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• W2039023048 abstract "Transforming growth factor-β (TGF-β) signaling requires a ligand-dependent interaction of TGF-β receptors ΤβR-I and ΤβR-II. It has been previously demonstrated that a soluble TGF-β type II receptor could be used as a TGF-β antagonist. Here we have generated and investigated the biochemical and signaling properties of a soluble TGF-β type I receptor (ΤβRIs-Fc). As reported for the wild-type receptor, the soluble ΤβR-I does not bind TGF-β1 on its own. Surprisingly, in the absence of TGF-β1, the ΤβRIs-Fc mimicked TGF-β1-induced transcriptional and growth responses in mink lung epithelial cells (Mv1Lu). Signaling induced by the soluble TGF-β type I receptor is mediated via the obligatory presence of both TGF-β type I and type II receptors at the cell surface since no signal was observed in Mv1Lu-derivated mutants for TGF-β receptors R-1B and DR-26. The comparison between the structures of TGF-βs and a three-dimensional model of the extracellular domain of ΤβRI has shown that five residues of the supposed binding site of TGF-β1 (Lys31, His34, Glu5, Tyr91, and Lys94) were found with equivalent biochemical properties and similar spatial positions. Transforming growth factor-β (TGF-β) signaling requires a ligand-dependent interaction of TGF-β receptors ΤβR-I and ΤβR-II. It has been previously demonstrated that a soluble TGF-β type II receptor could be used as a TGF-β antagonist. Here we have generated and investigated the biochemical and signaling properties of a soluble TGF-β type I receptor (ΤβRIs-Fc). As reported for the wild-type receptor, the soluble ΤβR-I does not bind TGF-β1 on its own. Surprisingly, in the absence of TGF-β1, the ΤβRIs-Fc mimicked TGF-β1-induced transcriptional and growth responses in mink lung epithelial cells (Mv1Lu). Signaling induced by the soluble TGF-β type I receptor is mediated via the obligatory presence of both TGF-β type I and type II receptors at the cell surface since no signal was observed in Mv1Lu-derivated mutants for TGF-β receptors R-1B and DR-26. The comparison between the structures of TGF-βs and a three-dimensional model of the extracellular domain of ΤβRI has shown that five residues of the supposed binding site of TGF-β1 (Lys31, His34, Glu5, Tyr91, and Lys94) were found with equivalent biochemical properties and similar spatial positions. transforming growth factor-β TGF-β type I receptor TGF-β type II receptor soluble TGF-β type I receptor fused with the Fc region of human immunoglobulin soluble TGF-β type II receptor fused with the Fc region of human immunoglobulin Sma- and Mad-related factor mink lung epithelial cells polymerase chain reaction polyacrylamide gel electrophoresis minimum essential medium extracellular domain bone morphogenetic protein bone morphogenetic protein receptor IA three-dimensional Transforming growth factor-β (TGF-β)1 is a multifunctional cytokine involved in the regulation of many biological processes (1Massague J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). In mammalian cells, responses to TGF-β are mediated by type I and type II cell surface receptors, which are expressed in most tissues (1Massague J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar, 2Vivien D. Bernaudin M. Buisson A. Divoux D. MacKenzie E.T. Nouvelot A. J. Neurochem. 1998; 70: 2296-2304Crossref PubMed Scopus (61) Google Scholar). TGF-β1, which is the prototype of the TGF-β family, elicits its effects by binding to a heteromeric complex of transmembrane Ser/Thr kinase receptors cloned as type I and type II receptors. The TGF-β type II receptor (ΤβR-II) binds TGF-β on its own and recruits and phosphorylates the TGF-β type I receptor (ΤβR-I) in a juxtamembrane domain rich in glycine and serine called the GS box (3Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar). Activation of the type I receptor leads to the phosphorylation of members of the Sma- and Mad- related factors (Smads) (4Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (591) Google Scholar, 5Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (809) Google Scholar). Following receptor-dependent phosphorylation, Smad2 and Smad3 interact with Smad4, a constitutively phosphorylated common mediator of all the members of TGF-β family (6Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (916) Google Scholar, 7Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar, 8Piek E. Heldin C.H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (749) Google Scholar), to translocate to the nucleus and mediate TGF-β responses. By these mechanisms, TGF-β1 activates the transcription of mammalian genes important for cell cycle regulation and for extracellular matrix formation and may also promote immunosuppressive responses. Based on these functions, the use of TGF-β agonists or antagonists could be of considerable interest in human therapy to prevent or exacerbate TGF-β responses. A better understanding of the mechanisms by which TGF-β could have beneficial or deleterious effects in various physiological processes is obtained through the generation of molecular tools that allow novel routes of investigation. The recent generation of transgenic mice either lacking the functional gene for TGF-β or overexpressing TGF-β in brain has greatly aided our understanding of the role that TGF-β plays in many systems. For example, targeted disruption of the TGF-β1 gene in mice causes enhanced inflammatory responses leading to early death (9Shull M.M. Ormsby I. Kier A.B. Pawlowski S. Diebold R.J. Yin M. Allen R. Sidman C. Proetzel G. Calvin D. et al.Nature. 1992; 359: 693-699Crossref PubMed Scopus (2653) Google Scholar, 10Kulkarni A.B. Huh C.G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 770-774Crossref PubMed Scopus (1662) Google Scholar). Overexpression of brain-derived TGF-β promotes vascular amyloidogenesis, a histopathological hallmark of Alzheimer's disease (11Wyss-Coray T. Masliah E. Mallory M. McConlogue L. Johnson-Wood K. Lin C. Mucke L. Nature. 1997; 389: 603-606Crossref PubMed Scopus (361) Google Scholar, 12Wyss-Coray T. Lin C. Sanan D.A. Mucke L. Masliah E. Am. J. Pathol. 2000; 156: 139-150Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). However, the molecular mechanisms that lead to these phenomena remain poorly understood. Recently a better understanding of the pathophysiological actions of TGF-β have been obtained through the use of recombinant soluble TGF-β type II receptors as TGF-β antagonists (13Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (51) Google Scholar, 14Krieglstein K. Richter S. Farkas L. Schuster N. Dunker N. Oppenheim R.W. Unsicker K. Nat. Neurosci. 2000; 3: 1085-1090Crossref PubMed Scopus (138) Google Scholar). In a previous study (13Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (51) Google Scholar), we have generated a soluble type II receptor for TGF-β fused with the Fc region of human immunoglobulin and characterized its ability to prevent TGF-β signaling in mink lung epithelial cells by sequestering the TGF-β peptide. By using this tool, we have demonstrated that blockage of the activity of endogenously produced TGF-β, in a model of cerebral ischemia in rats, enhanced the volume of the damaged tissue (15Ruocco A. Nicole O. Docagne F. Ali C. Chazalviel L. Komesli S. Yablonsky F. Roussel S. MacKenzie E.T. Vivien D. Buisson A. J. Cereb. Blood Flow Metab. 1999; 19: 1345-1353Crossref PubMed Scopus (113) Google Scholar). These data suggest that the potentiation of the TGF-β signaling in ischemic brain tissues may represent an innovative approach for the treatment of stroke in man. With the aim to develop agonists or antagonists for TGF-β, we have generated a soluble type I receptor for TGF-β and then tested its potential ability to bind TGF-β1 and to influence both TGF-β-induced transcriptional and cell growth responses. The cDNA encoding the extracellular domain of the human ΤβR-I was amplified by PCR from the plasmid pBlueScript (Stratagene), which contained a full-length cDNA of the receptor (with minimal 5′- and 3′-untranslated regions). The