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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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Decreased proliferation and accelerated differentiation of Sox8-deficient osteoblasts. (A) Analysis of Sox8 expression in primary calvarial osteoblast cultures. Northern blot expression analysis shows that Sox8 is expressed in nondifferentiated wild-type calvarial osteoblast cultures (d0), but not in the same cultures 5 and 10 d (d5 and d10) after the addition of ascorbic acid and β-glycerophosphate. (B) Analysis of osteoblast proliferation. A BrdU-incorporation assay with primary osteoblasts from wild-type (white bars) and Sox8-deficient (gray bars) mice reveals a strong reduction of osteoblast proliferation in the absence of Sox8, occurring before ascorbic acid and β-glycerophosphate were added to the culture medium (d0). (C) Analysis of ECM mineralization. Von Kossa staining reveals an accelerated mineralization of Sox8-deficient primary osteoblast cultures compared with wild-type cultures. n.d., not detectable. (D) X-Gal staining of tibia sections from Sox8-deficient mice at 1 wk old reveals LacZ expression in the growth plate, but also in cells of the primary spongiosa (arrowheads). Higher magnification reveals expression in groups of cells (indicated by the dotted red lines) at the bone surface which is characteristic of osteoblasts. (E) Analysis of osteoblast proliferation in vivo by BrdU-incorporation assays at 1 wk old. The percentage of BrdU-positive cells in calvarial sections is significantly decreased in Sox8-deficient mice compared with wild-type littermates. Note the flattened appearance of Sox8-deficient osteoblasts compared with wild-type controls, suggesting premature differentiation. (F) Toluidine blue staining of undecalcified vertebral bone sections shows that the teams of bone-forming osteoblasts (indicated by the dotted red lines) are shorter in Sox8-deficient mice. Quantification of the average numbers of osteoblasts within these bone-forming units (N.Ob/BFU) reveals a significant reduction in the absence of Sox8. (G) Fluorescent micrographs showing the calcein-labeled surfaces representing newly formed bone. The length of these surfaces is significantly decreased in Sox8-deficient mice. Values represent means ± SEM. Asterisks indicate statistically significant differences as determined by t test between the two groups (n = 6). **, P < 0.005.
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fig3: Decreased proliferation and accelerated differentiation of Sox8-deficient osteoblasts. (A) Analysis of Sox8 expression in primary calvarial osteoblast cultures. Northern blot expression analysis shows that Sox8 is expressed in nondifferentiated wild-type calvarial osteoblast cultures (d0), but not in the same cultures 5 and 10 d (d5 and d10) after the addition of ascorbic acid and β-glycerophosphate. (B) Analysis of osteoblast proliferation. A BrdU-incorporation assay with primary osteoblasts from wild-type (white bars) and Sox8-deficient (gray bars) mice reveals a strong reduction of osteoblast proliferation in the absence of Sox8, occurring before ascorbic acid and β-glycerophosphate were added to the culture medium (d0). (C) Analysis of ECM mineralization. Von Kossa staining reveals an accelerated mineralization of Sox8-deficient primary osteoblast cultures compared with wild-type cultures. n.d., not detectable. (D) X-Gal staining of tibia sections from Sox8-deficient mice at 1 wk old reveals LacZ expression in the growth plate, but also in cells of the primary spongiosa (arrowheads). Higher magnification reveals expression in groups of cells (indicated by the dotted red lines) at the bone surface which is characteristic of osteoblasts. (E) Analysis of osteoblast proliferation in vivo by BrdU-incorporation assays at 1 wk old. The percentage of BrdU-positive cells in calvarial sections is significantly decreased in Sox8-deficient mice compared with wild-type littermates. Note the flattened appearance of Sox8-deficient osteoblasts compared with wild-type controls, suggesting premature differentiation. (F) Toluidine blue staining of undecalcified vertebral bone sections shows that the teams of bone-forming osteoblasts (indicated by the dotted red lines) are shorter in Sox8-deficient mice. Quantification of the average numbers of osteoblasts within these bone-forming units (N.Ob/BFU) reveals a significant reduction in the absence of Sox8. (G) Fluorescent micrographs showing the calcein-labeled surfaces representing newly formed bone. The length of these surfaces is significantly decreased in Sox8-deficient mice. Values represent means ± SEM. Asterisks indicate statistically significant differences as determined by t test between the two groups (n = 6). **, P < 0.005.

Mentions: To determine whether the decreased bone formation of Sox8-deficient mice is caused by an intrinsic osteoblast differentiation defect, we next studied the behavior of wild-type and Sox8-deficient primary calvarial osteoblasts ex vivo. We first analyzed Sox8 expression in these cells by Northern blotting and found a strong expression in nondifferentiated wild-type cultures. Importantly, this expression was completely abolished 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate, two agents leading to terminal osteoblast differentiation and mineralization ex vivo (Fig. 3 A). We next determined the proliferation of wild-type and Sox8-deficient primary calvarial osteoblasts. Using a BrdU incorporation assay we found that the proliferation rate of Sox8-deficient cells was strongly reduced compared with wild-type cells, even before osteoblast differentiation was induced (Fig. 3 B). To analyze osteoblast differentiation we performed Von Kossa staining of the mineralized matrix from wild-type and Sox8-deficient cells formed before (d0) as well as 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate. Unexpectedly, we observed an accelerated mineralization of Sox8-deficient cells. In contrast to wild-type cultures, mineralized nodules were already detectable after 5 d of differentiation. After 10 d the mineralization was still much more pronounced (Fig. 3 C).


