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Endocrinology, doi:10.1210/en.2005-0053
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Endocrinology Vol. 146, No. 9 3875-3882
Copyright © 2005 by The Endocrine Society

Overexpression of Twisted Gastrulation Inhibits Bone Morphogenetic Protein Action and Prevents Osteoblast Cell Differentiation in Vitro

Elisabetta Gazzerro, Valerie Deregowski, Sergio Vaira and Ernesto Canalis

Department of Research, Saint Francis Hospital and Medical Center (E.G., V.D., S.V., E.C.), Hartford, Connecticut 06105-1299; and University of Connecticut School of Medicine (E.G., E.C.), Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: Dr. Ernesto Canalis, Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299. E-mail: ecanalis{at}stfranciscare.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic protein-2 (BMP-2) and BMP-4 and can display both BMP agonist and antagonist functions. Tsg acts as a BMP agonist in chondrocytes, but its expression and actions on the differentiation of cells of the osteoblastic lineage are not known. We investigated the effects of Tsg overexpression by transducing murine ST-2 stromal and MC3T3 cells with a retroviral vector where Tsg is under control of the cytomegalovirus promoter and compared them to cells transduced with the parental vector alone. ST-2 cells were cultured in osteoblastic differentiating conditions in the presence or absence of BMP-2. Tsg overexpression precluded the appearance of mineralized nodules induced by BMP-2, led to a delay in the expression of osteoblastic gene markers, and decreased the responsiveness of ST-2 differentiating cells to PTH. BMP-2 induced the phosphorylation of signaling mothers against decapentaplegic-1/5/8, but not ERK, c-Jun N-terminal kinase, and p38. ST-2 cells overexpressing Tsg displayed an inhibition of BMP/signaling mother against decapentaplegic signaling. Tsg action was specific to BMP, because Tsg overexpression did not affect TGF-ß or Wnt/ß-catenin signaling pathways. Tsg also opposed MC3T3 cell differentiation and the expression of a mature osteoblast phenotype. In conclusion, Tsg overexpression inhibits BMP action in stromal and preosteoblastic cells and, accordingly, arrests their differentiation toward the osteoblastic pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE BONE microenvironment, osteoblastic cell differentiation and function are regulated by autocrine and paracrine cytokines. Among the growth factors secreted by skeletal cells, bone morphogenetic proteins (BMP) have the unique function of inducing the differentiation of cells of the osteoblastic lineage toward mature cells and of enhancing the differentiated function of the osteoblast. The activity of BMPs is regulated by extracellular proteins, which modulate the binding of BMPs to their cognate receptors (1). Among these extracellular proteins, twisted gastrulation (Tsg) displays the particular feature of exhibiting BMP agonistic and antagonistic activities (1).

Tsg, a secreted glycoprotein with a molecular mass (Mr) of 23.5 kDa, was originally identified in Drosophila, where it is required for the proper establishment of the dorsal-ventral axis (2). Tsg is expressed during embryonic development as well as postnatally (3). The Tsg gene encodes a secreted protein with an amino-terminal, cysteine-rich (CR) domain, which is highly conserved and analogous to the CR domains of the BMP antagonist chordin. CR domains are probably the sites of primary interaction between BMPs and their antagonists and are presumed to be responsible for the interactions of Tsg with BMPs and for the BMP antagonist effect. As part of its antagonist activity, Tsg binds chordin and forms a ternary complex with BMP-2 or -4, in which the two antagonists are more efficient than either one alone in inhibiting BMP signaling (4, 5, 6).

The BMP agonist effect of Tsg involves activation of the metalloprotease BMP-1/tolloid and the consequent cleavage of chordin. This results in reactivation of BMP signals, possibly because of Tsg binding to chordin fragments competing with their residual anti-BMP activity (7, 8, 9). BMP-1 and tolloid are encoded by alternatively spliced transcripts and have a protease domain common to the astacin family of metalloproteases, and BMP-1 has procollagen C proteinase activity (10, 11, 12). BMP-1 is unrelated to other BMPs. Tsg, BMP-1/tolloid, chordin, and chordin-like/ventroptin, a chordin-related gene, are expressed by chondrocytes and osteoblasts and can regulate chondrocytic maturation (1, 13, 14, 15). The relative levels of chordin, Tsg, and BMP-1/tolloid appear to dictate whether Tsg acts as a BMP agonist or antagonist in a specific cell environment.

Targeted disruption of the mouse Tsg gene causes defects in craniofacial development, vertebral malformations due to delayed endochondral ossification, and dwarfism due to impaired chondrocyte proliferation in the growth plate (16, 17, 18). This indicates that Tsg acts as a BMP agonist in the regulation of endochondral bone formation. However, the expression of Tsg, BMP-1/tolloid, and chordin and the actions of Tsg on mesenchymal cells with the potential to differentiate to osteoblasts are not understood.