sense primer was 5′-CCGAAGCTTGGCGAGGCGAGGTTTGCTGGGGTGAGGCAGCG-3′ corresponding to a HindIII site (underlined) and to base pairs −76 to −46 of the cDNA preceding the start codon. The antisense primer had the sequence 5′-CGGGGATCCACTTACCTGTTTCCACAGGACCAAGGCC-3′ representing base pairs 357–375, which encoded residues Gly120 to Glu125 immediately preceded by a splice donator site and aBamHI site (underlined). The standard PCR conditions usingPfu DNA polymerase (Stratagene) were as follows: 92 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min for 30 cycles with a final extension at 72 °C for 7 min. The resulting amplified PCR product was digested with HindIII and BamHI and ligated into the respective sites in the polylinker of the vector pIg-Tail (R&D Systems). The pIg-Tail expression system enables the mammalian production of fusion proteins with a C-terminal Fc tail. The truncated cDNA derived from PCR was sequenced to confirm the fidelity of the reaction. The cDNA encoding the extracellular domain of human ΤβR-II was amplified by PCR and cloned in the polylinker between the EcoRI and BamHI sites of the pIg-Tail (R&D Systems) containing a hygromycin-selectable marker. As a first attempt, the chimeric protein ΤβRIs-Fc was expressed into the medium of transiently transfected COS-7 cells (American Type Culture Collection CRL1651). COS-7 cells were cultured in AIM V medium (Life Technologies, Inc.) and transfected as previously described (53Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (54) Google Scholar) with the recombinant pIg-Tail vector. Following biochemical characterization the chimeric receptor ΤβRIs-Fc was routinely expressed into the medium of cultured Chinese hamster ovary cells (American Type Culture Collection CRL 9618) after a stable double transfection with recombinant pIg-Tail vector and pTK-Hyg vector (CLONTECH). A stable clone was isolated by single-cell cloning. The selected clone was cultured in AIM V medium (Life Technologies, Inc.) in which it loses its adherent properties and forms spheroids. Stable transfected Chinese hamster ovary cells were adjusted to 1.3 × 106/ml, and 15 ml of the suspension were injected into the cell compartment of the CELLine production chamber (CL1000 Integra Biosciences). The nutrient medium reservoir was filled with 1 liter of AIM V medium without antibiotics supplemented with 4.5 g/liter glucose and 4 mm Glutamax (Life Technologies, Inc.). The CELLine was then placed into a 37 °C humidified 5% CO2 incubator. A silicone membrane, at the bottom of the production chamber, allowed gas exchange. The production chamber in which cells were grown was separated from the nutrient medium reservoir by a dialysis membrane permeable to nutrients smaller than 10 kDa. Low molecular weight metabolic products diffused from the 15-ml production chamber, which, in turn, provided nutrients for cell proliferation and protein synthesis. Nutrient medium was exchanged once every week. Cells were harvested 7 days after inoculation. The cell suspension (15 ml) was collected, and the concentration and percentage of viable cells were determined. Once more, 15 ml at 3 × 107 cells/ml were injected into the cell compartment. Cells were harvested three times per week. The human recombinant proteins ΤβRIs-Fc and TβRIIs-Fc were purified by one-step protein A affinity chromatography. To preserve the activity of the purified protein, the pH was immediately neutralized by the addition of a110 volume of 1 m Tris-HCl, pH 9.0 to the collected fractions. The eluted protein was dialyzed overnight against 0.1× phosphate-buffered saline and lyophilized. The purified protein was then analyzed by 10% SDS-PAGE using a precast gel (Novex) followed by silver staining (Silver Staining kit, Novex). The amount of protein was quantified using the Bio-Rad Protein Assay kit (microtiter plate format). Purified recombinant protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell). The membrane was blocked for 30 min with 10% dry fat milk in pH 7.2 NaCl/Pisupplemented with 