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• W2093595354 abstract "Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended β-sheet, whereas residues involved in type I binding are located in the α-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45–79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (∼4 nm). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-β members, activins, and BMPs. Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended β-sheet, whereas residues involved in type I binding are located in the α-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45–79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (∼4 nm). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-β members, activins, and BMPs. Activins and BMPs are members of the TGF-β 2The abbreviations used are: TGFtransforming growth factorBMPbone morphogenetic proteinALKactivin-like kinaseAct RIIactivin type II receptorECDextracellular domainFSHfollicle stimulating hormonewtwild typeMES4-morpholineethanesulfonic acidHPLChigh performance liquid chromatography.2The abbreviations used are: TGFtransforming growth factorBMPbone morphogenetic proteinALKactivin-like kinaseAct RIIactivin type II receptorECDextracellular domainFSHfollicle stimulating hormonewtwild typeMES4-morpholineethanesulfonic acidHPLChigh performance liquid chromatography. superfamily that comprises >40 encoded members in the human. At least five binding receptors (RII) and seven signaling receptors (RI) that bind the ligands of the family have been identified (1Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4693) Google Scholar, 2Vale W. Rivier C. Hsueh A. Campen C. Meunier H. Bicsak T. Vaughan J. Corrigan A. Bardin W. Sawchenko P. Petraglia F. Yu J. Plotsky P. Spiess J. Rivier J. Recent Prog. Horm. Res. 1988; 44: 1-34PubMed Google Scholar, 3Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1709) Google Scholar). The members of this superfamily are involved in a multitude of biological functions including development, differentiation, apoptosis, reproduction, tissue regeneration, immune responses, and bone growth (4Chen Y.G. Lui H.M. Lin S.L. Lee J.M. Ying S.Y. Exp. Biol. Med. 2002; 227: 75-87Crossref PubMed Scopus (197) Google Scholar, 5Risbridger G.P. Schmitt J.F. Robertson D.M. Endocr. Rev. 2001; 22: 836-858Crossref PubMed Scopus (150) Google Scholar, 6Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar, 7Wankell M. Werner S. Alzheimer C. Werner S. Ann. N. Y. Acad. Sci. 2003; 995: 48-58Crossref PubMed Scopus (19) Google Scholar). transforming growth factor bone morphogenetic protein activin-like kinase activin type II receptor extracellular domain follicle stimulating hormone wild type 4-morpholineethanesulfonic acid high performance liquid chromatography. transforming growth factor bone morphogenetic protein activin-like kinase activin type II receptor extracellular domain follicle stimulating hormone wild type 4-morpholineethanesulfonic acid high performance liquid chromatography. The ligands of TGF-β family share a distinct structural signature known as the “cysteine knot scaffold” (8Vitt U.A. Hsu S.Y. Hsueh A.J. Mol. Endocrinol. 2001; 15: 681-694Crossref PubMed Scopus (211) Google Scholar). Activins adopt this prototypical disulfide-linked dimeric structure and contain two subunits termed β-chains. Genes encoding four different β subunits, βA, βB, βC, and βE, have been identified in the human, theoretically offering a possibility of an array of activin dimers, although only activin A (βA-βA), activin-B (βB-βB), and activin AB (βA-βB) have been documented to be biologically active (9Phillips D.J. BioEssays. 