The purpose of this study was to examine the expression of Tsg and its partners in stromal cells and to investigate the direct effects of Tsg on the differentiation and function of cells of the osteoblastic lineage. For this purpose, we transduced ST-2 stromal and MC3T3 cell lines with a retroviral vector expressing Tsg under the control of the constitutive cytomegalovirus (CMV) promoter and determined their phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Retroviral vectors and packaging cell lines
After introduction of the Kozak sequence, a 669-bp DNA fragment containing the coding region of the mouse tsg gene (E. De Robertis, Los Angeles, CA) was epitope-tagged with a FLAG tag at the carboxyl-terminal region by cloning the tsg fragment into the pCMV-Tag1 vector (Invitrogen Life Technologies, Inc., Carlsbad, CA) (7). Tsg-FLAG was then subcloned into a bicistronic retroviral vector pMG1-internal ribosomal entry site-enhanced green fluorescent protein (EGFP; Vector Core, University of Connecticut Health Center, Farmington, CT) (19). The vector pMG1 contains a Moloney murine leukemia virus 5' long-terminal repeat to direct the packaging signal, a CMV promoter to direct the constitutive expression of Tsg-FLAG (pMG1-Tsg), and an internal ribosomal entry site-EGFP sequence for clone selection. pMG1 and pMG1-Tsg were transfected into 293-GPG packaging cells (American Type Culture Collection, Manassas, VA). Cells were grown to 70–80% density in DMEM (Invitrogen Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), and transfected with Transfast (Promega Corp., Madison, WI) in accordance with the manufacturer’s instructions. After transfection, EGFP-positive cells were isolated using cloning cylinders (Sigma-Aldrich Corp., St. Louis, MO). Forty-eight hours after confluence, the retroviral-containing conditioned medium was collected, filtered through a 0.2-µm pore size membrane, and used for the transduction of ST-2 and MC3T3 cells.

Cell culture, transduction, and selection
ST-2 cells, cloned stromal cells isolated from bone marrow of BC8 mice (S. Harris, Kansas City, MO), and MC3T3-E1, osteoblastic cells derived from mouse calvariae, were grown in a humidified 5% CO2 incubator at 37 C in {alpha}-MEM (Invitrogen Life Technologies, Inc.) supplemented with 10% FBS. ST-2 and MC3T3 cells were transduced with pMG1 control vector or with pMG1-Tsg by replacing the culture medium with retroviral conditioned medium from 293 GPG packaging cells in the presence of {alpha}-MEM and 8 mg/ml Polybrene (Sigma-Aldrich Corp.) and were incubated for 24 h at 37 C. The culture medium was replaced with fresh {alpha}-MEM, and cells were grown, trypsinized, replated, and selected for fluorescence by flow cytometry. In detail, ST-2 and MC3T3 cells transduced with pMG1 or pMG1-Tsg were washed in PBS and trypsinized. Cells were filtered through a 70-µm pore size strainer, resuspended in PBS supplemented with 2% FBS at a concentration of 2 x 107 cells/ml, and refiltered through a 35-µm pore size strainer. Flow cytometric selection was performed using a FACScan/Calibur (BD Biosciences, San Jose, CA) with a 488-nm excitation wavelength generated by a 15-megawatt argon ion laser. Emission was detected by a 500-nm long-pass filter for GFP detection (20).

Cytochemical assays, alkaline phosphatase activity, cAMP levels, and cell viability
To analyze the phenotypic impact of Tsg, untransduced ST-2 or MC3T3 cells (wild type) and cells transduced with pMG1 or pMG1-Tsg were plated at a density of 104 cells/cm2 and cultured in {alpha}-MEM supplemented with 10% FBS until reaching confluence (2–3 d). At confluence (experimental d 0), ST-2 or MC3T3 cells were transferred to {alpha}-MEM containing 10% FBS, 100 µg/ml ascorbic acid, and 5 mM ß-glycerophosphate (Sigma-Aldrich Corp.) and cultured for a period of up to 3 or 4 wk in the presence or absence of recombinant human BMP-2 (Wyeth Research, Collegeville, PA) as indicated in the text and legends. Cells were refed with fresh medium containing control or test solutions every 3–4 d. To determine mineralized nodule formation, ST-2 or MC3T3 cells were fixed with 3.7% formaldehyde and stained with 2% Alizarin Red (Sigma-Aldrich Corp.) (21). Alkaline phosphatase activity (APA) was determined in 0.5% Triton X-100 cell extracts by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol and was measured by spectroscopy at 410 nm after 15–30 min of incubation at 37 C according to the manufacturer’s instructions (Sigma-Aldrich Corp.). Data are expressed as picomoles of p-nitrophenol released per minute per microgram of protein. The total protein content was determined in cell extracts by the DC protein assay in accordance with the manufacturer’s instructions (Bio-Rad Laboratories, Richmond, CA). To quantify cAMP levels, cells were serum-deprived for 24 h and treated with 500 µM isobutylmethylxanthine (Sigma-Aldrich Corp.) for 10 min before the addition of recombinant rat PTH-(1–34) (Bachem, King of Prussia, PA) at 100 nM for 2 min. Cells were washed with cold PBS and extracted in 0.05 M HCl (22). An aliquot of the extract was used to determine cAMP levels with a specific RIA kit, according to the manufacturer’s instructions (Biomedical Technologies, Stoughton, MA). Total protein content was determined by the DC protein assay. Data are expressed as picomoles of cAMP per milligram of cellular protein. To estimate the number of viable cells, mitochondrial dehydrogenase activity was measured using the Cell Titer 96 Aqueous One cell proliferation assay in accordance with the manufacturer’s instructions (Promega Corp.). Metabolically active cells were estimated by their ability to reduce the tetrazolium compound 3-(4,5-dimethyl-thiazol-2-yl)-5(-3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt to a formazan product, which was quantified at an absorbance of 490 nm. Data are expressed in arbitrary units of absorbance at 490 nm.

