Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation
Sylviane Spinella-Jaegle1,*,
Georges Rawadi1,*,
Shinji Kawai1,
Sylvie Gallea1,
Chi Faucheu1,
Patrick Mollat1,
Brigitte Courtois1,
Brigitte Bergaud1,
Valérie Ramez1,
Anne Marie Blanchet1,
Guillaume Adelmant2,
Roland Baron3 and
Sergio Roman-Roman1,
1 Bone Diseases Group, Department of Biotechnology, Hoechst-Marion-Roussel, 111 route de Noisy, 93230 Romainville, France
2 Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115, USA
3 Departments of Cell Biology and Orthopaedics, Yale University School of Medicine, New Haven, CT 06510, USA
* Both authors equally contributed to this work
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Fig. 1. Shh enhances BMP-2-induced alkaline phosphatase activity. (A) MC3T3-E1, (B) C2C12, (C) C3H10T1/2 and (D) ST2 cells were stimulated with increasing concentrations of BMP-2 (15-500 ng/ml) in the presence or absence of Shh (10 µg/ml). The inset in C shows ALP activity obtained with C3H10T1/2 cells stimulated with Shh (10 µg/ml) in the absence of BMP-2. Cells were cultivated for 5 days and ALP activity was measured in cell lysates and normalized to protein contents. The results (mean±s.d.) are representative of three independent experiments performed in triplicate.
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Fig. 2. Shh increases the number of alkaline phosphatase-positive cells in response to BMP-2. C3H10T1/2 cells were stimulated with either BMP-2 (100 ng/ml) or BMP-2 and Shh (10 µ/ml). (A) After three days, cells were stained for plasma-membrane-associated ALP as indicated in Materials and Methods. (B) The percentage of ALP-positive cells was determined from six series of treatment. Results are expressed as mean±s.d. Statistically significant changes were detected using Student’s t-test (*P<0.001).
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Fig. 3. Overexpression of Shh induces osteocalcin and Osf2/Cbfa1 mRNA in C3H10T1/2 cells. C3H10T1/2 cells were transfected with a vector expressing N-Shh (pShh), as indicated in Materials and Methods. Transfected cells were cultured for 5 days, and total RNA was extracted. Control was performed by transfecting cells with empty vector. The mRNA expression level of alkaline phosphatase (ALP), Osf2/Cbfa1 and osteocalcin (OC) in both pShh transfected cells and control cells was determined by real-time TaqMan PCR. The expression level of tested markers was normalized on the basis of GAPDH expression. Results (mean±s.d.) are representative of two independent experiments, both performed in triplicate. Data are presented as the relative expression level of osteoblast markers in N-Shh expressing cells versus control cells.
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Fig. 4. Effect of Noggin overexpression on Shh activity. (A) Noggin blocks completely BMP-2 activation. C3H10T1/2 cells were transfected with either empty vector or Noggin expressing vector (pNog), as indicated in Materials and Methods. Transfected cells were cultured in the absence or presence of BMP-2 (100 ng/ml) for 5 days, then ALP activity was measured in cell lysates and normalized to protein concentration. (B,C) Shh or Gli1 induce ALP but their activity is not blocked by Noggin. C3H10T1/2 were co-transfected with pShh and pNog, or pGli1 and pNog and cultured for 5 days. In control wells, pGli1 or pShh were replaced by empty vector. ALP phosphatase activity was determined in cell lysates and normalized to protein content. Results (mean±s.d.) are representative of three independent experiments, each performed in triplicate.
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Fig. 5. A short time of Shh treatment is sufficient to enhance BMP-2 activity. C3H10T1/2 cells were treated for the time indicated with Shh (10 µg/ml) then washed with sterile PBS and cultured in the presence of BMP-2 (100 ng/ml) for additional 5 days. ALP activity was measured in cell lysates and normalized to protein content. Results (mean±s.d.) are representative of three independent experiments, each performed in triplicate.
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Fig. 6. Shh enhances BMP-2 mediated Smad1 activation. (A) C3H10T1/2 or ST2 cells were transiently cotransfected, as indicated in Materials and Methods, with an expression vector coding for Smad1 fused to the yeast Gal4 binding site (pGal4-Smad1) and a reporter plasmid driven by the Gal4 binding site (pG15E1b-luc). To normalize luciferase signal, pTK-RL was included in the transfection mix. Transfected cells were either left unstimulated (CTRL) or stimulated with BMP-2 (100 ng/ml) and Shh (10 µg/ml) for 24 hours. Cells were then lysated and luciferase activity was evaluated and normalized to Renilla activity obtained from pTK-RL plasmid. (B) C3H10T1/2 were co-transfected with Gal4-Smad1, pGli1 and pG15E1b-luc. In the control experiment, pGli1 was replaced by empty vector (pcDNA3). Transfected cells were cultured for 24 hours in the absence or presence of BMP-2 (100 ng/ml). Luciferase activity was determined in cell lysates and normalized to Renilla activity obtained from pTK-RL plasmid. Results (mean luciferase fold induction±s.d.) are representative of two independent experiments, each performed in triplicate.
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Fig. 7. Shh inhibits adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells were either left unstimulated (A) or treated with (B) BMP-2 (100 ng/ml), (C) Shh (10 µg/ml) or (D) both. Cells were then cultured for 5 days and stained with Oil Red O, as described in Materials and Methods. The photos are representative of three distinct series of treatment.
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Fig. 9. Gli1 inhibits aP2 promoter activity. C3H10T1/2 cells were transfected with paP2/luc reporter construct and then either left unstimulated or stimulated with Shh (10 µg/ml) for 24 hours. In a distinct set of experiments, C3H10T1/2 cells were co-transfected either with paP2/luc and pGli1, or with paP2/luc and empty vector (pcDNA3); after transfection, cells were cultured for 24 additional hours. In all transfections pTK-RL plasmid was included. Luciferase activity was determined in cell lysates and normalized to Renilla activity. Results (mean±s.d.) are representative of two independent experiments, both performed in triplicate.
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2. (A) C3H10T1/2 were stimulated with Shh (10 µg/ml)) and total RNA was extracted after 4-day stimulation. Control cells were cultured without any stimulation and total RNA was extracted at the same time as stimulated cells. (B) C3H10T1/2 cells were stimulated for 1 hour with Shh (10 µg/ml); cells were washed and cultured for 4-days and total RNA was extracted. Control cells were similarly treated but did not receive any stimulation. The mRNA expression level of aP2, leptin (Lep), C/EBP