0.1% Tween 20. For the analysis of fractions containing ΤβRIs-Fc, the membrane was incubated with goat anti-human IgG Fc (Caltag) then with swine anti-goat IgG conjugated to horseradish (Caltag) peroxidase. As indicated in the corresponding figure, some immunoblots were revealed either with a rabbit antibody raised against human TGF-β1 (a generous gift from P. ten Dijke) or a goat antibody raised against human IgG Fc (Caltag) then with an appropriate secondary antibody conjugated to horseradish peroxidase. The chemiluminescence immunoassay was performed with Renaissance Western Blot Chemiluminescence Reagent (PerkinElmer Life Sciences) following the procedure given by the manufacturer. The FlashPlate (PerkinElmer Life Sciences) is a white 96-well polystyrene microtiter plate with plastic scintillator-coated wells. The FlashPlates were precoated with protein A to allow the immobilization of the chimeric proteins (100 μl of affinity-purified protein at 2.5 μg/ml in pH 7.2 NaCl/Pi) by the Fc portion for 2 h at room temperature. After two washes with binding buffer (128 mmNaCl; 5 mm KCl; 5 mm MgSO4; 1.3 mm CaCl2; 50 mm Hepes, pH 7.6) the wells were treated for 2 h at room temperature with a blocking solution consisting of binding buffer containing 5% bovine serum albumin. After incubation, the wells were washed three times with additional binding buffer containing 1% bovine serum albumin.125I-labeled TGF-β1 (PerkinElmer Life Sciences) was diluted in binding buffer prior to its addition into the wells. The plates were incubated at room temperature for 2 h, then sealed, and counted on a Packard Top Count microplate scintillation counter. A 1-min counting period was used. All determinations were carried out at least in duplicate. Nonspecific binding was determined with an excess of unlabeled TGF-β1 (100-fold). For the competition binding assay, the final concentration of radiolabeled ligand was 600 pm(specific activity, 300–450 Ci/mmol) with final concentrations ranging over 0.001–3 μg/ml for ΤβRIs-Fc and ΤβRIIs-Fc. The Mv1Lu cell line (American Type culture collection CCL-64) was maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum (Life Technologies, Inc.). 125I-TGFβ1 (PerkinElmer Life Sciences) binding to monolayer cells was performed following the published procedures (2Vivien D. Bernaudin M. Buisson A. Divoux D. MacKenzie E.T. Nouvelot A. J. Neurochem. 1998; 70: 2296-2304Crossref PubMed Scopus (61) Google Scholar). For the competition binding assay, the final concentration of radiolabeled ligand was 40 pm (specific activity, 3000–4500 Ci/mmol) with final concentrations ranging over 0.001–10 μg/ml for ΤβRIs-Fc and ΤβRIIs-Fc alone or associated. Mv1Lu cells were stable transfected (by using the lipofection protocol Transfast (Promega)) with the construct (caga)12MLP-Luc (16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar) and the selection vector pTK-Hyg (CLONTECH). CAGA reporter vectors were generated using pGL3 basic plasmid (Promega). Transfected cells were plated into 96-well plates (5 × 104 cells/well) using MEM containing 10% fetal calf serum for one night before the assay. The wells were washed three times in MEM, 0% fetal calf serum then cells were incubated at 37 °C, 5% CO2 in MEM, 1% fetal calf serum. After 1 h, increasing concentrations of TGF-β1 (R&D Systems) (25–300 pg/ml) and ΤβRIs-Fc and ΤβRIIs-Fc (0.03–3 μg/ml) were added to the medium. Cells were then harvested 6 h later and assayed for luciferase activity using the Luc-Lite Packard kit as described by the manufacturer. The total light emission was measured using a Packard TopCount microplate luminescence counter. Similar experiments were performed following transient transfection of the (caga)12MLP-Luc in Mv1Lu, DR-26, and R-1B cell lines by using the lipofection protocol (Transfast, Promega). Cells were harvested 20 h after the addition of either TGF-β1 (1 ng/ml) or ΤβRIs-Fc (3 μg/ml) in serum-deficient MEM and assayed for luciferase activity by using a commercial system (luciferase reporter assay system, Promega). Cells