Northern blot analysis
Total cellular RNA was isolated using the RNeasy kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). Equal amounts of RNA were loaded on a formaldehyde agarose gel after denaturation. The RNA was blotted onto GeneScreen Plus charged nylon (PerkinElmer, Norwalk, CT). A 0.7-kb mouse Tsg cDNA, a 1.6-kb rat type I collagen cDNA (B. Kream, Farmington, CT), a 0.45-kb mouse osteocalcin, a 0.6-kb BMP-1, and a 0.7-kb 18S ribosomal RNA cDNAs (all three from American Type Culture Collection) were labeled with [{alpha}-32P]deoxy-CTP using the Ready-To-Go DNA labeling beads (-dCTP) Kit (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridizations and washes were carried out as described previously (23). The bound radioactive material was visualized by autoradiography on Kodak X-AR5 film, employing Cronex Lightning Plus or Biomax MS (both from Eastman Kodak Co., Rochester, NY) intensifying screens. Northern analyses are representative of three samples. Relative hybridization levels were determined by densitometry.

RT-PCR
For RT-PCR, 2 µg deoxyribonuclease I-treated total RNA were reverse transcribed with murine Moloney leukemia virus reverse transcriptase (Invitrogen Life Technologies, Inc.) in the presence of 20 µM random primers. Four microliters of RT reaction were amplified. The number of PCR amplification cycles used varied for each of the genes, so that they were quantitated in the exponential phase of the amplification. Conditions were as follows: for Tsg-FLAG, 28 cycles of PCR at 58 C annealing temperature, 5' primer (5'-AACCCTCGGAATTACAGC-3'), 3' primer (5'-AAGACGGCAATATGGTGG-3'); for endogenous Tsg, 24 cycles at 60 C annealing temperature, 5' primer (5'-GAAGAACCATGAAGTCACAC-3'), 3' primer (5'-AGGGGCAGTTCCCTTTCTC-3'); for chordin, 28 cycles at 53 C annealing temperature, 5' primer (5'-TTGTGACATCCACTCACC-3'), 3' primer (5'-CGAGCTGTTCCTGAATGT-3'); for chordin-like, 28 cycles at 56 C annealing temperature, 5' primer (5'-CCTGCCTTTGGTGAATGAG-3', 3' primer (5'-GGAGATAGAGGTTAGATAGTA-3'); for tolloid, tolloid-like-1 and tolloid-like-2, 35 cycles at 55 C annealing temperature, 5' primers (5'-TTGCACAAGCAAGAAAGC-3', 5'-TGGACAACAGAATCCACC-3', and 5'-CCAAACGATACAGCCTCTAACG-3', respectively), 3' primers (5'-GAAAGGTCAGTCCAACATGG-3', 5'-CGTCATTAGCCACTGTGC-3', and 5'-TCATGCCAGAACCCAACC-3', respectively); and for 18S, 20 cycles at 58 C annealing temperature, 5' primer (5'-ATGTCTAAGTACGCACGG-3') 3' primer (5'-AGCGACCAAAGGAACCATA-3'). PCRs were carried out in the presence of [{alpha}-32P]deoxy-CTP and 2.5 U Taq polymerase to yield products of a predicted size of 676 bp for Tsg-FLAG, 190 bp for endogenous Tsg, 730 bp for chordin, 750 bp for chordin-like, 504 bp for tolloid, 419 bp for tolloid-like-1, 440 bp for tolloid-like-2, and 76 bp for 18S. Products of PCRs were resolved by electrophoresis on a 6% polyacrylamide gel and exposed to Kodak XAR5 film overnight. RT-PCRs are representative of three samples. Relative levels of transcript expression were determined by densitometry.

Western immunoblot analysis
To detect Tsg-FLAG peptide, culture medium from ST-2 and MC3T3 cells transduced with pMG1 or pMG1-Tsg was precipitated with 5% trichloroacetic acid and fractionated by PAGE in 15% acrylamide gels under reducing conditions. Proteins were transferred to Immobilon P membranes (Millipore Corp., Bedford, MA), blocked with 2% BSA in PBS, and exposed overnight to 5 µg/ml of the mouse monoclonal antibody FLAG-M2 raised to FLAG fusion proteins (Sigma-Aldrich Corp.). Blots were exposed to antimouse IgG conjugated to horseradish peroxidase (1:10,000; Sigma-Aldrich Corp.) and developed with a chemiluminescence detection reagent (PerkinElmer). Tsg-FLAG was identified by migration at the expected Mr of 26 kDa (24).

