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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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Sox2 inactivation impairs proliferation of primary osteoblasts(A) PCR analysis of primary osteoblast DNA. Primary calvarial osteoblasts were prepared from Sox2flox/-;Cre (cko) mice and Sox2flox/+;Cre (control). Upon confluency, cells were passaged and DNA was extracted at each passage. The deleted flox and the flox allele were detected by PCR using specific primers to assess persistence of Sox2- cells. Numbers indicate passage number following isolation.(B) PCR analysis of GFP- and Cre-virus infected primary osteoblast DNA. Primary osteoblasts from Sox2 flox/+ (control) and Sox2 flox/- (cko) mice were infected with GFP or Cre-adenovirus and passaged after infection. DNA was extracted from each passage and the deleted flox allele was detected by PCR using specific primers to check for persistence of Sox2- cells.(C) Colony Assay. Primary calvarial osteoblasts from Sox2 flox/+ (control) and Sox2flox/- (cko) mice were infected with either GFP or Cre-adenovirus for 72 hours and plated in triplicate in 6-well plates. Colonies were counted after crystal violet staining. Percent of colonies obtained in Cre infection are plotted as a percentage of the colonies in the corresponding GFP infection in two independent Sox2 flox/- and Sox2 flox/+ osteoblast pools [(1) and (2)]. * = p<0.05. GFP Adenovirus infection of Sox2flox/- or Sox2flox/+ cells produced equivalent number of colonies (~120 colonies/plate).
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Figure 2: Sox2 inactivation impairs proliferation of primary osteoblasts(A) PCR analysis of primary osteoblast DNA. Primary calvarial osteoblasts were prepared from Sox2flox/-;Cre (cko) mice and Sox2flox/+;Cre (control). Upon confluency, cells were passaged and DNA was extracted at each passage. The deleted flox and the flox allele were detected by PCR using specific primers to assess persistence of Sox2- cells. Numbers indicate passage number following isolation.(B) PCR analysis of GFP- and Cre-virus infected primary osteoblast DNA. Primary osteoblasts from Sox2 flox/+ (control) and Sox2 flox/- (cko) mice were infected with GFP or Cre-adenovirus and passaged after infection. DNA was extracted from each passage and the deleted flox allele was detected by PCR using specific primers to check for persistence of Sox2- cells.(C) Colony Assay. Primary calvarial osteoblasts from Sox2 flox/+ (control) and Sox2flox/- (cko) mice were infected with either GFP or Cre-adenovirus for 72 hours and plated in triplicate in 6-well plates. Colonies were counted after crystal violet staining. Percent of colonies obtained in Cre infection are plotted as a percentage of the colonies in the corresponding GFP infection in two independent Sox2 flox/- and Sox2 flox/+ osteoblast pools [(1) and (2)]. * = p<0.05. GFP Adenovirus infection of Sox2flox/- or Sox2flox/+ cells produced equivalent number of colonies (~120 colonies/plate).

Mentions: To analyze the phenotype of Sox2 osteoblasts, we generated cultures of calvarial osteoblasts from the mutant mice. PCR analysis of DNA from freshly cultured cells showed that deletion of the floxed Sox2 allele had occurred. However, after culturing osteoblast pools from the P1 calvaria of the mutant Sox2flox/-;Cre mice for 3-4 passages, the deleted Sox2 allele could no longer be detected by PCR analysis, while it was maintained in cultures from Sox2flox/+;Cre mice, which carry a wild type Sox2 allele that cannot be excised by the Cre recombinase, suggesting that Sox2 deletion is incompatible with osteoblast survival (Fig. 2A). To directly determine whether Sox2 inactivation was deleterious to cell survival, we infected freshly cultured primary Sox2flox/- osteoblast populations with a Cre-expressing adenovirus (also expressing GFP linked to Cre by an IRES) to induce Sox2 excision. We could show that Sox2 excision had taken place in a majority of the cells by PCR analysis, but again, after 2-3 passages in culture Sox2-negative cells could no longer be detected (Fig. 2B). To better quantitate these experiments, we performed colony assays. We infected independent cultures of primary osteoblasts derived from Sox2flox/- mice with the Cre-expressing adenovirus and the corresponding control adenovirus expressing only GFP. While cells infected in the GFP-virus yielded a very similar number of colonies, Cre-virus infection drastically reduced the colony forming ability of these cultures. In contrast, cultures of primary osteoblasts from Sox2flox/+ mice were completely resistant to the effect of Cre-virus infection (Fig. 2C). Together these results strongly suggested that Sox2 inactivation abolished the ability of osteoblasts to sustain proliferation in culture


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)