were plated 24 h after treatment in 24-well plates containing medium with 10% fetal bovine serum. Medium was replaced with fresh medium containing 1% fetal bovine serum in the presence or absence of TGF-β1 (1 ng/ml) or ΤβRIs-Fc (3 μg/ml) for 20 h. Cells were labeled with [3H]thymidine (1 μCi/ml) (PerkinElmer Life Sciences) for the last 4 h of incubation. Cells were washed three times with cold phosphate-buffered saline, fixed with 5% trichloroacetic acid for 1 h at 4 °C, washed twice with cold 5% trichloroacetic acid, and extracted with 1 n NaOH for 30 min at room temperature. The extracts were collected and counted in a β-counter. The coordinates of the different three-dimensional (3D) structures are found in the Protein Data Bank (PDB) (17Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.E. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8182) Google Scholar, 18Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27906) Google Scholar). The 3D structure of the extracellular domain (ECD) of bone morphogenetic protein receptor IA (BRIA-ECD) (19Kirsch T. Sebald W. Dreyer M.K. Nat. Struct. Biol. 2000; 7: 492-496Crossref PubMed Scopus (276) Google Scholar) has been recently determined (PDB entry 1es7). The software TITO (20Labesse G. Mornon J. Bioinformatics. 1998; 14: 206-211Crossref PubMed Scopus (63) Google Scholar) has been used to check and optimize the alignment and to build the Cα chain of the extracellular domain of TβR-I (TβR-I-ECD) based on the coordinates of BRIA-ECD. The model has been built using Modeler (21Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10635) Google Scholar). The 3D structure of TGF-β is known for the three isoforms (PDB entries 1klc for TGF-β1 (22Hinck A.P. Archer S.J. Qian S.W. Roberts A.B. Sporn M.B. Weatherbee J.A. Tsang M.L. Lucas R. Zhang B.L. Wenker J. Torchia D.A. Biochemistry. 1996; 35: 8517-8534Crossref PubMed Scopus (155) Google Scholar), 2tgi for TGF-β2 (23Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (376) Google Scholar), and 1tgj for TGF-β3 (24Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (128) Google Scholar)). COS-7 cells were transfected with the expression plasmid pIg-Tail containing the cDNA encoding for a truncated TGF-β type I receptor (amino acid residues 1–124) as described in Fig. 1. After one-step purification by affinity chromatography on protein A-Sepharose, a soluble pure chimeric protein was recovered from the medium of cells transiently transfected with the pIg-Tail/ΤβRIs plasmid. Analysis of the purified chimeric receptor was performed by SDS-PAGE under either nonreducing or reducing conditions prior to silver staining or by immunoblotting performed with an antibody raised against human immunoglobulin (Fig. 2). A product of molecular mass of ∼40 kDa was visualized in reducing conditions corresponding to the expected molecular mass of the extracellular domain of ΤβR-I fused with the Fc region of the human immunoglobulin (11 + 30 kDa). However, in nonreducing conditions, immunoblotting revealed products of higher molecular mass (over 100 kDa), which may represent homomeric forms of a secreted chimeric soluble receptor due to the presence of the Fc fragment.Figure 2SDS-PAGE and Western blotting analyses of the recombinant soluble chimeric ΤβRIs-Fc. A andB, 10% SDS-PAGE of protein fractions was eluted from the Hitrap protein A column. The gel was either silver-stained or immunoblotted with goat anti-human Fc domain of immunoglobulin as indicated in the figure. Samples of the different elution steps and purified recombinant protein were separated under either nonreducing (A) or reducing conditions (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ligand binding activity of recombinant ΤβRIs-Fc receptor was tested and compared with the previously characterized ΤβRIIs-Fc (13Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (51) Google Scholar) in a protein A FlashPlate binding assay. Protein A-coated plastic surfaces within the wells were coated with the purified recombinant ΤβRIs-Fc chimeric