To determine the level of phosphorylation of signaling mothers against decapentaplegic (Smad)-1/5/8, and Smad-2 and of the MAPKs p44/42, ERK1/2, c-Jun N-terminal kinases (JNK)-1 and -2/3, and p38, the cell layer of ST-2 cells transduced with pMG1 or pMG1-Tsg was washed with cold PBS and extracted in TNE lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 1 mM EDTA] in the presence of protease and phosphatase inhibitors as previously described (25). The protein concentration was determined by DC protein assay, and 70 µg total cellular protein were fractionated by PAGE in 12% acrylamide gels under nonreducing conditions and transferred to Immobilon P membranes. For Smad-1/5/8, the membranes were blocked with 2% BSA in PBS and exposed to a rabbit polyclonal antibody, which recognizes Smad-1, -5, and -8 phosphorylated at the last two serine residues (C. H. Heldin, Uppsala, Sweden) or exposed to a rabbit polyclonal antibody to unphosphorylated Smad-5 (Cell Signaling Technologies, Beverly, MA) at a 1:1000 dilution. For Smad-2, the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/0.1% Tween (pH 7.6) and exposed to rabbit polyclonal antibodies to either phosphorylated Smad-2 (Ser465/467) or to unphosphorylated Smad-2 (Cell Signaling Technology) at a 1:1,000 dilution (26). To estimate MAPK activation, membranes were blocked with 2% BSA in PBS and exposed to rabbit polyclonal antibodies to phosphorylated p44 ERK1 (Thr183/185) and p42 ERK2 (Thr202/204), JNK-1 and -2/3 (Thr183/Tyr185), or p38 (Thr180/Tyr182) or to rabbit polyclonal antibodies to corresponding unphosphorylated proteins (all at 1:1,000 dilution; all from Cell Signaling Technology). Blots for Smads and MAPKs were exposed to antirabbit IgG conjugated to horseradish peroxidase (1:10,000; Sigma-Aldrich Corp.) and developed with a chemiluminescence detection reagent (PerkinElmer).

Transient transfections
To determine changes in BMP-2 signaling, a construct containing 12 copies of a Smad-1 consensus sequence linked to the osteocalcin basal promoter and a luciferase reporter gene (12xSBE-OC-pGL3; M. Zhao, San Antonio, TX) was tested in transient transfection experiments (27). To determine changes in TGF-ß signaling, a construct containing four copies of the Smad-3 consensus sequence, a minimal simian virus 40 promoter, and a luciferase reporter gene (pSBE4-luc; R. Derynk, San Francisco, CA) was used (28). To determine changes in Wnt/ß-catenin signaling, a construct containing 16 copies of the lymphoid enhancer-binding factor-1/T cell transcription factor 4 recognition sequence, cloned upstream of a minimal thymidine kinase promoter and a luciferase reporter gene (16xTCF-luc) was tested in transient cotransfections in the presence of a Wnt3/pUSE expression construct (both from J. Billiard, Wyeth Research) or pUSE vector (29). ST-2 cells were cultured to 70% confluence and transiently transfected with the indicated constructs using FuGene6 (3 µl FuGene/2 µg DNA) according to the manufacturer’s instructions (Roche, Indianapolis, IN). Cotransfections with a ß-galactosidase expression construct (BD Clontech, San Jose, CA) were used to control transfection efficiency. For 12xSBE-OC-pGL3 and pSBE4-luc activities, cells were exposed to the FuGene-DNA mix for 16 h and transferred to serum containing medium for 8 h. Cells were then cultured in medium supplemented with 0.1% FBS for 20 h, treated with BMP-2 at 0.03–3.3 nM or with TGF-ß at 40–200 pM for 24 h, and harvested. For 16xTCF-luc activity, cells were exposed to the FuGene-DNA mix for 16 h, transferred to serum-containing medium for 24 h, and harvested. Luciferase and ß-galactosidase activities were measured using an Optocomp luminometer (MGM Instruments, Hamden, CT) as previously described (25). Luciferase activity was corrected for ß-galactosidase activity to control transfection efficiency.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical significance was determined by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whether Tsg acts as a BMP agonist or as an antagonist is determined by the relative levels of expression of Tsg, chordin, and the metalloproteases BMP-1/tolloid and related genes in the cellular environment. Consequently, their expression patterns were analyzed in ST-2 cells under basal conditions and after treatment with BMP-2, which is known to regulate the synthesis of BMP antagonists (1). Tsg and the metalloprotease BMP-1 were expressed in ST-2 control cells, and BMP-2 decreased Tsg mRNA levels by 40%, but did not modify BMP-1 expression (Fig. 1AGo). In contrast, tolloid, tolloid like-1, and tolloid like-2 transcripts were not detected by Northern blot or RT-PCR analyses in ST-2 cells (data not shown). Chordin and chordin-like mRNA were not detected by Northern blot analysis, but their transcripts were detected by RT-PCR in ST-2 cells. BMP-2 caused a modest increase in chordin expression at 24 and 48 h of treatment, whereas it up-regulated chordin-like mRNA levels with a maximal effect at 6 h by 3.5 ± 0.8-fold (mean ± SEM; n = 3; P < 0.05; Fig. 1BGo). The pattern of expression and the regulation by BMP-2 of this group of genes was analogous in MC3T3 cells (data not shown).



