [PMC free article] [PubMed]
  • Lyons R.M., Miller D.A., Graycar J.L., Moses H.L., Derynck R. Mol. Endocrinol. 1991;5:1887–1896. [PubMed]
  • Massague J. Annu. Rev. Biochem. 1998;67:753–791. [PubMed]
  • Massague J., Seoane J., Wotton D. Genes Dev. 2005;19:2783–2810. [PubMed]
  • Mehra A., Wrana J.L. Biochem. Cell Biol. 2002;80:605–622. [PubMed]
  • Mizuno S., Matsumoto K., Kurosawa T., Mizuno-Horikawa Y., Nakamura T. Kidney Int. 2000;57:937–948. [PubMed]
  • Nakamura H., Siddiqui S.S., Shen X., Malik A.B., Pulido J.S., Kumar N.M., Yue B.Y. Mol. Vis. 2004;10:703–711. [PubMed]
  • Ohta M., Greenberger J.S., Anklesaria P., Bassols A., Massague J. Nature. 1987;329:539–541. [PubMed]
  • Park Y.H., Park H.J., Kim B.S., Ha E., Jung K.H., Yoon S.H., Yim S.V., Chung J.H. Cancer Lett. 2006;243:16–22. [PubMed]
  • Pepin M.C., Beauchemin M., Plamondon J., O’Connor-McCourt M.D. Proc. Natl Acad. Sci. USA. 1994;91:6997–7001. [PubMed]
  • Pepin M.C., Beauchemin M., Collins C., Plamondon J., O’Connor-McCourt M.D. FEBS Lett. 1995;377:368–372. [PubMed]
  • Perry K.T., Anthony C.T., Steiner M.S. Prostate. 1997;33:133–140. [PubMed]
  • Reiss M. Microbes Infect. 1999;1:1327–1347. [PubMed]
  • Rosenthal E., McCrory A., Talbert M., Young G., Murphy-Ullrich J., Gladson C. Mol. Carcinog. 2004;40:116–121. [PubMed]
  • Rudolph R., Lilie H. FASEB J. 1996;10:49–56. [PubMed]
  • Saed G.M., Collins K.L., Diamond M.P. Am. J. Reprod. Immunol. 2002;48:387–393. [PubMed]
  • Saunier E.F., Akhurst R.J. Curr. Cancer Drug Targets. 2006;6:565–578. [PubMed]
  • Sawyer J.S., et al. J. Med. Chem. 2003;46:3953–3956. [PubMed]
  • Schlingensiepen K.H., Schlingensiepen R., Steinbrecher A., Hau P., Bogdahn U., Fischer-Blass B., Jachimczak P. Cytokine Growth Factor Rev. 2006;17:129–139. [PubMed]
  • Sheppard D. Proc. Am. Thorac. Soc. 2006;3:413–417. [PMC free article] [PubMed]
  • Singh J., et al. Bioorg. Med. Chem. Lett. 2003;13:4355–4359. [PubMed]
  • Smith J.D., Bryant S.R., Couper L.L., Vary C.P., Gotwals P.J., Koteliansky V.E., Lindner V. Circ. Res. 1999;84:1212–1222. [PubMed]
  • Turley R.S., Finger E.C., Hempel N., How T., Fields T.A., Blobe G.C. Cancer Res. 2007;67:1090–1098. [PubMed]
  • Ueno H., Sakamoto T., Nakamura T., Qi Z., Astuchi N., Takeshita A., Shimizu K., Ohashi H. Hum. Gene Ther. 2000;11:33–42. [PubMed]
  • Verona E.V., Elkahloun A.G., Yang J., Bandyopadhyay A., Yeh I.T., Sun L.Z. Cancer Res. 2007;67:5737–5746. [PubMed]
  • Verrecchia F., Mauviel A. World J. Gastroenterol. 2007;13:3056–3062. [PubMed]
  • Vilchis-Landeros M.M., Montiel J.L., Mendoza V., Mendoza-Hernandez G., Lopez-Casillas F. Biochem. J. 2001;355:215–222. [PubMed]
  • Watkins S.J., Jonker L., Arthur H.M. Cardiovasc. Res. 2006;69:432–439. [PubMed]
  • Wiater E., Harrison C.A., Lewis K.A., Gray P.C., Vale W.W. J. Biol. Chem. 2006;281:17011–17022. [PubMed]
  • Yang Y.A., et al. J. Clin. Invest. 2002;109:1607–1615. [PMC free article] [PubMed]
  • Yingling J.M., Blanchard K.L., Sawyer J.S. Nat. Rev. Drug Discov. 2004;3:1011–1022. [PubMed]
  • Yu L., Border W.A., Huang Y., Noble N.A. Kidney Int. 2003;64:844–856. [PubMed]
  • Zhang F., Lee J., Lu S., Pettaway C.A., Dong Z. Clin. Cancer Res. 2005;11:4512–4520. [PubMed]
  • Zuniga J.E., et al. J. Mol. Biol. 2005;354:1052–1068. [PubMed]

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