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Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development.

Liu Z, Yao X, Yan G, Xu Y, Yan J, Zou W, Wang G - Nat Commun (2016)

Bottom Line: In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression.Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity.Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.

ABSTRACT
How lineage specifiers are regulated during development is an outstanding question, and the molecular regulation of osteogenic factor RUNX2 remains to be fully understood. Here we report that the Mediator subunit MED23 cooperates with RUNX2 to regulate osteoblast differentiation and bone development. Med23 deletion in mesenchymal stem cells or osteoblast precursors results in multiple bone defects similar to those observed in Runx2(+/-) mice. In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression. Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity. Moreover, Med23 deficiency in osteoprogenitor cells exacerbates the skeletal abnormalities observed in Runx2(+/-) mice. Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

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Med23 deficiency inhibits osteoblast differentiation.(a) Expression of Med23 and Runx2 in bone marrow-derived mesenchymal stem cells and calvarial osteoblasts from 6 to 8-week-old control and Med23MSC−/− mice. Isolated cells were expanded and analysed by western blotting. (b) c.f.u.-F assay for bone marrow cells from control and Med23MSC−/− littermates. Representative images of c.f.u.-Fs stained with crystal violet (left, scale bar, 0.5 cm) and quantification of c.f.u.-Fs (right, n=3 for each group.). Data represent means±s.d. t-test. (c) ALP staining of bone marrow mesenchymal stem cells after cultured for 7 days in osteogenic medium. Scale bar, 500 μm. (d) c.f.u.-ob assay for bone marrow of control and Med23MSC−/− littermates. Bone marrow cells were cultured in osteogenic medium for 21 days, followed by staining with Alizarin red (left, scale bar, 0.5 cm) and quantification (right, n=3 for each group). Data represent means±s.d. t-test.***P<0.001 (e) The relative mRNA levels of Med23, Runx2, Osx, Alp and Ocn were quantified by RT–PCR. Data represent means±s.d. All data represent means±s.d. t-test, **P<0.01, ***P<0.001.
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f3: Med23 deficiency inhibits osteoblast differentiation.(a) Expression of Med23 and Runx2 in bone marrow-derived mesenchymal stem cells and calvarial osteoblasts from 6 to 8-week-old control and Med23MSC−/− mice. Isolated cells were expanded and analysed by western blotting. (b) c.f.u.-F assay for bone marrow cells from control and Med23MSC−/− littermates. Representative images of c.f.u.-Fs stained with crystal violet (left, scale bar, 0.5 cm) and quantification of c.f.u.-Fs (right, n=3 for each group.). Data represent means±s.d. t-test. (c) ALP staining of bone marrow mesenchymal stem cells after cultured for 7 days in osteogenic medium. Scale bar, 500 μm. (d) c.f.u.-ob assay for bone marrow of control and Med23MSC−/− littermates. Bone marrow cells were cultured in osteogenic medium for 21 days, followed by staining with Alizarin red (left, scale bar, 0.5 cm) and quantification (right, n=3 for each group). Data represent means±s.d. t-test.***P<0.001 (e) The relative mRNA levels of Med23, Runx2, Osx, Alp and Ocn were quantified by RT–PCR. Data represent means±s.d. All data represent means±s.d. t-test, **P<0.01, ***P<0.001.

Mentions: Next, we asked whether Med23 deficiency affects the osteogenic potential of MSCs in a cell-autonomous manner. Primary MSCs were isolated from the bone marrow of control and Med23MSC−/− littermates, and the deficiency of Med23 in the MSCs from Med23MSC−/− mice was confirmed by western blot assay (Fig. 3a). Med23 deficiency in MSCs does not appear to alter the cell viability or growth rate, indicated by comparable numbers of colony-forming unit fibroblasts (c.f.u.-F) (Fig. 3b). However, Med23−/− MSCs displayed markedly decreased alkaline phosphatase (ALP) activity and mineralization (Fig. 3c,d). In addition, the mRNA levels of osteogenic genes, such as osteocalcin (Ocn), were significantly downregulated in Med23MSC−/− cells (Fig. 3e), while Runx2, a key regulator in bone development, did not change during osteogenesis (Fig. 3a,e). Similarly, impaired osteogenesis was observed in Med23-deficient osteoblasts too (Supplementary Fig. 6). These results indicated that Med23 functioned necessarily in osteogenesis in vitro.


Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development.