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  		FIG. 1. Expression of BMP-1, Tsg, chordin, and chordin-like mRNA in ST-2 stromal cells in the absence or presence of BMP-2. ST-2 cells were cultured to confluence, serum deprived, and treated with control medium (–) or with BMP-2 at 3.3 nM (+) for 2–48 h. A, Total RNA was subjected to Northern blot analysis and hybridized with {alpha}-32P-labeled mouse BMP-1, Tsg, and 18S cDNA. B, Two micrograms of total RNA were reverse transcribed, and 0.2 µg was amplified by PCR in the presence of [{alpha}-32P]deoxy-CTP and specific primers to detect chordin, chordin-like, and 18S genes. Northern blots and RT-PCRs are representative of three cultures.

 
To examine the impact of Tsg on ST-2 cell phenotype, ST-2 cells were transduced with the retroviral expression construct pMG1-Tsg and compared with cells transduced with pMG1 vector. Tsg overexpression was confirmed by RT-PCR with construct-specific primers and documented the presence of Tsg-FLAG transcripts only in Tsg-overexpressing cells (Fig. 2AGo). To determine the levels of Tsg expression in control and overexpressing cells, primers annealing to Tsg-coding sequences were used. The levels of Tsg mRNA were 12.0 ± 1-fold (n = 3; P < 0.01) higher in overexpressing cells compared with control pMG1 cells (Fig. 2BGo). Western immunoblot analysis confirmed the synthesis of the FLAG epitope-tagged Tsg peptide, which migrated on the gel with a Mr of 26 kDa, the predicted Mr of the glycosylated Tsg protein (Fig. 2CGo) (24). pMG1-Tsg, control pMG1, and wild-type ST-2 cells were cultured under osteoblastic differentiating conditions in the presence of 5 mM ß-glycerophosphate and 100 µg/ml ascorbic acid in the presence or absence of BMP-2 at 1 nM for 4 wk. The cellular phenotypes of wild-type cells and cells transduced with pMG1 vector were not different (not shown), and Tsg overexpression opposed osteoblastic cell maturation and the effects of BMP-2. Control vector cells expressed modest levels of type I collagen transcripts that declined as the cells progressed in culture, but in the presence of BMP-2, type I collagen mRNA levels were sustained. Tsg overexpression decreased type I collagen expression in the first 2 wk of culture (Fig. 3AGo). Osteocalcin mRNA levels and APA increased as ST-2 cells matured in culture, and BMP-2 increased APA levels. Tsg overexpression decreased osteocalcin mRNA levels and APA, and opposed the effect of BMP during the first 3 wk of culture (Fig. 3Go). The effect of BMP-2 on APA was dose dependent and was observed at doses of 0.03–3.3 nM. Tsg decreased the effect of BMP-2 at 0.03–1 nM by approximately 70–80%, whereas it reduced the activity of BMP-2 at 3.3 nM by about 30% (Fig. 4AGo). To ensure that ST-2 cells expressed the osteoblastic phenotype, their ability to respond to PTH with a change in cAMP levels was tested (30). In accordance with changes in osteocalcin mRNA expression, the effect of PTH at 100 nM on cellular cAMP levels was more pronounced after 2 wk of culture, and the PTH effect was reduced by Tsg overexpression (Fig. 4BGo). Tsg overexpression also precluded the formation of mineralized nodules induced by BMP-2 (Fig. 4CGo). The effects of Tsg were not dependent on cell replication, because its overexpression did not reduce the number of viable ST-2 cells over a 4-wk period (data not shown).



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  		FIG. 2. Overexpression of Tsg mRNA and protein in transduced ST-2 stromal cells. ST-2 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence, and total RNA was extracted, or conditioned medium was obtained for 24 h in the absence of serum for the determination of Tsg-FLAG protein. A and B, Two micrograms of total RNA were reverse transcribed, and 0.2 µg was amplified by PCR in the presence of [{alpha}-32P]deoxy-CTP. Specific primers to Tsg-FLAG (A), Tsg-coding sequence (B), and 18S genes were used. C, Proteins were resolved by gel electrophoresis, transferred to an Immobilon P membrane, exposed to an antibody to mouse FLAG, and visualized by chemiluminescence. RT-PCRs and Western immunoblots are representative of three cultures.

 


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  		FIG. 3. Effect of Tsg overexpression on the differentiation of ST-2 stromal cells. ST-2 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence (0) or for up to 4 wk in the absence or presence of BMP-2 (1 nM). A, Type I collagen [{alpha}1(I)coll], osteocalcin (Oc), and 18S mRNA levels were determined at the indicated times. The experiment shown is representative of three cultures. B, APA was quantified in total cell extracts from pMG1 cells in the absence ({blacksquare}) or presence ({cjs2112}) of BMP-2 (1 nM) and from pMG1-Tsg cells in the absence ({square}) or presence ({cjs2110}) of BMP-2 (1 nM). APA is expressed as picomoles of p-nitrophenol per minute per microgram of total protein. Bars represent the mean ± SEM for six observations. +, P < 0.05, BMP-2-treated vs. untreated cells; *, P < 0.05, pMG1-Tsg vs. pMG1 cells.

