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Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation.

Mansukhani A, Ambrosetti D, Holmes G, Cornivelli L, Basilico C - J. Cell Biol. (2005)

Bottom Line: Wnt signals promote osteoblast function and regulate bone mass.Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain.Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA. mansua01@med.nyu.edu

ABSTRACT
Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.

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Sox2 expression blocks osteoblast differentiation. (A) 105 cells per well of OB1/vector and OB1/Sox2-cl2 and OB1/Sox2-pool cells were plated in 6-well plates and placed in differentiation medium. Plates were stained for alkaline phosphatase (ALP) expression (purple-brown) on the indicated days. (B) Inhibition of ALP up-regulation upon differentiation (left) is evident in some OB1/Sox2 clones expressing Sox2 protein (right). White lines indicate that intervening lanes have been spliced out. (C) OB5 and OB5/Sox2 cells were seeded at 105 cells per plate and kept in differentiation medium. On the indicated days, cells were stained for ALP (purple) and for mineralization with Alizarin red (red).
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fig4: Sox2 expression blocks osteoblast differentiation. (A) 105 cells per well of OB1/vector and OB1/Sox2-cl2 and OB1/Sox2-pool cells were plated in 6-well plates and placed in differentiation medium. Plates were stained for alkaline phosphatase (ALP) expression (purple-brown) on the indicated days. (B) Inhibition of ALP up-regulation upon differentiation (left) is evident in some OB1/Sox2 clones expressing Sox2 protein (right). White lines indicate that intervening lanes have been spliced out. (C) OB5 and OB5/Sox2 cells were seeded at 105 cells per plate and kept in differentiation medium. On the indicated days, cells were stained for ALP (purple) and for mineralization with Alizarin red (red).

Mentions: To test whether Sox2 could interfere with osteoblast differentiation, we transfected OB1 cells with a murine Sox2 expression plasmid and isolated individual hygromycin-resistant clones as well as pools of resistant cells. Clones or pools were tested for their ability to differentiate and compared with a pool transfected with the empty vector (OB1/vector). Cells were plated and stained after 2 and 25 d for ALP (Fig. 4 A). Although at day 2 all the cells expressed very little ALP, after 25 d much fewer ALP-positive cells were visible in the Sox2-expressing cells relative to the control (Fig. 4 A). OB1/vector cells displayed the multilayering typical of differentiating cells and stained strongly for ALP. OB1/Sox2-pool cells that have a low level of Sox2 show much less differentiation than OB1/vector at day 25. The OB1/Sox2-cl2 cells that express high levels of Sox2 mRNA do not express ALP, do not form multilayers, and maintain the phenotype of undifferentiated cells (Fig. 4 A). In other clones that expressed relatively high levels of Sox2 protein, the block in differentiation was evident as measured by ALP staining (Fig. 4 B).


Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation.

Mansukhani A, Ambrosetti D, Holmes G, Cornivelli L, Basilico C - J. Cell Biol. (2005)

Sox2 expression blocks osteoblast differentiation. (A) 105 cells per well of OB1/vector and OB1/Sox2-cl2 and OB1/Sox2-pool cells were plated in 6-well plates and placed in differentiation medium. Plates were stained for alkaline phosphatase (ALP) expression (purple-brown) on the indicated days. (B) Inhibition of ALP up-regulation upon differentiation (left) is evident in some OB1/Sox2 clones expressing Sox2 protein (right). White lines indicate that intervening lanes have been spliced out. (C) OB5 and OB5/Sox2 cells were seeded at 105 cells per plate and kept in differentiation medium. On the indicated days, cells were stained for ALP (purple) and for mineralization with Alizarin red (red).
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fig4: Sox2 expression blocks osteoblast differentiation. (A) 105 cells per well of OB1/vector and OB1/Sox2-cl2 and OB1/Sox2-pool cells were plated in 6-well plates and placed in differentiation medium. Plates were stained for alkaline phosphatase (ALP) expression (purple-brown) on the indicated days. (B) Inhibition of ALP up-regulation upon differentiation (left) is evident in some OB1/Sox2 clones expressing Sox2 protein (right). White lines indicate that intervening lanes have been spliced out. (C) OB5 and OB5/Sox2 cells were seeded at 105 cells per plate and kept in differentiation medium. On the indicated days, cells were stained for ALP (purple) and for mineralization with Alizarin red (red).
Mentions: To test whether Sox2 could interfere with osteoblast differentiation, we transfected OB1 cells with a murine Sox2 expression plasmid and isolated individual hygromycin-resistant clones as well as pools of resistant cells. Clones or pools were tested for their ability to differentiate and compared with a pool transfected with the empty vector (OB1/vector). Cells were plated and stained after 2 and 25 d for ALP (Fig. 4 A). Although at day 2 all the cells expressed very little ALP, after 25 d much fewer ALP-positive cells were visible in the Sox2-expressing cells relative to the control (Fig. 4 A). OB1/vector cells displayed the multilayering typical of differentiating cells and stained strongly for ALP. OB1/Sox2-pool cells that have a low level of Sox2 show much less differentiation than OB1/vector at day 25. The OB1/Sox2-cl2 cells that express high levels of Sox2 mRNA do not express ALP, do not form multilayers, and maintain the phenotype of undifferentiated cells (Fig. 4 A). In other clones that expressed relatively high levels of Sox2 protein, the block in differentiation was evident as measured by ALP staining (Fig. 4 B).

Bottom Line: Wnt signals promote osteoblast function and regulate bone mass.Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain.Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA. mansua01@med.nyu.edu

ABSTRACT
Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.

Show MeSH
Related in: MedlinePlus