First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00965
Journal of Cell Science 117, 1457-1468 (2004)
Published by The Company of Biologists 2004
MyoD enhances BMP7-induced osteogenic differentiation of myogenic cell cultures
M. Komaki1,*,
A. Asakura2,
M. A. Rudnicki2,
J. Sodek1 and
S. Cheifetz1,
1 CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada
2 Center for Molecular Medicine, The Ottawa Hospital Research Institute, University of Ottawa, Ottawa, ON K1H 8L6, Canada
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Fig. 1. Dose-response effects of BMP-7 on muscle satellite cell differentiation. Primary muscle satellite cells derived from either wild-type (WT) or MyoD-/- mice were treated for 3 days with 0, 100 or 400 ng/ml of BMP-7. Upper panel, cultures stained for MHC (brown) or ALP activity (blue). Lower panel, the percentage area of MHC-positive cells was measured using NIH image software (n=6). Cell lysates from parallel cultures were assayed for total ALP activity using p-nitrophenylphosphate as a substrate. The ALP activity is expressed as OD405 nm/min/mg protein±s.d. (n=3). Scale bar, 250 µm.
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Fig. 2. Temporal effects of BMP-7 on muscle satellite cell differentiation. Twenty-four hours after seeding, at 2x104/cm2, cultures of primary muscle satellite cells were shifted to differentiation media (day 0, d0). BMP-7 (400 ng/ml) was added at various times over the 6-day culture period as indicated by the solid line in the outline of the experimental design. At the end of 6 days, the cultures were stained for the expression of MHC (black/brown) or for ALP activity (blue). Results from a representative experiment are shown in the upper panel. Scale bar, 250 µm.
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Fig. 3. RT-PCR analyses of muscle- and bone-associated genes. Upper panel, total RNA was purified from MyoD-/- and WT cells either before shifting them to differentiation media (day 0) or after a 3-day treatment with or without BMP-7 (400 ng/ml). Target mRNAs were analyzed by RT-PCR. ß-Actin levels indicate that similar amounts of RNA were used for each time point. Arrows indicate Runx2 transcripts. Lower panel, expression of Osx and the housekeeping gene ß-actin in a second cDNA synthesis from the RNA used in the upper panel.
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Fig. 4. Transfection with MyoD expression vector partially rescues the osteogenic response of MyoD-/- cells to BMP-7. The phenotype of MyoD-/- cells, stably transfected with either a MyoD-expression vector or its empty vector, was compared to wild-type cells after a 3-day incubation with BMP-7 (400 ng/ml). Cultures were double-stained for MHC expression (brown/black) and ALP activity (blue). Wild-type cells (A), MyoD-/- cells (B), MyoD-/- cells transfected with PGK-puro (C) or pEMSV-MyoD/PGK-puro vectors (D). Immunostaining analysis of the transfected cultures shows that only the MyoD transfected cultures are positive for MyoD expression (compare insets in C and D). Scale bar, 100 µm.
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Fig. 5. Transient transfection with pMyoD increases ALP activity in C3H10T1/2 cells. (A) C3H10T1/2 cells were transiently transfected with MyoD expression vector (pEMSV-MyoD/PGK-puro) and empty vector (PGK-puro) and treated for 0, 1, 3 and 6 days with or without BMP-7 (400 ng/ml). ALP activity in the cell lysate was measured using p-nitrophenylphosphate as a substrate. An increase in BMP-7 induced ALP activity in MyoD transfected cultures above that induced in the control cultures, was evident after 3 days and was further increased at 6 days post-transfection. (B) Representative cultures of transfected cells were double-stained for ß-Gal (green) and for ALP activity (red) after a 3-day incubation in differentiation media with or without BMP-7 (400 ng/ml). ß-Gal staining was used to identify the transfected cells independently of any response to BMP-7 treatment. Double-stained cells are evident in the cultures transfected with MyoD and treated with BMP-7. Scale bar, 80 µm.
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Fig. 6. Stable transfection of MyoD enhances osteogenic response of C3H10T1/2 cells to BMP. (A) Isolated clones of C3H10T1/2 cells, stably transfected with either MyoD-expression vector (10T-MD) or its empty vector (10T-puro), were cultured for 3 days in differentiation media with or without BMP-7 (400 ng/ml) and then double-stained for MyoD expression (black/brown) and ALP activity (blue). MyoD was only detected in the 10TMD lines incubated without BMP. BMP-treatment decreases MyoD expression and increases ALP-positive cells. More ALP-positive cells are evident in 10TMD cell lines than in the 10Tpuro cell lines (arrows). Scale bar, 80 µm. (B) RT-PCR analyses of selected genes in clonal cell lines incubated for 3 days with or without BMP-7 (400 ng/ml). BMP-treatment decreased the expression of MyoD and AchR in 10TMD-12 and increased the expression of ALP, consistent with the inhibition of myogenesis and the stimulation of osteogenesis. Higher ALP activity (A, BMP) is reflected in the increased ALP mRNA in the MyoD-transfected culture incubated with BMP-7.
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Fig. 7. Effect of co-transfection of MyoD with Runx2/Cbfa1 on ALP activity. (A) C3H10T1/2 cells were transiently transfected with MyoD- or Runx2/Cbfa1-expression vectors alone or in combination. Co-transfection with ß-Gal-expression vector allowed identification of transfected cells. Cultures were double-stained for ß-Gal and ALP activity after a 3-day incubation in differentiation media. MyoD and Runx2/Cbfa1 transfected cells tended to show more double-stained cells than cells transfected with Runx2/Cbfa1 alone (arrow in A); however, the difference did not reach statistical significance. (B) Immunohistochemical analyses of Runx2/Cbfa1 expression in C3H10T1/2 cells at day 0 and after 3 days in low mitogen media containing 400 ng/ml of BMP-7, revealed protein expression in BMP-7 treated cultures.
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Fig. 8. MyoD affects transcriptional activity of Runx2/Cbfa1. (A) The effect of Runx2/Cbfa1 on MyoD transcriptional activity was determined by using the 4Rtk-luc reporter construct transiently transfected into C3H10T1/2 cells. Luciferase activity in extracts of cultures incubated for 48 hours with or without BMP-7 (400 ng/ml) was normalized to ß-Gal activity to correct for transfection efficiency. The results are expressed relative to the activity in cultures transfected with empty vectors and incubated without BMP-7. Each transfection was done in triplicate. In the absence of BMP-7, Runx2/Cbfa1 has little effect on MyoD transactivation. However in the presence of BMP-7 the transactivation was decreased, consistent with the inhibition of myogenesis. (B) The effect of bHLH-factors on Runx2/Cbfa1 transcriptional activity was determined using the p6OSE-luc reporter construct as described in A. MyoD transfection increased the Runx2/Cbfa1 transactivation and the effect was markedly increased by incubating the transfected cultures with BMP-7. None of the other bHLH factors could synergize with Runx2/Cbfa1 to increase luciferase expression. (C) The effect of MyoD on transactivation of a reporter construct containing the -147/+13 fragment of the osteocalcin promoter.
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© The Company of Biologists Ltd 2004