Sox2 inactivation impairs proliferation of primary osteoblasts(A) PCR analysis of primary osteoblast DNA. Primary calvarial osteoblasts were prepared from Sox2flox/-;Cre (cko) mice and Sox2flox/+;Cre (control). Upon confluency, cells were passaged and DNA was extracted at each passage. The deleted flox and the flox allele were detected by PCR using specific primers to assess persistence of Sox2- cells. Numbers indicate passage number following isolation.(B) PCR analysis of GFP- and Cre-virus infected primary osteoblast DNA. Primary osteoblasts from Sox2 flox/+ (control) and Sox2 flox/- (cko) mice were infected with GFP or Cre-adenovirus and passaged after infection. DNA was extracted from each passage and the deleted flox allele was detected by PCR using specific primers to check for persistence of Sox2- cells.(C) Colony Assay. Primary calvarial osteoblasts from Sox2 flox/+ (control) and Sox2flox/- (cko) mice were infected with either GFP or Cre-adenovirus for 72 hours and plated in triplicate in 6-well plates. Colonies were counted after crystal violet staining. Percent of colonies obtained in Cre infection are plotted as a percentage of the colonies in the corresponding GFP infection in two independent Sox2 flox/- and Sox2 flox/+ osteoblast pools [(1) and (2)]. * = p<0.05. GFP Adenovirus infection of Sox2flox/- or Sox2flox/+ cells produced equivalent number of colonies (~120 colonies/plate).
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Figure 2: Sox2 inactivation impairs proliferation of primary osteoblasts(A) PCR analysis of primary osteoblast DNA. Primary calvarial osteoblasts were prepared from Sox2flox/-;Cre (cko) mice and Sox2flox/+;Cre (control). Upon confluency, cells were passaged and DNA was extracted at each passage. The deleted flox and the flox allele were detected by PCR using specific primers to assess persistence of Sox2- cells. Numbers indicate passage number following isolation.(B) PCR analysis of GFP- and Cre-virus infected primary osteoblast DNA. Primary osteoblasts from Sox2 flox/+ (control) and Sox2 flox/- (cko) mice were infected with GFP or Cre-adenovirus and passaged after infection. DNA was extracted from each passage and the deleted flox allele was detected by PCR using specific primers to check for persistence of Sox2- cells.(C) Colony Assay. Primary calvarial osteoblasts from Sox2 flox/+ (control) and Sox2flox/- (cko) mice were infected with either GFP or Cre-adenovirus for 72 hours and plated in triplicate in 6-well plates. Colonies were counted after crystal violet staining. Percent of colonies obtained in Cre infection are plotted as a percentage of the colonies in the corresponding GFP infection in two independent Sox2 flox/- and Sox2 flox/+ osteoblast pools [(1) and (2)]. * = p<0.05. GFP Adenovirus infection of Sox2flox/- or Sox2flox/+ cells produced equivalent number of colonies (~120 colonies/plate).
Mentions: To analyze the phenotype of Sox2 osteoblasts, we generated cultures of calvarial osteoblasts from the mutant mice. PCR analysis of DNA from freshly cultured cells showed that deletion of the floxed Sox2 allele had occurred. However, after culturing osteoblast pools from the P1 calvaria of the mutant Sox2flox/-;Cre mice for 3-4 passages, the deleted Sox2 allele could no longer be detected by PCR analysis, while it was maintained in cultures from Sox2flox/+;Cre mice, which carry a wild type Sox2 allele that cannot be excised by the Cre recombinase, suggesting that Sox2 deletion is incompatible with osteoblast survival (Fig. 2A). To directly determine whether Sox2 inactivation was deleterious to cell survival, we infected freshly cultured primary Sox2flox/- osteoblast populations with a Cre-expressing adenovirus (also expressing GFP linked to Cre by an IRES) to induce Sox2 excision. We could show that Sox2 excision had taken place in a majority of the cells by PCR analysis, but again, after 2-3 passages in culture Sox2-negative cells could no longer be detected (Fig. 2B). To better quantitate these experiments, we performed colony assays. We infected independent cultures of primary osteoblasts derived from Sox2flox/- mice with the Cre-expressing adenovirus and the corresponding control adenovirus expressing only GFP. While cells infected in the GFP-virus yielded a very similar number of colonies, Cre-virus infection drastically reduced the colony forming ability of these cultures. In contrast, cultures of primary osteoblasts from Sox2flox/+ mice were completely resistant to the effect of Cre-virus infection (Fig. 2C). Together these results strongly suggested that Sox2 inactivation abolished the ability of osteoblasts to sustain proliferation in culture

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
Related in: MedlinePlus