2000; 22: 689-696Crossref PubMed Scopus (58) Google Scholar). In each monomer two pairs of antiparallel β-strands stretch out from the cysteine core of the dimer to form short and long fingers. The characteristic curvature of these fingers creates concave and convex surfaces on the ligand. At the base of the fingers, each monomer has an α-helix, which together with the pre-helix loop and the inner concave surface of the fingers of the other monomer form the “wrist” region (10Greenwald J. Vega M.E. Allendorph G.P. Fischer W.H. Vale W. Choe S. Mol. Cell. 2004; 15: 485-489Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 11Thompson T.B. Woodruff T.K. Jardetzky T.S. EMBO J. 2003; 22: 1555-1566Crossref PubMed Scopus (173) Google Scholar). Bone morphogenetic proteins (BMPs) were first identified as molecules that induce bone and cartilage formation in rodents (12Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3307) Google Scholar). BMPs are a large family (more than 20 members) with extremely complex and diverse roles both in development and adult life (12Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3307) Google Scholar, 13Whitman M. Genes Dev. 1998; 12: 2445-2462Crossref PubMed Scopus (441) Google Scholar, 14Matzuk M.M. Kumar T.R. Shou W. Coerver K.A. Lau A.L. Behringer R.R. Finegold M.J. Recent Prog. Horm. Res. 1996; 51 (discussion 155–157): 123-154PubMed Google Scholar). For example, BMP7 has a role in kidney morphogenesis and bone formation during development and is involved in regulating gonadal function in the adult (15Luo G. Hofmann C. Bronckers A.L. Sohocki M. Bradley A. Karsenty G. Genes Dev. 1995; 9: 2808-2820Crossref PubMed Scopus (864) Google Scholar, 16Dudley A.T. Lyons K.M. Robertson E.J. Genes Dev. 1995; 9: 2795-2807Crossref PubMed Scopus (946) Google Scholar, 17Lee W.S. Otsuka F. Moore R.K. Shimasaki S. Biol. Reprod. 2001; 65: 994-999Crossref PubMed Scopus (192) Google Scholar, 18Zhao G.Q. Chen Y.X. Liu X.M. Xu Z. Qi X. Dev. Biol. 2001; 240: 212-222Crossref PubMed Scopus (71) Google Scholar). BMPs, like activins, signal via type II and type I receptors (19Nohe A. Hassel S. Ehrlich M. Neubauer F. Sebald W. Henis Y.I. Knaus P. J. Biol. Chem. 2002; 277: 5330-5338Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar) and subsequently activate Smad proteins 1, 5, and 8, which in turn transmit signals into the cell nucleus (6Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar, 20Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar). Although BMP type I receptors (ALK3, ALK6, and ALK2) are largely specific for BMP family, it is not true for type II receptors. BMP RII binds only BMPs (21Kawabata M. Chytil A. Moses H.L. J. Biol. Chem. 1995; 270: 5625-5630Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 22Liu V.F. Boubnov N.V. Weaver D.T. Stem Cells. 1995; 13: 117-128PubMed Google Scholar, 23Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (472) Google Scholar), but activin type II receptors can bind both BMPs and activins (24Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (457) Google Scholar). The receptor activation mechanism for activin involves initial binding to its type II receptor (Act RII or IIB), which leads to the recruitment, phosphorylation, and activation of its type I receptor (ALK4) followed by activation of intracellular signaling molecules, Smad2 and -3 (25Massague J. Chen Y.G. Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 26Lebrun J.J. Vale W.W. Mol. Cell. Biol. 1997; 17: 1682-1691Crossref PubMed Scopus (144) Google Scholar, 27Attisano L. Wrana J.L. Montalvo E. Massague J. Mol. Cell. Biol. 1996; 16: 1066-1073Crossref PubMed Scopus (283) Google Scholar). The specific amino acid residues of activin A involved in the underlying intermolecular interactions within the activin/Act RII-ECD/ALK4-ECD complex can be deduced now based on relevant structural and functional data. The activin A/Act RIIB interface involves hydrophobic (Ile-30, Ala-31, Pro-32, Pro-88, Leu-92, Tyr-94, and Ile-100) and ionic/polar (Arg-87, Ser-90, Lys-102, Glu-111) residues on activin A and overlaps the interface determined in the BMP7/Act RII structure (11Thompson T.B. Woodruff T.K. Jardetzky T.S. EMBO J. 2003; 22: 1555-1566Crossref PubMed Scopus (173) Google Scholar, 28Greenwald J. Groppe J. Gray P. Wiater E. Kwiatkowski W. Vale W. Choe S. Mol. Cell. 2003; 11: 605-617Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The activin A/ALK4 interface is not very well characterized. Based on the crystal structure of BMP2 in complex with ALK3 (29Kirsch T. Sebald W. Dreyer M.K. Nat. Struct. Biol. 2000; 7: 492-496Crossref PubMed Scopus (268) Google Scholar), it has been predicted that all type I receptors bind to the wrist epitope of their respective ligands irrespective of affinity. Substantiating this model, an allosteric conformational change was observed in