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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 affects the Wnt pathway. (A) Schematic representation of Sox2 constructs. (B) Sox 2 interferes with the β-catenin–mediated activation of a TCF/LEF reporter construct. 293 cells were transiently cotransfected with TOPFLASH, a stable β-catenin mutant (ΔN89), and increasing amounts of wild-type Sox 2 and Sox2 mutants (40 ng) as indicated. (C) Expression of the Sox2 constructs and of β-catenin was verified by Western blotting of cell lysates using antibodies against β-catenin, Sox2, or tubulin. (D) Sox2 associates with β-catenin in osteoblasts. 1 mg of whole cell extracts (WCE) from untreated (−) or cells treated with 10 ng/ml FGF1 for 24 h (+) were immunoprecipitated (IP) with antibodies against Sox2 or β-catenin and run on SDS PAGE. Western blots were immunoblotted (IB) with the indicated antibodies. 20 μg of whole cell extracts from CR cells was run as a control.
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fig6: Sox2 affects the Wnt pathway. (A) Schematic representation of Sox2 constructs. (B) Sox 2 interferes with the β-catenin–mediated activation of a TCF/LEF reporter construct. 293 cells were transiently cotransfected with TOPFLASH, a stable β-catenin mutant (ΔN89), and increasing amounts of wild-type Sox 2 and Sox2 mutants (40 ng) as indicated. (C) Expression of the Sox2 constructs and of β-catenin was verified by Western blotting of cell lysates using antibodies against β-catenin, Sox2, or tubulin. (D) Sox2 associates with β-catenin in osteoblasts. 1 mg of whole cell extracts (WCE) from untreated (−) or cells treated with 10 ng/ml FGF1 for 24 h (+) were immunoprecipitated (IP) with antibodies against Sox2 or β-catenin and run on SDS PAGE. Western blots were immunoblotted (IB) with the indicated antibodies. 20 μg of whole cell extracts from CR cells was run as a control.

Mentions: To test whether or not Sox2 could repress Wnt–β-catenin–induced gene expression, we assessed the effect of increasing amounts of Sox2 on the expression of the TOPFLASH Wnt reporter plasmid. When TOPFLASH is cotransfected with a plasmid encoding a constitutively active form of β-catenin (Δ N89), basal luciferase activity was strongly increased in 293 cells (Fig. 6 A). Cotransfection with increasing amounts of the Sox2 encoding plasmid reduced the β-catenin–induced activity of TOPFLASH in a dose-dependent manner, whereas Sox2 alone had no effect on the basal activity of TOPFLASH (Fig. 6 A). A control plasmid (FOPFLASH) containing mutated TCF/LEF sites was unaffected by cotransfection of active β-catenin or Sox2 (unpublished data).


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 affects the Wnt pathway. (A) Schematic representation of Sox2 constructs. (B) Sox 2 interferes with the β-catenin–mediated activation of a TCF/LEF reporter construct. 293 cells were transiently cotransfected with TOPFLASH, a stable β-catenin mutant (ΔN89), and increasing amounts of wild-type Sox 2 and Sox2 mutants (40 ng) as indicated. (C) Expression of the Sox2 constructs and of β-catenin was verified by Western blotting of cell lysates using antibodies against β-catenin, Sox2, or tubulin. (D) Sox2 associates with β-catenin in osteoblasts. 1 mg of whole cell extracts (WCE) from untreated (−) or cells treated with 10 ng/ml FGF1 for 24 h (+) were immunoprecipitated (IP) with antibodies against Sox2 or β-catenin and run on SDS PAGE. Western blots were immunoblotted (IB) with the indicated antibodies. 20 μg of whole cell extracts from CR cells was run as a control.
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Related In: Results  -  Collection

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fig6: Sox2 affects the Wnt pathway. (A) Schematic representation of Sox2 constructs. (B) Sox 2 interferes with the β-catenin–mediated activation of a TCF/LEF reporter construct. 293 cells were transiently cotransfected with TOPFLASH, a stable β-catenin mutant (ΔN89), and increasing amounts of wild-type Sox 2 and Sox2 mutants (40 ng) as indicated. (C) Expression of the Sox2 constructs and of β-catenin was verified by Western blotting of cell lysates using antibodies against β-catenin, Sox2, or tubulin. (D) Sox2 associates with β-catenin in osteoblasts. 1 mg of whole cell extracts (WCE) from untreated (−) or cells treated with 10 ng/ml FGF1 for 24 h (+) were immunoprecipitated (IP) with antibodies against Sox2 or β-catenin and run on SDS PAGE. Western blots were immunoblotted (IB) with the indicated antibodies. 20 μg of whole cell extracts from CR cells was run as a control.
Mentions: To test whether or not Sox2 could repress Wnt–β-catenin–induced gene expression, we assessed the effect of increasing amounts of Sox2 on the expression of the TOPFLASH Wnt reporter plasmid. When TOPFLASH is cotransfected with a plasmid encoding a constitutively active form of β-catenin (Δ N89), basal luciferase activity was strongly increased in 293 cells (Fig. 6 A). Cotransfection with increasing amounts of the Sox2 encoding plasmid reduced the β-catenin–induced activity of TOPFLASH in a dose-dependent manner, whereas Sox2 alone had no effect on the basal activity of TOPFLASH (Fig. 6 A). A control plasmid (FOPFLASH) containing mutated TCF/LEF sites was unaffected by cotransfection of active β-catenin or Sox2 (unpublished data).

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