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The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

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A threshold of Sox2 is required to maintain osteoblast survival(A) Western Blot for Sox2 expression. OB1-Apert cells were infected with pBabe-Hygro retrovirus expressing the indicated shRNAs against Sox2 or scrambled (sc) shRNA. Pools of cells were selected in Hygromycin B and protein was extracted. Sox2 expression was determined by immunoblotting.(B) Colony formation by Sox2 shRNA-expressing osteoblasts. OB5, OB1 and OB1-Apert cells infected with pBabe-Hygro retro-virus expressing either scrambled (sc) or sh900, sh998 and sh1043 were plated in triplicate and selected in Hygromycin B for 7-10 days followed by staining with Crystal Violet. Pictures of representative plates of OB1 and OB1-Apert cells expressing shRNA are shown and quantified in adjoining Table(C) Growth of Sox2 shRNA-expressing OB1-Apert osteoblasts. OB1-Apert cells (0.1 × 106 cells/well) stably expressing either scrambled (sc) or sh900, sh998 or sh1043 were plated in triplicate and growth of cells assessed by counting cells in a Z1 Beckman Coulter counter. * = p<0.05
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Figure 5: A threshold of Sox2 is required to maintain osteoblast survival(A) Western Blot for Sox2 expression. OB1-Apert cells were infected with pBabe-Hygro retrovirus expressing the indicated shRNAs against Sox2 or scrambled (sc) shRNA. Pools of cells were selected in Hygromycin B and protein was extracted. Sox2 expression was determined by immunoblotting.(B) Colony formation by Sox2 shRNA-expressing osteoblasts. OB5, OB1 and OB1-Apert cells infected with pBabe-Hygro retro-virus expressing either scrambled (sc) or sh900, sh998 and sh1043 were plated in triplicate and selected in Hygromycin B for 7-10 days followed by staining with Crystal Violet. Pictures of representative plates of OB1 and OB1-Apert cells expressing shRNA are shown and quantified in adjoining Table(C) Growth of Sox2 shRNA-expressing OB1-Apert osteoblasts. OB1-Apert cells (0.1 × 106 cells/well) stably expressing either scrambled (sc) or sh900, sh998 or sh1043 were plated in triplicate and growth of cells assessed by counting cells in a Z1 Beckman Coulter counter. * = p<0.05

Mentions: While our findings with osteoblasts from Sox2 mutant mice highlighted the role of Sox2 in maintaining proliferation potential in osteoblasts, all of our experiments had been performed with osteoblasts from mutant mice carrying a floxed Sox2 allele, and it was of interest to be able to extend these observations to independent osteoblastic cell lines. Furthermore, we wished to rule out the effect of excision of any Sox2 flanking sequences such as those that may encode regulatory microRNAs. To address these questions, we used two immortalized osteoblast cell lines that our laboratory had previously characterized: OB1, a PyLT immortalized immature osteoblast line, and OB5, a spontaneously immortalized cell line derived from p53+/- calvaria (6, 27). We also tested OB1-Apert cells, an OB1-derived clone carrying the activated FGFR2 bearing the S252W mutation found in Apert syndrome, that express high levels of Sox2, at least 10-fold more than the parental OB1 cells (8). We infected these cell lines with pBabe-hygro retrovirus expressing three shRNAs directed against murine Sox2, and a scrambled shRNA control and selected for Hygromycin resistant cell clones. These three shRNAs were found to differentially reduce Sox2 expression in the OB1-Apert cells (Figure 5A), ranging from 60 to 90% inhibition of Sox2 protein expression. As seen in Figure 5B, infection of OB1 or OB5 cells with Sox2 shRNA vectors completely abrogated colony formation. On the other hand, OB1-Apert cells were still capable of forming colonies, with an efficiency ranging between 25-85% of the colonies produced by the cells expressing the scrambled control shRNA (Fig. 5B). The differential ability of the shRNAs to inhibit Sox2 protein expression was reflected in the ability to form colonies, as the shRNA (1043) that caused the least reduction in Sox2 levels produced the highest number of colonies. The OB1-Apert cells expressing Sox2 shRNA 900 and 998 that could form colonies grew considerably more slowly than the parental cells (Fig. 5C), while the growth of cells expressing shRNA 1043 was not affected, as expected. The proliferation of NIH3T3 mouse fibroblasts, that do not express Sox2, was unaffected by Sox2 shRNAs as infection with all Sox2-specific shRNA viruses resulted in a number of hygromycin resistant colonies essentially identical to that produced by the scrambled shRNA control (not shown). These results demonstrate that the requirement for Sox2 expression is not limited to osteoblasts from Sox2 mutant mice and suggest that a threshold level of Sox2 expression is necessary to maintain osteoblast self-renewal.


