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The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2

Andrea Hoffmann^1,*, Stefan Czichos^1,2,*, Christian Kaps¹, Dietmar Bächner², Hubert Mayer¹, Yoram Zilberman³, Gadi Turgeman³, Gadi Pelled³, Gerhard Gross^1, and Dan Gazit³

¹ Osteo-Angiogenesis Group, Gesellschaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
² Institute of Cellbiochemistry and Clinical Neurobiology, University Hospital Eppendorf, 22529 Hamburg, Germany
³ Skeletal Biotech. Laboratory, Hebrew University, Jerusalem, Israel

View larger version (39K): [in a new window]   Fig. 1. FGFR3 mediates chondrocytic differentiation in mesenchymal stem cell line C3H10T1/2. (A) RT-PCR analyses of BMP2-dependent expression of FGF- and PTH/PTHrP-receptors in mesenchymal stem cell line C3H10T1/2 incubated for various times in the presence or absence of recombinantly expressed BMP2. (B) Cycloheximide pretreatment of C3H10T1/2 cells does not prevent BMP-induction of the FGFR3 gene. Cells were mock-treated (Control) or treated with BMP2 (50 ng/ml) for various times. Preincubation with cycloheximide (CHX) was for 30 minutes (50 µg/ml). RT-PCR analyses of FGFR3 mRNA levels were as described in Materials and Methods. (C) Western immunoblotting for the detection of BMP2-dependent FGFR3 and FGFR2 expression in cellular extracts of C3H10T1/2 lines. Immunoblotting was performed with polyclonal antibodies SC-123 (FGFR3; Santa Cruz, left) and SC-122 (FGFR2; Santa Cruz, right). (D) (Left) Western immunoblotting to document the forced expression of FGFR3 in mesenchymal progenitors C3H10T1/2. (Right) The recombinant expression of FGFR3 in C3H10T1/2 leads to enhanced levels of activated MAP-kinases pERK-1 and pERK-2 during cultivation (New England Biolabs MAPK Detection Kit 9100). Cell lysates were prepared (at confluence/0) and 4 days post-confluence. (E) The forced expression of FGFR3 in parental C3H10T1/2 cells is sufficient for the induction of the chondrogenic lineage. (Bottom) RT-PCR analyses of expression levels of chondrogenic and ostesteogenic markers were performed as described in Materials and Methods. Collagen 1a1 and osteocalcin are markers predominantly for osteogenic and collagen 2a1 for chondrogenic differentiation, respectively. The PTH/PTHrP-receptor is a marker for early osteogenic and late chondrogenic development. HPRT was used to standardize PCR conditions. (Top) Osteoblast-like cells stably expressing BMP2 or FGFR3 were identified by alkaline phosphatase staining (6 days post-confluence). (Middle) Chondrocyte-like cells secreting proteoglycans were histologically stained with Alcian Blue (12 days post-confluence). Cellular confluence has been arbitrarily termed day 0.

View larger version (32K): [in a new window]   Fig. 5. FGFR3 and Brachyury are involved in an autoregulatory loop. (A) RT-PCR analyses of FGFR3 and Brachyury mRNA levels in mesenchymal progenitors C3H10T1/2 expressing recombinant FGFR3 (C3H10T1/2-FGFR3) or Brachyury (C3H10T1/2-Brachyury). (B) Smad1-signaling is not sufficient for Brachyury and FGFR3 but is sufficient for osteocalcin expression, as shown by RT-PCR analyses of FGFR3 and Brachyury mRNA levels in mesenchymal progenitors C3H10T1/2 expressing the biologically active Smad1-MH2 domain (C3H10T1/2-Smad1-MH2).

