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The high mobility group transcription factor Sox8 is a negative regulator of osteoblast differentiation.

Schmidt K, Schinke T, Haberland M, Priemel M, Schilling AF, Mueldner C, Rueger JM, Sock E, Wegner M, Amling M - J. Cell Biol. (2005)

Bottom Line: This is achieved through a balanced activity of bone-resorbing osteoclasts and bone-forming osteoblasts.In this study, we identify the high mobility group transcription factor Sox8 as a physiologic regulator of bone formation.Together, these data demonstrate a novel function of Sox8, whose tightly controlled expression is critical for bone formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nürnberg, Erlangen 91054, Germany.

ABSTRACT
Bone remodeling is an important physiologic process that is required to maintain a constant bone mass. This is achieved through a balanced activity of bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we identify the high mobility group transcription factor Sox8 as a physiologic regulator of bone formation. Sox8-deficient mice display a low bone mass phenotype that is caused by a precocious osteoblast differentiation. Accordingly, primary osteoblasts derived from these mice show an accelerated mineralization ex vivo and a premature expression of osteoblast differentiation markers. To confirm the function of Sox8 as a negative regulator of osteoblast differentiation we generated transgenic mice that express Sox8 under the control of an osteoblast-specific Col1a1 promoter fragment. These mice display a severely impaired bone formation that can be explained by a strongly reduced expression of runt-related transcription factor 2, a gene encoding a transcription factor required for osteoblast differentiation. Together, these data demonstrate a novel function of Sox8, whose tightly controlled expression is critical for bone formation.

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Premature expression of osteoblast differentiation markers in Sox8-deficient osteoblasts. (A) RT-PCR expression analysis of primary osteoblast cultures from wild-type (WT) and Sox8-deficient (−/−) mice at d0, d5, and d10 of differentiation. Note the premature expression of Bsp, Phex, Runx2, and Osx in Sox8-deficient cultures. Tnsalp (tissue nonspecific alkaline phosphatase), Osc (osteocalcin), Bsp (bone sialoprotein), Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome), Runx2 (runt-related transcription factor 2), Osx (osterix), Lrp5 (low density lipoprotein receptor-related protein 5), Gapdh (Glycerinaldehydephosphate dehydrogenase). (B) Analysis of Runx2 DNA-binding activity. An electrophoretic mobility shift assay using the Runx2-binding site (OSE-2; Ducy et al., 1997) reveals the presence of Runx2 in nuclear extracts from nondifferentiated Sox8-deficient osteoblasts, but not from wild-type osteoblasts (d0). Binding was not observed with a mutated binding site (mutOSE-2) or in the presence of an anti–Runx2-antibody. (C) Analysis of osteoblast differentiation markers in vivo. A Northern blot expression analysis using calvaria and femur RNA from wild-type and Sox8-deficient mice at 6 wk old reveals an elevated expression of Osc, Bsp, Phex, and Runx2 in the absence of Sox8.
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fig4: Premature expression of osteoblast differentiation markers in Sox8-deficient osteoblasts. (A) RT-PCR expression analysis of primary osteoblast cultures from wild-type (WT) and Sox8-deficient (−/−) mice at d0, d5, and d10 of differentiation. Note the premature expression of Bsp, Phex, Runx2, and Osx in Sox8-deficient cultures. Tnsalp (tissue nonspecific alkaline phosphatase), Osc (osteocalcin), Bsp (bone sialoprotein), Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome), Runx2 (runt-related transcription factor 2), Osx (osterix), Lrp5 (low density lipoprotein receptor-related protein 5), Gapdh (Glycerinaldehydephosphate dehydrogenase). (B) Analysis of Runx2 DNA-binding activity. An electrophoretic mobility shift assay using the Runx2-binding site (OSE-2; Ducy et al., 1997) reveals the presence of Runx2 in nuclear extracts from nondifferentiated Sox8-deficient osteoblasts, but not from wild-type osteoblasts (d0). Binding was not observed with a mutated binding site (mutOSE-2) or in the presence of an anti–Runx2-antibody. (C) Analysis of osteoblast differentiation markers in vivo. A Northern blot expression analysis using calvaria and femur RNA from wild-type and Sox8-deficient mice at 6 wk old reveals an elevated expression of Osc, Bsp, Phex, and Runx2 in the absence of Sox8.

Mentions: We next analyzed the expression of osteoblast differentiation markers in wild-type and Sox8-deficient primary osteoblasts by RT-PCR. In wild-type cultures expression of tissue-nonspecific alkaline phosphatase (Tnsalp), osteocalcin (Osc), bone sialoprotein (Bsp), phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome (Phex), Runx2 and Osx was observed only 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate (Fig. 4 A). In Sox8-deficient cells however, we observed a different expression pattern for all of these genes except Osc. Whereas Tnsalp was prematurely down-regulated in the absence of Sox8, Bsp, and Phex, two genes associated with ECM mineralization, were expressed in Sox8-deficient cells even without the addition of ascorbic acid and β-glycerophosphate (Fig. 4 A). A premature expression in Sox8-deficient cells was also observed for Runx2 and Osx, two genes encoding transcription factors required for osteoblast differentiation. In contrast, we did not observe changes in the expression pattern of low density lipoprotein receptor-related protein 5 (Lrp5), a gene that was recently identified to play a major role in bone formation in a Runx2-independent manner (Kato et al., 2002).


