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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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Expression of Sox2 in osteoblasts. (A) mRNA for Sox2 is induced by FGF. OB1, AP, CR, or NIH3T3 cells were grown in the presence of serum and treated with 10 ng/ml FGF1 and 5 μg/ml heparin for the indicated times and analyzed by Northern blot with a 32P-labeled Sox2 cDNA (Yuan et al., 1995). Gapdh probe was used as a control. (B) Northern analysis of Sox2 expression in AP cells treated with DMSO (D) or SU5402 (SU) for 24 h. (C) Sox2 mRNA is induced by FGF1 in murine primary calvarial osteoblasts. (D and E) Western analysis of Sox2 induction by 24-h FGF1 treatment (+) in OB1 and OB5 osteoblasts and in U2OS and primary calvarial osteoblasts. (F) Serial sagittal sections through the coronal suture of a wild-type mouse at P1 is shown (frontal bone at left, parietal bone at right; of, osteogenic front). ALP expression was detected by enzymatic histochemistry, whereas Sox2 and Osteopontin RNA expression was detected by in situ hybridization using anti-sense riboprobes labeled with digoxigenin-UTP. ALP is present in immature and maturing osteoblasts, Sox2 expression is restricted to the immature osteoblasts of the osteogenic fronts. Osteopontin is expressed in maturing osteoblasts.
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fig3: Expression of Sox2 in osteoblasts. (A) mRNA for Sox2 is induced by FGF. OB1, AP, CR, or NIH3T3 cells were grown in the presence of serum and treated with 10 ng/ml FGF1 and 5 μg/ml heparin for the indicated times and analyzed by Northern blot with a 32P-labeled Sox2 cDNA (Yuan et al., 1995). Gapdh probe was used as a control. (B) Northern analysis of Sox2 expression in AP cells treated with DMSO (D) or SU5402 (SU) for 24 h. (C) Sox2 mRNA is induced by FGF1 in murine primary calvarial osteoblasts. (D and E) Western analysis of Sox2 induction by 24-h FGF1 treatment (+) in OB1 and OB5 osteoblasts and in U2OS and primary calvarial osteoblasts. (F) Serial sagittal sections through the coronal suture of a wild-type mouse at P1 is shown (frontal bone at left, parietal bone at right; of, osteogenic front). ALP expression was detected by enzymatic histochemistry, whereas Sox2 and Osteopontin RNA expression was detected by in situ hybridization using anti-sense riboprobes labeled with digoxigenin-UTP. ALP is present in immature and maturing osteoblasts, Sox2 expression is restricted to the immature osteoblasts of the osteogenic fronts. Osteopontin is expressed in maturing osteoblasts.

Mentions: To further analyze the FGF-mediated induction of Sox2 in osteoblasts, we treated OB1, AP, and CR cells with FGF1 for various times and examined the expression of Sox2 mRNA by Northern analysis. Fig. 3 A shows that Sox2 mRNA was strongly induced by FGF treatment in the OB1 cells, whereas the induction is modest in AP and CR cells that have a high basal level. Treatment of AP cells with an inhibitor of FGFR kinase activity (SU5402) drastically reduced the level of Sox2 mRNA, indicating that Sox2 expression is dependent on FGF signaling (Fig. 3 B). We examined whether or not this response was specific to osteoblasts. The induction of Sox2 mRNA by FGF was not observed in NIH3T3 fibroblasts (Fig. 3 A), RCS chondrocytes, or in mouse embryo fibroblasts (not depicted). To ensure that the specificity of Sox2 induction was not a peculiarity of OB1 cells, we also determined that Sox2 mRNA is induced by FGF treatment of primary murine calvarial osteoblasts (Fig. 3 C).


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)

Expression of Sox2 in osteoblasts. (A) mRNA for Sox2 is induced by FGF. OB1, AP, CR, or NIH3T3 cells were grown in the presence of serum and treated with 10 ng/ml FGF1 and 5 μg/ml heparin for the indicated times and analyzed by Northern blot with a 32P-labeled Sox2 cDNA (Yuan et al., 1995). Gapdh probe was used as a control. (B) Northern analysis of Sox2 expression in AP cells treated with DMSO (D) or SU5402 (SU) for 24 h. (C) Sox2 mRNA is induced by FGF1 in murine primary calvarial osteoblasts. (D and E) Western analysis of Sox2 induction by 24-h FGF1 treatment (+) in OB1 and OB5 osteoblasts and in U2OS and primary calvarial osteoblasts. (F) Serial sagittal sections through the coronal suture of a wild-type mouse at P1 is shown (frontal bone at left, parietal bone at right; of, osteogenic front). ALP expression was detected by enzymatic histochemistry, whereas Sox2 and Osteopontin RNA expression was detected by in situ hybridization using anti-sense riboprobes labeled with digoxigenin-UTP. ALP is present in immature and maturing osteoblasts, Sox2 expression is restricted to the immature osteoblasts of the osteogenic fronts. Osteopontin is expressed in maturing osteoblasts.
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Related In: Results  -  Collection

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fig3: Expression of Sox2 in osteoblasts. (A) mRNA for Sox2 is induced by FGF. OB1, AP, CR, or NIH3T3 cells were grown in the presence of serum and treated with 10 ng/ml FGF1 and 5 μg/ml heparin for the indicated times and analyzed by Northern blot with a 32P-labeled Sox2 cDNA (Yuan et al., 1995). Gapdh probe was used as a control. (B) Northern analysis of Sox2 expression in AP cells treated with DMSO (D) or SU5402 (SU) for 24 h. (C) Sox2 mRNA is induced by FGF1 in murine primary calvarial osteoblasts. (D and E) Western analysis of Sox2 induction by 24-h FGF1 treatment (+) in OB1 and OB5 osteoblasts and in U2OS and primary calvarial osteoblasts. (F) Serial sagittal sections through the coronal suture of a wild-type mouse at P1 is shown (frontal bone at left, parietal bone at right; of, osteogenic front). ALP expression was detected by enzymatic histochemistry, whereas Sox2 and Osteopontin RNA expression was detected by in situ hybridization using anti-sense riboprobes labeled with digoxigenin-UTP. ALP is present in immature and maturing osteoblasts, Sox2 expression is restricted to the immature osteoblasts of the osteogenic fronts. Osteopontin is expressed in maturing osteoblasts.
Mentions: To further analyze the FGF-mediated induction of Sox2 in osteoblasts, we treated OB1, AP, and CR cells with FGF1 for various times and examined the expression of Sox2 mRNA by Northern analysis. Fig. 3 A shows that Sox2 mRNA was strongly induced by FGF treatment in the OB1 cells, whereas the induction is modest in AP and CR cells that have a high basal level. Treatment of AP cells with an inhibitor of FGFR kinase activity (SU5402) drastically reduced the level of Sox2 mRNA, indicating that Sox2 expression is dependent on FGF signaling (Fig. 3 B). We examined whether or not this response was specific to osteoblasts. The induction of Sox2 mRNA by FGF was not observed in NIH3T3 fibroblasts (Fig. 3 A), RCS chondrocytes, or in mouse embryo fibroblasts (not depicted). To ensure that the specificity of Sox2 induction was not a peculiarity of OB1 cells, we also determined that Sox2 mRNA is induced by FGF treatment of primary murine calvarial osteoblasts (Fig. 3 C).

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