View larger version (54K): [in a new window]   Fig. 2. The T-box transcription factor Brachyury mediates chondrogenic differentiation in MSCs in vitro and ectopically in vivo. (A) Schematic representation of Brachyury according to Kispert et al. (Kispert et al., 1995). Western immunoblotting of recombinant HA-tagged Brachyury (aa 1-436) in cellular extracts of C3H10T1/2 (C3H10T1/2-Brachyury) with HA-antibody SC-805 (Santa Cruz) Brachyury is constitutively expressed under the control of the murine PGK-promoter. Expression of Brachyury is indicated (triangle). The molecular mass marker (M) shown is ovalbumin (43 kDa). NLS, nuclear localization signal. (B) Histological characterization of C3H10T1/2-Brachyury cells in culture. Top: at day 4 post-confluency cells develop alkaline-phosphatase-positive osteoblast-like cells. Below right: Alcian Blue histology of C3H10T1/2 cells stably expressing Brachyury show secreted proteoglycans and efficient differentiation into the chondrogenic lineage. Below left: Collagen-immunohistochemistry of C3H10T1/2-Brachyury cells in culture 7 days post-confluency was performed as described in Materials and Methods. Monoclonal anti-collagen 2 antibodies and anti-collagen X antibodies were used for collagen histology. The red precipitate of AEC (aminoethylcarbazole) indicates a positive signal. Cells secrete chondrocyte-specific collagen 2 but not collagen X, which is specific for hypertrophic chondrocytes. (C) RT-PCR analysis of the expression at various time intervals of chondrogenic and osteogenic marker genes in C3H10T1/2 cells recombinantly expressing Brachyury. (D) The forced expression of the T-box factor Brachyury in C3H10T1/2 cells leads to differentiation into chondrocytes and cartilage development at murine ectopic sites after intramuscular transplantation. Transplantations were performed as outlined in Materials and Methods. C3H10T1/2-Brachyury cells differentiate into chondrocytes after ectopic implantation at murine intramuscular sites (10 days and 20 days after implantation; HE, Alcian Blue and Safranin O staining). C3H10T1/2-Brachyury cells develop chondrocytes (20 days after implantation). Day 0, 1st day of cellular confluency.

View larger version (59K): [in a new window]   Fig. 3. Dominant-negative Brachyury (dnBrachyury; T-box domain) blocks BMP2-mediated chondrogenic development in C3H10T1/2 MSCs in vitro and ectopically in vivo. (A) Brachyury's T-box domain interferes with the transcriptional activity of full-length Brachyury. For the assessment of Brachyury's transcriptional activity, the Brachyury Binding Element (BBE) (see Materials and Methods) was placed twice in front of an HSV TK minimal promoter fused to a choloramphenicol-acetyltransferase reporter gene. These constructs were transfected into HEK293T cells with a full-length Brachyury expression construct and increasing amounts of an expression construct encoding the T-box domain (dnBrachyury). The total amount of Brachyury expression constructs was kept constant or filled up with empty vector (control). The ratios of full-length BrachyurydnBrachyury were increasing from 1:1 to 1:2 and 1:3, as indicated. Values represent -fold activation in relation to an empty vector and are the average of at least three experiments done in triplicate. (B) Expression of dnBrachyury (T-box domain) in C3H10T1/2-BMP2 during cultivation (day 0; cellular confluence) The T-box domain (aa 1-229) was subcloned and HA-tagged in expression vector pMT7T3 and constitutively expressed in C3H10T1/2-BMP2 cells. Western immunoblotting was with HA-antibody SC-805 (Santa Cruz). The recombinantly expressed T-box domain (dnBrachyury) is indicated (triangle). The marker was carbonic anhydrase (29 kDa). (C) RT-PCR experiments with osteo/chondrogenic marker genes show that T-box domain (dnBrachyury) expression in C3H10T1/2-BMP2 cells interferes with the BMP2-dependent FGFR2 but not FGFR3 expression. (D) dnBrachyury (T-box) interferes with BMP2-mediated FGFR2 expression, as analyzed by western immunoblotting with antiFGFR3 and antiFGFR2 antibodies, as described Fig. 1. (E) The forced expression of the dominant-negative acting T-box domain in C3H10T1/2-BMP2 cells interferes with BMP-2 mediated osteo/chondrogenic development. dnBrachyury interferes with expression of alkaline phosphatase-positive and with Alcian Blue-positive matrix synthesis of chondrocyte-like cells at days 6 and 11 post-confluence, respectively. dnBrachyury also interferes with BMP2-dependent osteo/chondrogenic development at murine ectopic sites after intramuscular transplantation (20 days after implantation; HE-staining) resulting in connective tissue formation at intramuscular sites, only.