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)

Decreased proliferation and accelerated differentiation of Sox8-deficient osteoblasts. (A) Analysis of Sox8 expression in primary calvarial osteoblast cultures. Northern blot expression analysis shows that Sox8 is expressed in nondifferentiated wild-type calvarial osteoblast cultures (d0), but not in the same cultures 5 and 10 d (d5 and d10) after the addition of ascorbic acid and β-glycerophosphate. (B) Analysis of osteoblast proliferation. A BrdU-incorporation assay with primary osteoblasts from wild-type (white bars) and Sox8-deficient (gray bars) mice reveals a strong reduction of osteoblast proliferation in the absence of Sox8, occurring before ascorbic acid and β-glycerophosphate were added to the culture medium (d0). (C) Analysis of ECM mineralization. Von Kossa staining reveals an accelerated mineralization of Sox8-deficient primary osteoblast cultures compared with wild-type cultures. n.d., not detectable. (D) X-Gal staining of tibia sections from Sox8-deficient mice at 1 wk old reveals LacZ expression in the growth plate, but also in cells of the primary spongiosa (arrowheads). Higher magnification reveals expression in groups of cells (indicated by the dotted red lines) at the bone surface which is characteristic of osteoblasts. (E) Analysis of osteoblast proliferation in vivo by BrdU-incorporation assays at 1 wk old. The percentage of BrdU-positive cells in calvarial sections is significantly decreased in Sox8-deficient mice compared with wild-type littermates. Note the flattened appearance of Sox8-deficient osteoblasts compared with wild-type controls, suggesting premature differentiation. (F) Toluidine blue staining of undecalcified vertebral bone sections shows that the teams of bone-forming osteoblasts (indicated by the dotted red lines) are shorter in Sox8-deficient mice. Quantification of the average numbers of osteoblasts within these bone-forming units (N.Ob/BFU) reveals a significant reduction in the absence of Sox8. (G) Fluorescent micrographs showing the calcein-labeled surfaces representing newly formed bone. The length of these surfaces is significantly decreased in Sox8-deficient mice. Values represent means ± SEM. Asterisks indicate statistically significant differences as determined by t test between the two groups (n = 6). **, P < 0.005.
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fig3: Decreased proliferation and accelerated differentiation of Sox8-deficient osteoblasts. (A) Analysis of Sox8 expression in primary calvarial osteoblast cultures. Northern blot expression analysis shows that Sox8 is expressed in nondifferentiated wild-type calvarial osteoblast cultures (d0), but not in the same cultures 5 and 10 d (d5 and d10) after the addition of ascorbic acid and β-glycerophosphate. (B) Analysis of osteoblast proliferation. A BrdU-incorporation assay with primary osteoblasts from wild-type (white bars) and Sox8-deficient (gray bars) mice reveals a strong reduction of osteoblast proliferation in the absence of Sox8, occurring before ascorbic acid and β-glycerophosphate were added to the culture medium (d0). (C) Analysis of ECM mineralization. Von Kossa staining reveals an accelerated mineralization of Sox8-deficient primary osteoblast cultures compared with wild-type cultures. n.d., not detectable. (D) X-Gal staining of tibia sections from Sox8-deficient mice at 1 wk old reveals LacZ expression in the growth plate, but also in cells of the primary spongiosa (arrowheads). Higher magnification reveals expression in groups of cells (indicated by the dotted red lines) at the bone surface which is characteristic of osteoblasts. (E) Analysis of osteoblast proliferation in vivo by BrdU-incorporation assays at 1 wk old. The percentage of BrdU-positive cells in calvarial sections is significantly decreased in Sox8-deficient mice compared with wild-type littermates. Note the flattened appearance of Sox8-deficient osteoblasts compared with wild-type controls, suggesting premature differentiation. (F) Toluidine blue staining of undecalcified vertebral bone sections shows that the teams of bone-forming osteoblasts (indicated by the dotted red lines) are shorter in Sox8-deficient mice. Quantification of the average numbers of osteoblasts within these bone-forming units (N.Ob/BFU) reveals a significant reduction in the absence of Sox8. (G) Fluorescent micrographs showing the calcein-labeled surfaces representing newly formed bone. The length of these surfaces is significantly decreased in Sox8-deficient mice. Values represent means ± SEM. Asterisks indicate statistically significant differences as determined by t test between the two groups (n = 6). **, P < 0.005.
Mentions: To determine whether the decreased bone formation of Sox8-deficient mice is caused by an intrinsic osteoblast differentiation defect, we next studied the behavior of wild-type and Sox8-deficient primary calvarial osteoblasts ex vivo. We first analyzed Sox8 expression in these cells by Northern blotting and found a strong expression in nondifferentiated wild-type cultures. Importantly, this expression was completely abolished 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate, two agents leading to terminal osteoblast differentiation and mineralization ex vivo (Fig. 3 A). We next determined the proliferation of wild-type and Sox8-deficient primary calvarial osteoblasts. Using a BrdU incorporation assay we found that the proliferation rate of Sox8-deficient cells was strongly reduced compared with wild-type cells, even before osteoblast differentiation was induced (Fig. 3 B). To analyze osteoblast differentiation we performed Von Kossa staining of the mineralized matrix from wild-type and Sox8-deficient cells formed before (d0) as well as 5 and 10 d after the addition of ascorbic acid and β-glycerophosphate. Unexpectedly, we observed an accelerated mineralization of Sox8-deficient cells. In contrast to wild-type cultures, mineralized nodules were already detectable after 5 d of differentiation. After 10 d the mineralization was still much more pronounced (Fig. 3 C).

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