First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02758
Journal of Cell Science 119, 581-591 (2006)
Published by The Company of Biologists 2006
Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation
Jennifer E. Phillips1,2,
Charles A. Gersbach1,2,
Abigail M. Wojtowicz1,2 and
Andrés J. García2,3,*
1 Wallace H. Coulter Department of Biomedical Engineering and Georgia Tech/Emory Department of Biomedical Engineering, 313 Ferst Drive, Atlanta, GA 30332, USA
2 Parker H. Petit Institute for Bioengineering and Bioscience and Georgia Tech/Emory Center for the Engineering of Living Tissues, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA
3 George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 801 Ferst Drive, Atlanta, GA 30332, USA
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Fig. 1. DEX enhances Runx2-induced osteoblastic gene expression. Primary dermal fibroblasts were transduced with Runx2 or empty vector retrovirus, and cultured in osteogenic media with and without 10 nM DEX. mRNA expression was investigated by quantitative RT-PCR at 1, 3 and 7 days post-transduction [mean + s.e.m., n=16; ANOVA: P<1E-11 (i.e. 1x10-11); *different from empty vector control, **different from empty vector and DEX controls,  different from Runx2 (P< 0.05)]. Relative gene expression is expressed on a logarithmic scale. Detection limits for each gene were determined by reactions without cDNA and are shown as a dotted line. Graphs without dotted lines have detection limits that fall below y-axis minimum value.
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Fig. 3. DEX decreases Runx2 serine phosphorylation. Primary dermal fibroblasts were transduced with Runx2 retrovirus or left unmodified for controls and cultured in osteogenic media with and without 10 nM DEX. (A) Runx2 protein levels were examined at 7 days post-transduction by western blotting of whole-cell lysates with a polyclonal antibody against Runx2. GAPDH was used as a loading control. (B) Quantification of Runx2 band intensities [mean ± s.e.m., n=9; ANOVA: P<0.05; **different from unmodified and DEX-only controls (P<0.05)]. (C) Runx2 phosphoserine levels were assessed by immunoprecipitation of whole-cell lysates with an antibody against Runx2 and western blotting with antibodies against Runx2 and phosphoserine. (D) Quantification of Runx2 phosphoserine band intensities [mean ± s.e.m., n=9; ANOVA: P<0.05;  different from Runx2+DEX (P< 0.05)].
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Fig. 4. Site-directed mutagenesis of the Runx2 retroviral vector. (A) Schematic diagram of the Runx2 retroviral expression vector and its mutated derivatives, including (a) Runx2-WT control, (b) Runx2-125Gly (mimicking constitutive dephosphorylation), and (c) Runx2-125Glu (mimicking constitutive phosphorylation). Primary dermal fibroblasts were transduced with Runx2-WT, Runx2-125Gly or Runx2-125Glu retrovirus and cultured in osteogenic media with (+) and without (–) 10 nM DEX. (B) Retroviral transduction efficiency was determined at 3 days post-transduction by flow cytometry detection of eGFP expression. Unmodified cells were used to detect autofluorescence. (C) Runx2 mRNA expression was assessed by quantitative RT-PCR at 3 days post-transduction and expressed on a logarithmic scale [mean + s.e.m., n=12; ANOVA: P<1E-11; **different from unmodified and DEX-only controls (P< 0.05)]. Detection limit determined by reactions without cDNA is shown as a dotted line. (D) Runx2 protein levels were examined at 7 days post-transduction by western blotting of whole-cell lysates with a polyclonal antibody against Runx2. GAPDH was used as a loading control. Blot is representative of data from two separate experiments in triplicate.
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Fig. 5. Mutation of Ser125 decreases Runx2 serine phosphorylation. Primary dermal fibroblasts were transduced with Runx2-WT or Runx2-125Gly retrovirus and cultured in osteogenic media with (+) and without (–) 10 nM DEX. (A) Runx2 phosphoserine levels were assessed by immunoprecipitation of whole-cell lysates with an antibody against Runx2 and western blotting with antibodies against Runx2 and phosphoserine (pSerine). (B) Quantification of normalized Runx2 phosphoserine band intensities [mean ± s.e.m., n=3; ANOVA: P<0.05; different from Runx2+DEX, ¥different from 125Gly-DEX, ##different from 125Gly+DEX (P< 0.05); representative data from three separate experiments in triplicate].
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Fig. 7. Constitutive phosphorylation of Runx2-Ser125 inhibits osteoblastic differentiation. Cells were transduced with Runx2-WT, Runx2-125Gly or Runx2-125Glu retrovirus and cultured in osteogenic media with (+) and without (–) 10 nM DEX. (A) ALP activity was examined by a biochemical assay at 7 days post-transduction [mean ± s.e.m., n=6; ANOVA: P<1E-11; **different from unmodified and DEX-only controls, different from Runx2-DEX,  different from 125Glu-DEX and 125Glu+DEX, £different from 125Glu-DEX only (P< 0.05)]. (B) Mineralized matrix deposition was assessed by von Kossa staining for phosphate-positive regions and (C) quantified by image analysis at 14 days post-transduction [mean ± s.e.m., n=6; ANOVA: P<1E-11, **different from unmodified and DEX-only controls, different from Runx2-DEX, ¥different from 125Gly-DEX, different from 125Glu-DEX and 125Glu+Dex, £different from 125Glu-DEX only (P< 0.05)]. Bar, 2 cm.
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Fig. 8. Runx2 phosphorylation regulates osteoblastic differentiation in primary bone marrow stromal cells (BMSCs). BMSCs were transduced with Runx2-WT, Runx2-125Gly, or Runx2-125Glu retrovirus and cultured in osteogenic media with (+) and without (–) 10 nM DEX. (A) ALP activity was examined by a biochemical assay at 7 days post-transduction [mean ± s.e.m., n =3; ANOVA: P<1E-4; *different from unmodified cell control, **different from unmodified and DEX-only controls, different from Runx2-DEX, £different from 125Glu-DEX only (P< 0.05)]. (B) Mineralized matrix deposition was assessed by von Kossa staining for phosphate-positive regions and image analysis at 14 days post-transduction. Image represents data from two separate experiments in triplicate. Bar, 2 cm.
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Fig. 10. Inhibition of MKP-1 attenuates the DEX-mediated decrease in Runx2 serine phosphorylation. Primary dermal fibroblasts were transduced with Runx2 retrovirus or left unmodified for controls and cultured in osteogenic media with (+) and without (–) 10 nM DEX. After 7 days in culture, cells were treated with vehicle (ethanol), vehicle+DEX (10 nM), sanguinarine (50 µM), and sanguinarine (50 µM)+DEX (10 nM) for 30 minutes. (A) Western blot analysis of whole-cell lysates after 7 days in culture was conducted with antibodies against MKP-1, MKP-3, phospho-ERK (phospho-p44 ERK, phospho-p42 ERK) and ERK (p44 ERK and p42 ERK). GAPDH was used as a loading control. Blot are representative of data from three separate experiments in triplicate. (B) Runx2 phosphoserine levels were assessed by immunoprecipitation of whole-cell lysates with an antibody against Runx2 and western blotting with antibodies against Runx2 and phosphoserine (pSerine). Blots are representative of data from two separate experiments in triplicate.
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© The Company of Biologists Ltd 2006