Liu Z, Yao X, Yan G, Xu Y, Yan J, Zou W, Wang G - Nat Commun (2016)

Med23 deficiency inhibits osteoblast differentiation.(a) Expression of Med23 and Runx2 in bone marrow-derived mesenchymal stem cells and calvarial osteoblasts from 6 to 8-week-old control and Med23MSC−/− mice. Isolated cells were expanded and analysed by western blotting. (b) c.f.u.-F assay for bone marrow cells from control and Med23MSC−/− littermates. Representative images of c.f.u.-Fs stained with crystal violet (left, scale bar, 0.5 cm) and quantification of c.f.u.-Fs (right, n=3 for each group.). Data represent means±s.d. t-test. (c) ALP staining of bone marrow mesenchymal stem cells after cultured for 7 days in osteogenic medium. Scale bar, 500 μm. (d) c.f.u.-ob assay for bone marrow of control and Med23MSC−/− littermates. Bone marrow cells were cultured in osteogenic medium for 21 days, followed by staining with Alizarin red (left, scale bar, 0.5 cm) and quantification (right, n=3 for each group). Data represent means±s.d. t-test.***P<0.001 (e) The relative mRNA levels of Med23, Runx2, Osx, Alp and Ocn were quantified by RT–PCR. Data represent means±s.d. All data represent means±s.d. t-test, **P<0.01, ***P<0.001.
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f3: Med23 deficiency inhibits osteoblast differentiation.(a) Expression of Med23 and Runx2 in bone marrow-derived mesenchymal stem cells and calvarial osteoblasts from 6 to 8-week-old control and Med23MSC−/− mice. Isolated cells were expanded and analysed by western blotting. (b) c.f.u.-F assay for bone marrow cells from control and Med23MSC−/− littermates. Representative images of c.f.u.-Fs stained with crystal violet (left, scale bar, 0.5 cm) and quantification of c.f.u.-Fs (right, n=3 for each group.). Data represent means±s.d. t-test. (c) ALP staining of bone marrow mesenchymal stem cells after cultured for 7 days in osteogenic medium. Scale bar, 500 μm. (d) c.f.u.-ob assay for bone marrow of control and Med23MSC−/− littermates. Bone marrow cells were cultured in osteogenic medium for 21 days, followed by staining with Alizarin red (left, scale bar, 0.5 cm) and quantification (right, n=3 for each group). Data represent means±s.d. t-test.***P<0.001 (e) The relative mRNA levels of Med23, Runx2, Osx, Alp and Ocn were quantified by RT–PCR. Data represent means±s.d. All data represent means±s.d. t-test, **P<0.01, ***P<0.001.
Mentions: Next, we asked whether Med23 deficiency affects the osteogenic potential of MSCs in a cell-autonomous manner. Primary MSCs were isolated from the bone marrow of control and Med23MSC−/− littermates, and the deficiency of Med23 in the MSCs from Med23MSC−/− mice was confirmed by western blot assay (Fig. 3a). Med23 deficiency in MSCs does not appear to alter the cell viability or growth rate, indicated by comparable numbers of colony-forming unit fibroblasts (c.f.u.-F) (Fig. 3b). However, Med23−/− MSCs displayed markedly decreased alkaline phosphatase (ALP) activity and mineralization (Fig. 3c,d). In addition, the mRNA levels of osteogenic genes, such as osteocalcin (Ocn), were significantly downregulated in Med23MSC−/− cells (Fig. 3e), while Runx2, a key regulator in bone development, did not change during osteogenesis (Fig. 3a,e). Similarly, impaired osteogenesis was observed in Med23-deficient osteoblasts too (Supplementary Fig. 6). These results indicated that Med23 functioned necessarily in osteogenesis in vitro.

Bottom Line: In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression.Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity.Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.

ABSTRACT
How lineage specifiers are regulated during development is an outstanding question, and the molecular regulation of osteogenic factor RUNX2 remains to be fully understood. Here we report that the Mediator subunit MED23 cooperates with RUNX2 to regulate osteoblast differentiation and bone development. Med23 deletion in mesenchymal stem cells or osteoblast precursors results in multiple bone defects similar to those observed in Runx2(+/-) mice. In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression. Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity. Moreover, Med23 deficiency in osteoprogenitor cells exacerbates the skeletal abnormalities observed in Runx2(+/-) mice. Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

Show MeSH
Related in: MedlinePlus