the wrist region of BMP7 after binding to the Act RII-ECD, and this may allow for the cooperative type I/type II receptor assembly induced by TGF-β superfamily members (30Wuytens G. Verschueren K. de Winter J.P. Gajendran N. Beek L. Devos K. Bosman F. de Waele P. Andries M. van den Eijnden-van Raaij A.J. Smith J.C. Huylebroeck D. J. Biol. Chem. 1999; 274: 9821-9827Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Ligands that exhibit high affinity for type II receptors while being deficient in their ability to recruit type I receptors can act as antagonists. For example, an activin A/C chimera retaining its type II receptor binding was shown to be devoid of activin-like activity and, consequently, possessed activin and myostatin antagonistic properties (31Muenster U. Harrison C.A. Donaldson C. Vale W. Fischer W.H. J. Biol. Chem. 2005; 280: 36626-36632Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, a point mutation in the finger region (M108A) of activin yielded a ligand that binds the type II receptor and has a biological activity 3 orders of magnitude lower than wt and antagonized activin A in 293T cells (32Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In the present study we highlight the switch in affinity toward BMP type I receptor by introducing multiple residues of BMP2 or BMP7 into the activin A wrist region and the antagonistic properties of these chimeras. The ActA/BMP chimeras presented in this study were characterized with respect to their binding affinities for Act RII, their ability to disrupt activin signaling, and their ability to switch to BMP receptor I activation as well as their antagonistic properties. SDS-PAGE gels (4–12%) used were from Bio-Rad, 125I-labeled activin A was prepared using chloramine T method as described previously (33Gray P.C. Greenwald J. Blount A.L. Kunitake K.S. Donaldson C.J. Choe S. Vale W. J. Biol. Chem. 2000; 275: 3206-3212Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) by Ezra Wiater, Peptide Biology Laboratories, Salk Institute, La Jolla, CA. The ActA/BMP constructs used in this study were in the pCDNA3 expression vector (Invitrogen). Recombinant BMP2, BMP7, and myostatin were purchased from R & D Systems (Minneapolis, MN). Phospho-Smad1 and Phospho-Smad2 antibodies were purchased from Cellular Signaling Technologies (Beverly, MA). Construction of Chimeras–Chimeras were generated by two-step introduction of BMP base pairs into the activin A sequence. First, plasmids containing sequences for ActA/BMP2 45–52, ActA/BMP2 73–79, ActA/BMP7 45–52, and ActA/BMP7 73–79 were generated by “long PCR” (initial denaturation, 5 min at 94 °C; 12 cycles of 1 min at 94 °C, 2 min at 58 °C, and 3 min at 72 °C; final extension, 10 min at 72 °C) using a pGem vector containing the wt activin A sequence with a FLAG tag inserted at the N terminus of the mature activin region as template and primers introducing base pairs encoding for the respective homologous BMP2 or BMP7 residues (45–52 and 73–79). Blunt linear products were ligated overnight at 16 °C using T4 ligase (Invitrogen). To subclone chimeras from pGem into pCDNA, another PCR was performed (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; final extension, 10 min at 72 °C) using the pGem plasmids containing the DNA of the chimeras as a template and a forward primer containing an NheI site 169 bp upstream of the N-terminal FLAG sequence (NheI pr) together with a reverse primer containing an XhoI site annealing to the C terminus of the chimeras (XhoI pr). The resulting products were cut with NheI and XhoI and then ligated overnight at 16 °C into an NheI-XhoI cut pCDNA cassette containing the remaining wt activin A sequence. To obtain the cassette an NheI site was introduced by silent mutation 169 bp upstream of the mature activin A in pCDNA. The Act/BMP2 45–79 and ActA/BMP7 45–79 chimeras were constructed by “overlapping PCR” using the above produced plasmids as templates. In four separate PCRs (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; final extension, 10 min at 72 °C) using pCDNA containing the sequence for either ActA/BMP2 45–52, ActA/BMP2 73–79, ActA/BMP7 45–52, and ActA/BMP7 73–79 as template together with NheI pr or XhoI pr, respectively, as well as primers