receptor allowing the immobilization of the chimeric protein by its Fc portion. Then 125I-TGF-β1 was added in the presence of increasing concentrations of soluble type II receptor. As shown in Fig.3, increasing concentrations of soluble TGF-β type I receptor (from 3 ng/ml to 3 μg/ml) failed to influence125I-TGF-β1 binding (500 pm). However, co-incubation in the presence of similar concentrations of the previously characterized ΤβRIIs-Fc (13Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (51) Google Scholar) revealed125I-TGF-β1 binding to the coated type I receptor with a typical saturation curve. The kinetics of 125I-labeled TGF-β1 binding were performed at room temperature in conditions showing equilibrium binding, achieved after 2–3 h of incubation in the case of the ΤβRIIs-Fc-coated FlashPlate (data not shown). Nonspecific binding, obtained upon ΤβRIs-Fc coating, was determined for each condition in the presence of 200 nm cold TGF-β1 and subtracted from total binding. Similarly, TGF-β1 binding activity to the recombinant TβRIIs-Fc was tested in a precoated protein A FlashPlate binding assay as described under “Experimental Procedures.” The addition of increasing concentrations of the soluble type I receptor cannot compete binding of iodinated TGF-β1 to the precoated TβRIIs-Fc. Based on these observations, two concepts could explain these data. The first one could be that TβRIs-Fc is not capable of binding TGF-β1 on its own, and the second one may be that the TβRIs-Fc is actually binding type II and does not directly contact the ligand. To further characterize the binding properties of the soluble TGF-β type I receptor, 125I-TGF-β1 binding was evaluated in Mv1Lu cells in the presence of increasing concentrations of soluble TGF-β type I (Fig. 4). As expected, increasing concentrations of the soluble TGF-β type II receptor inhibited the binding ability of iodinated TGF-β1 on the Mv1Lu cell line with an apparent IC50 of 0.15 μg/ml. In contrast, increasing concentrations of the soluble type I receptor failed to influence the binding of TGF-β1 in Mv1Lu cells. These data show that extracellular domains of both ΤβR-I and ΤβR-II display the same binding properties that the full-length transmembrane receptors expressed in cell lines (3Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar). To test the properties of the recombinant chimeric TGF-β type I receptor on TGF-β signaling, this component was tested on a model of TGF-β-responsive luciferase reporter gene assay in the presence of exogenous TGF-β1. Following stable transfection in the Mv1Lu cell line, increasing concentrations of TGF-β1 were evaluated for their ability to induce the activation of the previously proposed (16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar) TGF-β-inducible element, named CAGA box (Fig.5). As shown in Fig. 5 A, TGF-β1 enhances the activity of the CAGA-luciferase reporter gene in the Mv1Lu cell line in a dose-dependent manner. As expected, the soluble TGF-β type II receptor induced a dose-dependent inhibition of the TGF-β1 response (TGF-β1 at 80 pg/ml) (Fig. 5 B). Surprisingly, although ΤβRIs-Fc did not influence the TGF-β-dependent transcriptional activity for a range of concentrations between 30 ng/ml and 1 μg/ml, at the highest concentrations tested (3 μg/ml) it induced a reproducible enhancement of the TGF-β-induced signal (Fig.5 B). Based on these results, similar experiments were performed in the absence of exogenous TGF-β1. As observed in Fig.5 C, the ΤβRIs-Fc induced a marked dose-dependent activation of the TGF-β1-inducible element CAGA box. Altogether these data demonstrate that while a soluble type II receptor could be characterized as a TGF-β antagonist, a soluble TGF-β type I receptor mimicked TGF-β transcriptional activity. Moreover, to address the possibility that immunoglobulin could drive TGF-β receptor heteromerization and subsequent signaling, increasing concentrations of purified whole human immunoglobulin were tested in a TGF-β1-responsive reporter gene assay. As shown in