The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

A threshold of Sox2 is required to maintain osteoblast survival(A) Western Blot for Sox2 expression. OB1-Apert cells were infected with pBabe-Hygro retrovirus expressing the indicated shRNAs against Sox2 or scrambled (sc) shRNA. Pools of cells were selected in Hygromycin B and protein was extracted. Sox2 expression was determined by immunoblotting.(B) Colony formation by Sox2 shRNA-expressing osteoblasts. OB5, OB1 and OB1-Apert cells infected with pBabe-Hygro retro-virus expressing either scrambled (sc) or sh900, sh998 and sh1043 were plated in triplicate and selected in Hygromycin B for 7-10 days followed by staining with Crystal Violet. Pictures of representative plates of OB1 and OB1-Apert cells expressing shRNA are shown and quantified in adjoining Table(C) Growth of Sox2 shRNA-expressing OB1-Apert osteoblasts. OB1-Apert cells (0.1 × 106 cells/well) stably expressing either scrambled (sc) or sh900, sh998 or sh1043 were plated in triplicate and growth of cells assessed by counting cells in a Z1 Beckman Coulter counter. * = p<0.05
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Figure 5: A threshold of Sox2 is required to maintain osteoblast survival(A) Western Blot for Sox2 expression. OB1-Apert cells were infected with pBabe-Hygro retrovirus expressing the indicated shRNAs against Sox2 or scrambled (sc) shRNA. Pools of cells were selected in Hygromycin B and protein was extracted. Sox2 expression was determined by immunoblotting.(B) Colony formation by Sox2 shRNA-expressing osteoblasts. OB5, OB1 and OB1-Apert cells infected with pBabe-Hygro retro-virus expressing either scrambled (sc) or sh900, sh998 and sh1043 were plated in triplicate and selected in Hygromycin B for 7-10 days followed by staining with Crystal Violet. Pictures of representative plates of OB1 and OB1-Apert cells expressing shRNA are shown and quantified in adjoining Table(C) Growth of Sox2 shRNA-expressing OB1-Apert osteoblasts. OB1-Apert cells (0.1 × 106 cells/well) stably expressing either scrambled (sc) or sh900, sh998 or sh1043 were plated in triplicate and growth of cells assessed by counting cells in a Z1 Beckman Coulter counter. * = p<0.05
Mentions: While our findings with osteoblasts from Sox2 mutant mice highlighted the role of Sox2 in maintaining proliferation potential in osteoblasts, all of our experiments had been performed with osteoblasts from mutant mice carrying a floxed Sox2 allele, and it was of interest to be able to extend these observations to independent osteoblastic cell lines. Furthermore, we wished to rule out the effect of excision of any Sox2 flanking sequences such as those that may encode regulatory microRNAs. To address these questions, we used two immortalized osteoblast cell lines that our laboratory had previously characterized: OB1, a PyLT immortalized immature osteoblast line, and OB5, a spontaneously immortalized cell line derived from p53+/- calvaria (6, 27). We also tested OB1-Apert cells, an OB1-derived clone carrying the activated FGFR2 bearing the S252W mutation found in Apert syndrome, that express high levels of Sox2, at least 10-fold more than the parental OB1 cells (8). We infected these cell lines with pBabe-hygro retrovirus expressing three shRNAs directed against murine Sox2, and a scrambled shRNA control and selected for Hygromycin resistant cell clones. These three shRNAs were found to differentially reduce Sox2 expression in the OB1-Apert cells (Figure 5A), ranging from 60 to 90% inhibition of Sox2 protein expression. As seen in Figure 5B, infection of OB1 or OB5 cells with Sox2 shRNA vectors completely abrogated colony formation. On the other hand, OB1-Apert cells were still capable of forming colonies, with an efficiency ranging between 25-85% of the colonies produced by the cells expressing the scrambled control shRNA (Fig. 5B). The differential ability of the shRNAs to inhibit Sox2 protein expression was reflected in the ability to form colonies, as the shRNA (1043) that caused the least reduction in Sox2 levels produced the highest number of colonies. The OB1-Apert cells expressing Sox2 shRNA 900 and 998 that could form colonies grew considerably more slowly than the parental cells (Fig. 5C), while the growth of cells expressing shRNA 1043 was not affected, as expected. The proliferation of NIH3T3 mouse fibroblasts, that do not express Sox2, was unaffected by Sox2 shRNAs as infection with all Sox2-specific shRNA viruses resulted in a number of hygromycin resistant colonies essentially identical to that produced by the scrambled shRNA control (not shown). These results demonstrate that the requirement for Sox2 expression is not limited to osteoblasts from Sox2 mutant mice and suggest that a threshold level of Sox2 expression is necessary to maintain osteoblast self-renewal.

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Show MeSH