 


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  		FIG. 4. Effect of Tsg overexpression on the differentiation of ST-2 stromal cells. A, ST-2 cells transduced with pMG1 or pMG1-Tsg were cultured for 1 wk in the absence or presence of BMP-2 at 0.03–3.3 nM. APA was quantified in total cell extracts from pMG1 cells ({blacksquare}) and from pMG1-Tsg cells ({square}) and is expressed as picomoles of p-nitrophenol per minute per microgram of total protein. Bars represent the mean ± SEM for four observations. +, P < 0.05, BMP-2-treated vs. untreated cells; *, P < 0.05, pMG1-Tsg vs. pMG1 cells. B, ST-2 cells transduced with pMG1 or pMG1-Tsg were cultured to confluence (0) and for 4 wk after confluence. At each time point, cells were serum deprived for 24 h, pretreated with isobutylmethylxanthine (500 µM) for 10 min, and stimulated with PTH (100 nM) for 2 min. cAMP levels for pMG1 cells ({blacksquare}) and pMG1-Tsg cells ({square}) were determined by RIA in cell lysates. Data are expressed as picomoles of cAMP per milligram of total protein. Bars represent the mean ± SEM for four observations. *, P < 0.05, pMG1-Tsg vs. pMG1 cells. C, ST-2 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured for 4 wk in the presence of BMP-2 (1 nM) and stained with Alizarin Red to detect mineralized nodules.

 
To elucidate the mechanism of Tsg action in ST-2 cells, we analyzed the effect of Tsg overexpression on downstream events of BMP-2 signaling. The signaling pathway used by BMPs is cell line dependent, and in differentiated osteoblasts, BMP-2 signals by activating Smad-1, -5, and -8, or the MAPKs, ERK1/2, JNK1–2/3, and p38 (31, 32, 33). In ST-2 cells, BMP-2 at 3.3 nM induced the phosphorylation of Smad-1, -5, and -8. The effect was maximal after 15 min, was sustained for 6 h, and was observed at BMP-2 concentrations of 0.3–3.3 nM (Fig. 5Go, A–C). In contrast, BMP-2 at 3.3 nM did not induce the phosphorylation of c-JNKs or p38 and caused a modest increase in ERK1/2 phosphorylation (Fig. 5DGo).



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  		FIG. 5. Effect of BMP-2 on the phosphorylation of Smad-1/5/8 and MAPKs, ERK1/2, JNK1–2/3, and p38, in ST-2 stromal cells. A–C, ST-2 cells were cultured to confluence, serum deprived overnight, and tested with BMP-2 at 3.3 nM for 15 min to 6 h (A and D) and with BMP-2 at 0.03–3.3 nM for 15 min (B; depicted as a graph in C). Total cell lysates were resolved by gel electrophoresis and transferred to Immobilon P membranes. The membranes were incubated with antibodies to phosphorylated Smad-1/5/8 or to unphosphorylated Smad-5. For MAPK phosphorylation, membranes were incubated with antibodies to phosphorylated ERK1/2, JNK1–2/3, or p38 or to the respective unphosphorylated proteins. Western immunoblots are representative of three cultures.

 
In accordance with the cellular phenotype observed, Tsg overexpression reduced the effect of BMP-2 at 1 nM on Smad-1/5/8 phosphorylation and on the transactivation of the Smad-1-responsive 12xSBE-Oc-pGL3 construct, so that the effects of 0.3 and 1 nM BMP-2 were about 50% lower in pMG1-Tsg cells compared with pMG1 control cells (Fig. 6Go). BMP-2 at 3.3 nM overcame the inhibitory effect of Tsg on Smad-1/5/8 phosphorylation and on the transactivation of the 12xSBE-OC-pGL3 construct. To determine whether the effect of Tsg was specific for BMP-2, we investigated Tsg actions on TGF-ß signaling. Tsg overexpression did not modify the TGF-ß-induced phosphorylation of Smad-2 or the transactivation of the TGF-ß-responsive construct pSBE4-luc in ST-2 cells (Fig. 7Go).



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  		FIG. 6. Effect of Tsg overexpression, in the absence or presence of BMP-2, on Smad-1/5/8 phosphorylation and transactivation of the 12xSBE-Oc-pGL3 construct in ST-2 stromal cells. A, ST-2 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence, switched to 0.1% FBS {alpha}-MEM for 20 h, and treated with BMP-2 (0.03–3.3 nM) for 15 min. Total cell lysates were resolved by gel electrophoresis and transferred to Immobilon P membranes. Membranes were incubated with an antibody to phosphorylated Smad-1/5/8 or to unphosphorylated Smad-5. B, ST-2 cells transduced with pMG1 or pMG1-Tsg were cultured to subconfluence and transiently transfected with 12xSBE-Oc-pGL3 or pGL3 plasmid (not shown) and a CMV/ß-galactosidase expression vector. After 16 h, cells were switched to 0.1% FBS {alpha}-MEM for 20 h and treated with BMP-2 (0.03–3.3 nM) for 24 h. The data shown represent luciferase activity/ß-galactosidase activity for pMG1 ({blacksquare}) or pMGI-Tsg ({square}) cells. Bars are the mean ± SEM for six to 12 observations. *, P < 0.05 vs. pMG1 control cells. The data shown are representative of two independent experiments.