View larger version (20K): [in a new window]   Fig. 7. Model of BMP2-dependent osteo/chondrogenic development in mesenchymal stem cells. BMP2 predominantly determines osteo/chondrogenic development via BMPR-IA, while BMPR-IB only exerts maturing functions on osteoblast development in C3H10T1/2 cells (in preparation). Therefore, Smad1 is a signaling mediator of osteogenesis rather than of chondrogenesis (Ju et al., 2000). Osteogenesis is controlled by R-Smad-signaling during the entire osteoblast-developmental sequence, also by recruiting CBFA1 into an heteromeric complex (Hanai et al., 1999). BMP2-dependent determination of chondrogenesis in mesenchymal progenitors C3H10T1/2 seems to involve the immediate upregulation of FGFR3 by an R-Smad-independent mechanism. After this triggering event, chondrogenesis is then predominantly controlled by BMP-independent mechanisms. Therefore, FGF-mediated signaling leads to MAPK-activation and upregulation of T-box factor (Brachyury) expression that is sufficient for de novo chondrogenesis in mesenchymal progenitors C3H10T1/2. Brachyury is able to maintain FGFR3 and FGFR2 expression in an autoregulatory loop. FGFR-mediated MAPK-signaling also upregulates mRNA levels of transcription factor Sox9, which is necessary for chondrogenic development (Murakami et al., 2000).

View larger version (55K): [in a new window]   Fig. 4. Dominant-negative FGFR3 (dnFGFR3) interferes with osteo/chondrogenic development, with FGFR2 and with Brachyury expression in C3H10T1/2-BMP2. (A) Forced expression of dnFGFR3 in C3H10T1/2-BMP2 cells interferes with BMP-2-mediated development of alkaline phosphatase-positive and Alcian Blue-positive chondrocyte-like cells, respectively. (B) dnFGFR3 interferes with BMP2-dependent FGFR2 and Brachyury but not with FGFR3 expression in C3H10T1/2-BMP2 cells. FGFR3, dnFGFR3, FGFR2 and Brachyury mRNA levels in mesenchymal progenitors C3H10T1/2-BMP2 and C3H10T1/2-BMP2 with forced expression of dnFGFR3 were analysed by RT-PCR. Vector-borne transcripts encoding dnFGFR3 were analyzed by primer pairs for vector- and FGFR3-specific sequences.

View larger version (95K): [in a new window]   Fig. 6. Brachyury is expressed at skeletal sites during late murine embryonic development (18.5 d.p.c.). Comparative expression analysis of murine Brachyury (Bra), Collagen 1a1 (Col 1a1) and Collagen 2a1 (Col 2a1) in embryonic development 18.5 d.p.c. (A) Intervertebral disc development. Consecutive sagittal (a-g) and transverse (h-j) sections of 18.5 d.p.c. mouse embryos were hybridized with riboprobes as indicated. Expression of Brachyury is enhanced in the nucleus pulposus (np) (a,d), Col 1a1 in the outer annulus (oa) (arrowheads in b,e), and Col 2a1 in the cartilage primordium (cp) of the vertebrae (c,f). No signals were obtained using RNase pre-incubated sections (g). With transverse sections at the level of the upper lumbar vertebra, expression of Brachyury is also detectable in distinct cells of the neural arch (h), whereas Col 1a1 is expressed in the outer annulus (i) and Col 2a1 in the cartilage primordium (j). (B) Limb bud development. Consecutive transverse sections of an 18.5 d.p.c. mouse hind limb at the level of the metatarsals, hybridized with riboprobes as indicated. Expression of Brachyury is evident in distinct chondrogenic cells of the forming metatarsal bones (a), better visible at higher magnification (b,c). In contrast Col 1a1 is expressed in the outer periosteal layer (d-f) and Col 2a1 expression is enhanced in differentiating chondrocytes (g-i). As was evident for the intervertebral disc formation, expression of Brachyury is only seen in chondrocyte-like cells that do not express Col 2a1. Bar, 100 µm.