introducing base pairs encoding for BMP2 or BMP7 residues 53–72, two pieces of overlapping DNA were generated for each chimera. In a second PCR (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; and final extension, 10 min at 72 °C), the two overlapping DNAs of BMP2 (to generate ActA/BMP2 45–79) as well as the two overlapping DNAs of BMP7 (to generate ActA/BMP7 45–79) were combined, and NheI pr as well as XhoI pr were used as primers. The resulting products were cut with NheI and XhoI and ligated into the pCDNA cassette described above. All of the PCRs were performed using 2.5 units of Takara DNA polymerase (Takara, Madison, WI) along with 0.2 unit of Pfu polymerase (Stratagene, La Jolla, CA). PCR products were separated on 1% agarose gels (Bio-Rad). To amplify the constructs, the plasmids were transformed into Top 10 competent bacteria by chemical transformation. Mini- and maxipreps as well as gel purifications were carried out using Qiagen (Valencia, CA) kits. Expression and Purification of Chimeric Proteins–For protein expression, ActA/BMP2 and ActA/BMP7 chimeric plasmids were transfected into 293T cells using polyethyleneimine as described (34Durocher Y. Perret S. Kamen A. Nucleic Acids Res. 2002; 30: E9Crossref PubMed Scopus (816) Google Scholar). In brief, 293T cells were grown to 70–80% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and l-glutamine in polylysine-coated 15-cm cell culture plates. After removal of the media, cells were washed with serum-free media, and 11 ml of serum-free medium was added to the cells. A solution of 36 μg of polyethyleneimine and 24 μg of plasmid DNA in 1.2 ml of serum-free medium was prepared, incubated for 10 min at room temperature, and then added to each cell culture dish. Cells were grown at 37 °C, 5% CO2. After 3 h, fetal calf serum was added to the cells to a final concentration of 10%. After 72 h, crude media containing chimeric proteins were harvested and filtered through a 5-μm nylon filter to separate cell debris. Then 1 m MES buffer, pH 6.2, was added to the filtrate to a final concentration of 50 mm along with 0.5 ml of M2 anti-FLAG-agarose bead suspension (Sigma). The media were left overnight at 4 °C under mild shaking conditions to allow protein binding to the beads. Then the suspension was poured into columns (10 cm × 1 cm; Bio-Rad) equipped with a one-way stop cock, the flow-through and 15 ml of additionally added 50 mm MES washing buffer were discarded, and ActA/BMP chimeras bound to the M2 anti-FLAG-agarose beads were eluted in 5 fractions of each 1 ml of glycine-HCl buffer, pH 2.8. Fractions were neutralized with 100 μl of Tris-HCl, pH 8, and then subjected to HPLC purification. Chimeras were separated in a single gradient run using a C4 column (2.1 × 150 mm, particle size, 5 μm; pore size, 300 Å; Vydac, Hesperia, CA) on an HP1100 HPLC machine (HP1100; Hewlett Packard). 0.05% trifluoroacetic acid (solvent A) as well as 0.05% trifluoroacetic acid dissolved in 90% acetonitrile (solvent B) were used as solvents. The gradient used was: min 0, 20% solvent B; min 40, 50% solvent B; min 41, 100% solvent B; min 45, 20% B, followed by a 12-min post-run with 20% solvent B at a flow rate of 0.2 ml/min. Chromatograms showed single peaks at 35.1 and 33.4 min for ActA/BMP2 and ActA/BMP7 chimeras, respectively. Peak fractions were collected, quantified by comparing the peak areas of the chimeras with those of known activin A amounts, dried down in the presence of bovine serum albumin, and redissolved in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 200 mm glutamine to a final chimera concentration of 10 mg/ml and 0.01% bovine serum albumin. Proteins were stored at –80 °C. Western Blot and Silver Staining–Crude medium, anti-FLAG column eluate, and HPLC fractions were checked for ActA/BMP chimera expression by Western blot. The samples were run under reducing and nonreducing conditions on 4–12% gradient SDS-polyacrylamide gels (Bio-Rad) along with known amounts of wt activin A as well as multi-marker (Sigma). Affinity-purified primary antibodies raised in rabbit against activin A residues 81–113 (kindly provided by Joan Vaughan, Peptide Biology Lab, Salk Institute, La Jolla, CA) were used in combination with an alkaline phosphatase-conjugated