Fig.5 D, whole IgG failed to induce TGF-β1-like signaling. Moreover, the specificity of the TβRIs-Fc-induced transcriptional response was confirmed by data showing that the same type of molecular construct containing the extracellular domain of the type II receptor did not induce any signal (Fig. 5 C). Similar results to those obtained for the transcriptional activity of the TGF-β1-dependent luciferase reporter gene were observed with the antimitogenic effect of TGF-β1 in mink lung epithelial cells. Although TGF-β1 (1 ng/ml) induced a marked decrease of the [3H]thymidine incorporation into the DNA (90% of the control value) (Fig. 6) incubation in the presence of the TβRIs-Fc (3 μg/ml) led to the same effect. These data show that the addition of the soluble TGF-β type I receptor mimicked the growth inhibition induced by TGF-β1 in the Mv1Lu cell line. To further characterize the signaling properties of the ΤβRIs-Fc we used R1-B and DR-26 cell lines subjected to transient transfection of the luciferase reporter gene driven by the TGF-β-responsive element (CAGA box). DR-26 cells are resistant to TGF-β due to a non-sense mutation in the transmembrane region of the endogenous ΤβR-II (3Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar). DR-26 cells lack ΤβR-II at their surface and are unable to bind TGF-β with endogenous ΤβR-I. As shown in Fig. 7, although both TGF-β1 and ΤβRIs-Fc enhance the CAGA-luciferase reporter gene activity in the Mv1Lu cell line (Fig. 7 A), ΤβRIs-Fc is devoid of this activity in the DR-26 cell line (Fig. 7 B). R1-B cells are unable to respond to TGF-β because of the absence of functional TGF-β type I receptor. As observed in Fig. 7 C, the soluble hΤβRIs-Fc failed to restore TGF-β signaling in this model. Overall these results suggest that the soluble TGF-β type I receptor requires functional ΤβR-II and ΤβR-I to transduce its signal. The soluble TGF-β type I receptor is able to form a stable complex with both ΤβR-II and ΤβR-I and to mimic TGF-β1-mediated responsiveness. To further understand this process, we have generated a three-dimensional model of the extracellular domain of TβR-I based on common sequence and structural features with the already known structure of BRIA-ECD (19Kirsch T. Sebald W. Dreyer M.K. Nat. Struct. Biol. 2000; 7: 492-496Crossref PubMed Scopus (276) Google Scholar). A model of TβR-I-ECD (PDB entry 1tbi) was constructed using protectin CD59 as a template (25Jokiranta T.S. Tissari J. Teleman O. Meri S. FEBS Lett. 1995; 376: 31-36Crossref PubMed Scopus (13) Google Scholar). The alignment between TβR-I-ECD and CD59 was based on a conserved cysteine pairing pattern and a highly conserved cysteine box (sequence CCXXXXCN). No other similarities between theses two sequences emerged from the alignment. The alignment with BRIA-ECD shows a better conservation of hydrophobic properties and less gaps and insertions than the alignment with CD59 even if only 9 of the 10 cysteine residues are conserved. Four disulfide bridges among the five have been preserved. The new disulfide bridge is easily rebuilt within the model without important distortions. The potential of mean force and the surface potential appear correct according to the validation software Verify3D (data not shown) (26Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2612) Google Scholar). A comparative analysis of the proposed TβR-I-ECD model and the structures of TGF-βs has shown interesting similarities. Although sequences and structures of TGF-βs and TβR-I-ECD are dissimilar and cannot be aligned, five residues with similar properties can be spatially superimposed (Fig.8, A and B). We were able to superimpose an arginine (a lysine in TGF-β2) on a lysine, a tyrosine on a phenylalanine, a histidine on a histidine, a glutamate on an aspartate, and a lysine on a lysine in TGF-βs and in TβR-I-ECD, respectively. In the present study, we have reported that a soluble TGF-β type I receptor could mimic TGF-β1 responses in mink lu" @default.
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