The high mobility group transcription factor Sox8 is a negative regulator of osteoblast differentiation.

Schmidt K, Schinke T, Haberland M, Priemel M, Schilling AF, Mueldner C, Rueger JM, Sock E, Wegner M, Amling M - J. Cell Biol. (2005)

Premature expression of osteoblast differentiation markers in Sox8-deficient osteoblasts. (A) RT-PCR expression analysis of primary osteoblast cultures from wild-type (WT) and Sox8-deficient (−/−) mice at d0, d5, and d10 of differentiation. Note the premature expression of Bsp, Phex, Runx2, and Osx in Sox8-deficient cultures. Tnsalp (tissue nonspecific alkaline phosphatase), Osc (osteocalcin), Bsp (bone sialoprotein), Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome), Runx2 (runt-related transcription factor 2), Osx (osterix), Lrp5 (low density lipoprotein receptor-related protein 5), Gapdh (Glycerinaldehydephosphate dehydrogenase). (B) Analysis of Runx2 DNA-binding activity. An electrophoretic mobility shift assay using the Runx2-binding site (OSE-2; Ducy et al., 1997) reveals the presence of Runx2 in nuclear extracts from nondifferentiated Sox8-deficient osteoblasts, but not from wild-type osteoblasts (d0). Binding was not observed with a mutated binding site (mutOSE-2) or in the presence of an anti–Runx2-antibody. (C) Analysis of osteoblast differentiation markers in vivo. A Northern blot expression analysis using calvaria and femur RNA from wild-type and Sox8-deficient mice at 6 wk old reveals an elevated expression of Osc, Bsp, Phex, and Runx2 in the absence of Sox8.
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Related In: Results  -  Collection

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fig4: Premature expression of osteoblast differentiation markers in Sox8-deficient osteoblasts. (A) RT-PCR expression analysis of primary osteoblast cultures from wild-type (WT) and Sox8-deficient (−/−) mice at d0, d5, and d10 of differentiation. Note the premature expression of Bsp, Phex, Runx2, and Osx in Sox8-deficient cultures. Tnsalp (tissue nonspecific alkaline phosphatase), Osc (osteocalcin), Bsp (bone sialoprotein), Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome), Runx2 (runt-related transcription factor 2), Osx (osterix), Lrp5 (low density lipoprotein receptor-related protein 5), Gapdh (Glycerinaldehydephosphate dehydrogenase). (B) Analysis of Runx2 DNA-binding activity. An electrophoretic mobility shift assay using the Runx2-binding site (OSE-2; Ducy et al., 1997) reveals the presence of Runx2 in nuclear extracts from nondifferentiated Sox8-deficient osteoblasts, but not from wild-type osteoblasts (d0). Binding was not observed with a mutated binding site (mutOSE-2) or in the presence of an anti–Runx2-antibody. (C) Analysis of osteoblast differentiation markers in vivo. A Northern blot expression analysis using calvaria and femur RNA from wild-type and Sox8-deficient mice at 6 wk old reveals an elevated expression of Osc, Bsp, Phex, and Runx2 in the absence of Sox8.
Mentions: We next analyzed the expression of osteoblast differentiation markers in wild-type and Sox8-deficient primary osteoblasts by RT-PCR. In wild-type cultures expression of tissue-nonspecific alkaline phosphatase (Tnsalp), osteocalcin (Osc), bone sialoprotein (Bsp), phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome (Phex), Runx2 and Osx was observed only 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate (Fig. 4 A). In Sox8-deficient cells however, we observed a different expression pattern for all of these genes except Osc. Whereas Tnsalp was prematurely down-regulated in the absence of Sox8, Bsp, and Phex, two genes associated with ECM mineralization, were expressed in Sox8-deficient cells even without the addition of ascorbic acid and β-glycerophosphate (Fig. 4 A). A premature expression in Sox8-deficient cells was also observed for Runx2 and Osx, two genes encoding transcription factors required for osteoblast differentiation. In contrast, we did not observe changes in the expression pattern of low density lipoprotein receptor-related protein 5 (Lrp5), a gene that was recently identified to play a major role in bone formation in a Runx2-independent manner (Kato et al., 2002).

Bottom Line: This is achieved through a balanced activity of bone-resorbing osteoclasts and bone-forming osteoblasts.In this study, we identify the high mobility group transcription factor Sox8 as a physiologic regulator of bone formation.Together, these data demonstrate a novel function of Sox8, whose tightly controlled expression is critical for bone formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nürnberg, Erlangen 91054, Germany.

ABSTRACT
Bone remodeling is an important physiologic process that is required to maintain a constant bone mass. This is achieved through a balanced activity of bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we identify the high mobility group transcription factor Sox8 as a physiologic regulator of bone formation. Sox8-deficient mice display a low bone mass phenotype that is caused by a precocious osteoblast differentiation. Accordingly, primary osteoblasts derived from these mice show an accelerated mineralization ex vivo and a premature expression of osteoblast differentiation markers. To confirm the function of Sox8 as a negative regulator of osteoblast differentiation we generated transgenic mice that express Sox8 under the control of an osteoblast-specific Col1a1 promoter fragment. These mice display a severely impaired bone formation that can be explained by a strongly reduced expression of runt-related transcription factor 2, a gene encoding a transcription factor required for osteoblast differentiation. Together, these data demonstrate a novel function of Sox8, whose tightly controlled expression is critical for bone formation.

Show MeSH
Related in: MedlinePlus