 


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  		FIG. 7. Effect of Tsg overexpression, in the absence or presence of TGF-ß, on Smad-2 phosphorylation and transactivation of the pSBE4-luc construct in ST-2 stromal cells. A, ST-2 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence, switched to 0.1% FBS {alpha}-MEM for 20 h, and treated with TGF-ß at 100 or 200 pM for 15 and 30 min. Total cell lysates were resolved by gel electrophoresis and transferred to Immobilon P membranes. Membranes were incubated with an antibody to phosphorylated Smad-2 or to unphosphorylated Smad-2. B, ST-2 cells transduced with pMG1 or pMG1-Tsg were cultured to subconfluence and transiently transfected with pSBE4-luc and a CMV/ß-galactosidase expression vector. After 16 h, cells were switched to medium supplemented with 0.1% FBS {alpha}-MEM for 20 h and treated with TGF-ß (40–200 pM) for 24 h. The data shown represent luciferase activity/ß-galactosidase activity for pMG1 ({blacksquare}) or pMG1-Tsg ({square}) cells. Bars are the mean ± SEM for six to 12 observations. The data shown are representative of two independent experiments.

 
To explore other possible mechanisms involved in the effects of Tsg on stromal cell differentiation, we tested whether it modified the canonical Wnt/ß-catenin signaling pathway, which plays a critical role in osteoblastogenesis and acts in a coordinated fashion with BMP signaling (34). For this purpose, the Wnt/ß-catenin-responsive construct 16xTCF-luc was cotransfected with a Wnt-3 expression or a control vector. Tsg overexpression did not modify the basal activity or the stimulatory effect of Wnt-3 on the 16xTCF-luc construct, suggesting the absence of a direct effect of Tsg on Wnt/ß-catenin signaling (Fig. 8Go).



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  		FIG. 8. Effect of Tsg overexpression on transactivation of the Wnt/ß-catenin-dependent 16xTCF-luc construct in ST-2 stromal cells. ST-2 cells transduced with pMG1 or pMG1-Tsg were cultured to subconfluence and transiently transfected with 16xTCF-luc and a CMV/ß-galactosidase expression vector. Where indicated, Wnt3 expression constructs were added to the transfection mix. After 16 h, cells were switched to serum-containing medium for 24 h and harvested. The data shown represent luciferase activity/ß-galactosidase activity for pMG1 ({blacksquare}) or pMG1-Tsg ({square}) cells. Bars are the mean ± SEM for six observations. The data shown are representative of three independent experiments.

 
The results obtained in ST-2 cells were confirmed in MC3T3 cells. RT-PCR demonstrated that Tsg mRNA levels were 2.5 ± 0.04-fold (n = 3; P < 0.05) higher in MC3T3 pMG1-Tsg cells than in vector control cells, and Western immunoblot analysis showed synthesis of Tsg-FLAG peptide in transduced cells (Fig. 9Go). Control pMG1 MC3T3 cell cultures mineralized after 3 wk and expressed osteocalcin transcripts, whereas Tsg-overexpressing cells did not. BMP-2 at 1 nM up-regulated osteocalcin expression and enhanced the formation of mineralized nodules, and Tsg overexpression opposed the BMP effect (Fig. 10Go).



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  		FIG. 9. Overexpression of Tsg mRNA and protein in transduced MC3T3 cells. MC3T3 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence, and total RNA was extracted, or conditioned medium was obtained for 24 h in the absence of serum for the determination of Tsg-FLAG protein. A and B, Two micrograms of total RNA were reverse transcribed, and 0.2 µg was amplified by PCR in the presence of [{alpha}-32P]deoxy-CTP. Specific primers to Tsg-FLAG (A), Tsg-coding sequence (B), and 18S genes were used. C, Proteins were resolved by gel electrophoresis, transferred to an Immobilon P membrane, exposed to an antibody to mouse FLAG, and visualized by chemiluminescence. RT-PCRs and Western immunoblots are representative of two cultures.

 


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  		FIG. 10. Effect of Tsg overexpression on the differentiation of MC3T3 cells. MC3T3 cells transduced with pMG1 (–) or pMG1-Tsg (+) were cultured to confluence (0) or for up to 3 wk after confluence in the absence or presence of BMP-2 (1 nM). A, Osteocalcin (Oc) and 18S mRNA levels were determined at the indicated times. B, MC3T3 cells transduced with pMGI or pMGI-Tsg were cultured for 3 wk and stained with Alizarin Red to detect mineralized nodules. The experiment shown is representative of three cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that overexpression of Tsg in ST-2 stromal and MC3T3 cells causes an inhibition of BMP-2 signaling and prevents the differentiation of cells toward functional osteoblasts. Wild-type ST-2 and MC3T3 cells expressed Tsg and BMP-1, but displayed low levels of chordin and chordin-like/ventroptin transcripts, which were detectable by RT-PCR, but not by Northern blot analysis. In contrast to the stimulatory effects of BMP-2 on the expression of other BMP antagonists, such as noggin and gremlin, BMP-2 inhibited the levels of Tsg mRNA in the two cell lines studied (1).