secondary goat anti-rabbit antibody (Bio-Rad). The proteins were visualized using alkaline phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. A protein silver staining method was used for the visualization of impurities not detectable by the βA antibody. The samples were run on 4–12% gradient SDS gels, then fixed for 15 min with 50% methanol followed by treatment for 15 min with 50 mm dithiothreitol and for 20 min with 0.1% silver nitrate solution. The excess of silver nitrate was sequestered by the addition of 25 ml of a 3% Na2CO3 solution containing 0.1% formaldehyde for 30 s. Protein bands were then visualized by the addition of another 50 ml of the 3% Na2CO3 solution containing 0.1% formaldehyde. Competitive Binding Studies of Chimeras–The Act RII binding assay was performed as previously described (32Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, HEK 293T cells grown in 24-well plates coated with polylysine and seeded at a density of 150,000 cells/well were transfected with mouse Act RII using Perfectin® (GenLantis, San Diego, CA). After 48 h the cells were incubated with increasing doses of the wt activin A or chimeras in the presence of a constant amount of iodinated wt activin A tracer (200,000 cpm/well) for 2 h in HDB (12.5 mm Hepes, pH 7.4, 140 mm NaCl, 5 mm KCl) containing 1.5 mm CaCl2, 0.1% bovine serum albumin, and 5 mm MgSO4. After incubation, binding buffer was removed, and the cells were washed 3 times with HEPES dissociation buffer and then lysed with 1% SDS solution for 20 min. Radioactivity in the cell lysates was measured using a γ counter (APEX Micromedic Systems, Horsham, PA). Transfection and Luciferase Assays in HepG2 Cells–HepG2 cells were grown at 37 °C in a 5% CO2 humidified incubator in α-modification of Eagle's medium (Fisher Mediatech, Pittsburgh, PA) supplemented with 10% fetal bovine serum and l-glutamine. Cells grown in 24-well plates (surface area ∼1.75 cm2) were transfected with the bone morphogenetic protein-responsive BRE-Luc reporter plasmid (35Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), Rous sarcoma virus β-galactosidase, and pCDNA3 empty vector in a ratio of 0.9 μg/0.1 μg/0.05 μg using SuperFect transfection reagent (Qiagen) according to the manufacturer's recommendations. After 24 h, fresh medium was added, and cells were treated with increasing doses of activin A, BMP-2, BMP-7, ActA/BMP2 chimera, or ActA/BMP7 chimera as indicated for 16–24 h. Then cells were harvested in solubilization buffer (1% Triton X-100, 25 mm HEPES, pH 7.8, 15 mm MgSO4, 5 mm EGTA), and luciferase reporter activity was measured and normalized to β-galactosidase activities using standard methods. Transfection and Luciferase Assays in HEK 293T Cells–293T cells were seeded into 24-well plates coated with polylysine at a density of 150,000 cells/well. After 24 h cells were transfected overnight with a mixture of A3 Lux (25 ng) and β-galactosidase (25 ng) reporter plasmids, the transcription factor FAST2 (50 ng), and empty pCDNA3 vector (400 ng) using Perfectin® transfection reagent (GenLantis) according to the manufacturer's recommendations. Then the cells were treated with increasing doses of wt activin A or ActA/BMP chimeras for 16–24 h. The cells were harvested in ice-cold lysis buffer (1% Triton X-100 in 25 mm glycylglycine, 4 nm EGTA, 15 mm MgSO4 containing 1 mm dithiothreitol) and assayed for luciferase and β-galactosidase activities using standard methods. To assess antagonistic properties of the ActA/BMP chimeras, the cells were treated alternatively either with 100 pm wt activin A or 500 pm myostatin in the presence of increasing doses of the chimeras for 16–24 h. Inhibition of Follicle Stimulating Hormone (FSH) Release from Rat Interior Pituitary Cells–The assay was performed as previously described (32Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, freshly isolated cells from male Sprague-Dawley rat interior pituitaries from several animals were combined and seeded into 96-well plates at a density of 50,000 cells/well in βPJ medium (as detailed in Ref. 36Vale W. Vaughan J. Yamamoto G. Bruhn T. Douglas C. Dalton D. Rivier C. Rivier J. Methods Enzymol. 