Studies conducted in Drosophila, Zebrafish, and Xenopus indicate that by promoting the formation of a stable ternary BMP/chordin/Tsg complex, Tsg can act as a BMP antagonist and prevent BMP binding to its receptor (4, 5, 6). Tsg and chordin, once associated, are more efficient than either component alone in repressing BMP signaling. However, Tsg can also have BMP agonist activity by promoting the cleavage of chordin by the metalloprotease BMP-1 (7). A BMP antagonist activity prevailed in ST-2 and MC3T3 cells overexpressing Tsg, because they displayed a decrease in BMP activity and, accordingly, an inhibition of osteoblastogenesis. The lack of a BMP-2 agonist effect could be explained by the high Tsg/chordin ratio present in our model, or, alternatively, by a lack of BMP-1 metalloprotease activity under the experimental conditions used (6, 35). Although transient up-regulation of chordin expression was reported in an osteogenic model of primary bone marrow stromal cells, chordin transcripts were low and up-regulated by BMP-2 only modestly (36). BMP-2 caused a significant stimulation of the expression of chordin-like/ventroptin, a chordin-related gene that also binds BMP-2. However, chordin-like transcripts were detected only by RT-PCR, not by Northern blot analysis, suggesting a limited level of expression.

Studies in tsg null mice have confirmed the dual action of Tsg; it acts as a BMP antagonist in thymocytes, where its expression is tightly coordinated with stages of T lymphocyte differentiation, and as a BMP agonist in endochondral bone formation and the development of forebrain structures (16, 17, 18, 24). The BMP agonist activity in endochondral bone is in contrast with the antagonist effect observed in ST-2 stromal or MC3T3 cells and could be dependent on the different levels of expression of Tsg, chordin, and BMP-1 in the models examined. It is important to note that chordin and BMP-1 are expressed at high levels in growth plate chondrocytes and in the articular perichondrium, possibly leading to the formation of chordin cleavage products and explaining the BMP agonist role of Tsg in this tissue.

To analyze the mechanism of action of Tsg, we tested its effect on BMP-2 signaling. In contrast to other stromal cell lines, in ST-2 cells, BMP-2 induced phosphorylation of BMP-2-dependent Smad-1/5/8, but did not activate MAPK signaling (37). The reason for this difference is not known, but it may be related to the degree of cell maturation or to the presence or absence of preformed heteromeric complexes of type I and II BMP receptors on the cell surface before BMP binding. BMP binding to preformed BMP type I and type II heteromeric complexes activates Smad signaling, whereas BMP binding to a homomeric receptor leading to the secondary formation of a heteromeric complex activates MAPK signaling (38, 39). Tsg overexpression decreased BMP-induced Smad-1/5/8 phosphorylation and the activity of a reporter construct containing 12 copies of a Smad recognition sequence directing luciferase expression, confirming the inhibitory effect of Tsg on BMP action.

Tsg is a member of a multigene superfamily of secreted CR peptides, which also includes IGF-binding proteins and members of the CCN family, cysteine-rich protein-61, connective tissue growth factor (CTGF) nephroblastoma overexpressed gene, and Wnt-inducible secreted protein-1, -2, and -3. These proteins have a degree of structural homology and one or more CR domains that interact with members of the TGF-ß family of peptides (40). CCN peptides also bind to extracellular matrix proteins and matrix metalloproteases, and CTGF enhances TGF-ß activity and inhibits BMP-2 and Wnt/ß-catenin signaling (41, 42). Although Tsg shares structural properties with secreted CR peptides, it did not alter TGF-ß and Wnt canonical signaling pathways, suggesting that its action is BMP specific and that it displays distinct activities from members of the CCN family.

In conclusion, this study demonstrates that Tsg, when overexpressed, acts as a specific BMP antagonist in ST-2 stromal and MC3T3 cells and, accordingly, inhibits their differentiation toward a mature osteoblastic phenotype.


   Acknowledgments
 
We thank Dr. E. De Robertis for Tsg cDNA; Dr. B. Kream for type I collagen cDNA; Dr. C. H. Heldin for phospho-Smad-1/5/8 antibody; Dr. R. Derynk for TGFß/Smad responsive construct; Dr. M. Zhang for 12xSBE-Oc-pGL3 construct; Wyeth Research for BMP-2, Wnt 3 expression, and 16xTCF-1uc construct; and Ms. Nancy Wallach for helpful secretarial assistance.


   Footnotes
 
This work was supported by Grants AR-21707 and P30-AR-46026 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases and a fellowship award from the Arthritis Foundation.

First Published Online May 26, 2005

Abbreviations: APA, Alkaline phosphatase activity; BMP, bone morphogenetic protein; CCN, cysteine-rich protein 61, connective tissue growth factor, nephroblastoma; CMV, cytomegalovirus; CR, cysteine-rich; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; Mr, molecular mass; Smad, signaling mother against decapentaplegic; Tsg, Twisted gastrulation.

Received January 14, 2005.

Accepted for publication May 10, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
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