1983; 103: 565-577Crossref PubMed Scopus (278) Google Scholar) supplemented with 2% fetal bovine serum and appropriate growth factors (36Vale W. Vaughan J. Yamamoto G. Bruhn T. Douglas C. Dalton D. Rivier C. Rivier J. Methods Enzymol. 1983; 103: 565-577Crossref PubMed Scopus (278) Google Scholar). After 24 h cells were treated with increasing doses of wt activin A or ActA/BMP chimera (0–40 nm) in the presence or absence of 100 pm wt activin A. 72 h later media were harvested, and the concentration of the secreted FSH was determined by radioimmunoassay. Differentiation and Alkaline Phosphatase Assays in C2C12 Cells–C2C12 cells were maintained at 37 °C in a 5% CO2 humidified incubator in Dulbecco's modified Eagle's medium (Fisher Mediatech) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and l-glutamine. Cells were subjected to differentiation under low serum conditions. In brief, C2C12 cells were plated at 3 × 10 4 cells/well in 94-well plates in 50 μl of differentiation media (Dulbecco's modified Eagle's medium with 1% horse serum, penicillin, streptomycin, and l-glutamine). Three hours later, cells were treated with differentiation media containing 10 nm wt activin A or wt BMPs or ActA/BMP chimeras as shown to a total volume of 100 μl/well. Cells were allowed to differentiate for 5 days at 37 °C in a 5% CO2 humidified incubator and assayed for alkaline phosphatase activity using standard methods (37Kirsch T. Nickel J. Sebald W. EMBO J. 2000; 19: 3314-3324Crossref PubMed Scopus (209) Google Scholar). Briefly, for quantitative alkaline phosphatase assays, triplicate wells were washed once in HDB and lysed for 60 min in 100 μl of 1% Nonidet P-40, 100 mm glycine, pH 9.6, 1 mm MgCl2, 1 mm ZnCl2. Lysates were incubated with 100 μl of 1 mg/ml p-nitrophenyl phosphate in 100 mm glycine, pH 9.6, 1 mm MgCl2, 1 mm ZnCl2 until color developed and was measured by absorbance at 405 nm. Pictures of differentiated cells were taken in low magnification (20×), bright field conditions without fixation using a Canon EOS Elan II camera. Phospho-Smad Analysis in C2C12 Cells–C2C12 cells were maintained at 37 °C in a 5% CO2 humidified incubator in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and l-glutamine. C2C12 cells were plated in 6-well plates. After 24 h cells were treated with 1 or 5 nm wt activin A, wt BMP-2, wt BMP7, or ActA/BMP chimera for 30 and 60 min. Cells were lysed on ice in radioimmune precipitation assay buffer (150 mm NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) in the presence of protease inhibitors. The protein concentration was determined in the lysates. Total protein (67 μg) was loaded onto 10% SDS gels and subjected to SDS-PAGE. Gels were blotted onto polyvinylidene difluoride membranes and probed with anti-phosphorylated Smad1 (1:1000) or anti-phosphorylated Smad2 (1: 1000) by Western blotting. Bands were detected by horseradish peroxidase chemiluminescence and exposure to BioMax Light Kodak film. Selection of the Region for Chimera Construction–Based on available structural information, residues involved in ALK3 binding of BMP2 are located in both wrist and finger regions of BMP2 (29Kirsch T. Sebald W. Dreyer M.K. Nat. Struct. Biol. 2000; 7: 492-496Crossref PubMed Scopus (268) Google Scholar). Recently two activin A mutants containing a point mutation in finger 2 (32Harrison C.A. Gray P.C. Fischer W.H. Donaldson C. Choe S. Vale W. J. Biol. Chem. 2004; 279: 28036-28044Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) as well as a chimera, in which the entire wrist of activin A was changed to corresponding residues of activin C (A/C46–78) (31Muenster U. Harrison C.A. Donaldson C. Vale W. Fischer W.H. J. Biol. Chem. 2005; 280: 36626-36632Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) were shown to retain their affinity for the type II receptor, Act RII. Concurrently, these mutants were devoid of activin-like activity and consequently possessed activin and myostatin antagonistic properties. Following our hypothesis that TGF-β fami" @default.
• W2093595354 created "2016-06-24" @default.
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• W2093595354 title "Activin A/Bone Morphogenetic Protein (BMP) Chimeras Exhibit BMP-like Activity and Antagonize Activin and Myostatin" @default.
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